Methods In Stem Cell Biology Part B Ilio Vitale Gwenola Manic
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Methods In Stem Cell Biology Part B Ilio Vitale Gwenola Manic
Methods In Stem Cell Biology Part B Ilio Vitale Gwenola Manic
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Series Editor
Lorenzo Galluzzi
Weill Cornell Medical College,
New York, NY, United States
Lorenzo Galluzzi
Weill Cornell Medical College,
New York, NY, United States
Academic Press is an imprint of Elsevier
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
525 B Street, Suite 1650, San Diego, CA 92101, United States
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
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First edition 2022
Copyright © 2022 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
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(other
than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our understanding, changes in research methods, professional practices, or medical treatment may
become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds, or experiments described herein. In using such information
or methods they should be mindful of their own safety and the safety of others, including parties for whom
they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any
liability for any injury and/or damage to persons or property as a matter of products liability, negligence or
otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the
material herein.
ISBN: 978-0-323-90018-8
ISSN: 0091-679X
For information on all Academic Press publications
visit our website athttps://www.elsevier.com/books-and-journals
Publisher:Zoe
Kruze
Developmental Editor:Naiza Ermin Mendoza
Production Project Manager:Vijayaraj Purushothaman
Cover Designer:Matthew Limbert
Typeset by STRAIVE, India
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
525 B Street, Suite 1650, San Diego, CA 92101, United States
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
125 London Wall, London, EC2Y 5AS, United Kingdom
First edition 2022
Copyright © 2022 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about
the Publisher’s permissions policies and our arrangements with organizations such as the Copyright
Clearance Center and the Copyright Licensing Agency, can be found at our website:www.elsevier.com/
permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher
(other
than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our understanding, changes in research methods, professional practices, or medical treatment may
become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds, or experiments described herein. In using such information
or methods they should be mindful of their own safety and the safety of others, including parties for whom
they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any
liability for any injury and/or damage to persons or property as a matter of products liability, negligence or
otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the
material herein.
ISBN: 978-0-323-90018-8
ISSN: 0091-679X
For information on all Academic Press publications
visit our website athttps://www.elsevier.com/books-and-journals
Publisher:Zoe
Kruze
Developmental Editor:Naiza Ermin Mendoza
Production Project Manager:Vijayaraj Purushothaman
Cover Designer:Matthew Limbert
Typeset by STRAIVE, India
Contents
Contributors.............................................................................................................xi
CHAPTER 1 Multiple gene knockdown strategies for
investigating the properties of human leukemia
stem cells and exploring new therapies.....................1
Andrew Wu, Aysha S. Ansari, Hasan Uludag, and
Xiaoyan Jiang
1.Introduction....................................................................................2
2.Materials.........................................................................................6
3.Methods..........................................................................................8
4.Notes............................................................................................13
5.Concluding remarks.....................................................................18
Acknowledgments.............................................................................19
Disclosures.........................................................................................19
References.........................................................................................19
CHAPTER 2 Evaluation of cancer stem cells markers
expression in HCC trough real-time polymerase
chain reaction............................................................23
Barbara Bueloni, Esteban Fiore, Manuel Gidekel,
Juan Bayo, and Guillermo Mazzolini
1.Introduction and rationale............................................................24
2.Materials.......................................................................................25
3.Methods........................................................................................25
4.Results and discussion.................................................................31
Disclosures.........................................................................................31
References.........................................................................................32
CHAPTER 3 Reverse Phase Protein Arrays in cancer stem cells...33
Michele Signore and Valeria Manganelli
1.Introduction..................................................................................34
2.Materials.......................................................................................36
3.Methods........................................................................................40
4.Notes............................................................................................48
5.Concluding remarks.....................................................................55
Disclosures.........................................................................................56
References.........................................................................................57
v
Contributors.............................................................................................................xi
CHAPTER 1 Multiple gene knockdown strategies for
investigating the properties of human leukemia
stem cells and exploring new therapies.....................1
Andrew Wu, Aysha S. Ansari, Hasan Uludag, and
Xiaoyan Jiang
1.Introduction....................................................................................2
2.Materials.........................................................................................6
3.Methods..........................................................................................8
4.Notes............................................................................................13
5.Concluding remarks.....................................................................18
Acknowledgments.............................................................................19
Disclosures.........................................................................................19
References.........................................................................................19
CHAPTER 2 Evaluation of cancer stem cells markers
expression in HCC trough real-time polymerase
chain reaction............................................................23
Barbara Bueloni, Esteban Fiore, Manuel Gidekel,
Juan Bayo, and Guillermo Mazzolini
1.Introduction and rationale............................................................24
2.Materials.......................................................................................25
3.Methods........................................................................................25
4.Results and discussion.................................................................31
Disclosures.........................................................................................31
References.........................................................................................32
CHAPTER 3 Reverse Phase Protein Arrays in cancer stem cells...33
Michele Signore and Valeria Manganelli
1.Introduction..................................................................................34
2.Materials.......................................................................................36
3.Methods........................................................................................40
4.Notes............................................................................................48
5.Concluding remarks.....................................................................55
Disclosures.........................................................................................56
References.........................................................................................57
v
CHAPTER 4 Isolation of cancer stem cells from skin
squamous cell carcinoma..........................................63
Priyanka Joshi, Dnyanada S. Ghadi, and
Sanjeev K. Waghmare
1.Introduction..................................................................................64
2.Materials.......................................................................................69
3.Methods........................................................................................70
4.CSCs assays.................................................................................75
5.Precautions to be taken during CSC isolation to avoid
commonly faced problems...........................................................76
6.Concluding remarks.....................................................................77
Disclosures.........................................................................................78
References.........................................................................................78
CHAPTER 5 Immunoblotting-assisted assessment
of JAK/STAT and PI3K/Akt/mTOR signaling
in myeloproliferative neoplasms CD34+ stem
cells............................................................................81
Laura Calabresi, Manjola Balliu, and Niccolo`Bartalucci
1.Introduction..................................................................................82
2.Materials.......................................................................................85
3.Isolation of mononuclear cells (MNCs) from human
peripheral blood by density gradient centrifugation...................89
4.Immunomagnetic separation of hematopoietic stem cells
(CD34+) from MNCs..................................................................90
5.Protein extraction and quantification from CD34+ cells............91
6.Sample preparation and SDS-PAGE Western blotting...............92
7.Membranes probing to detect phosphorylated proteins of
JAK/STAT and PI3K/mTOR/Akt pathway.................................94
8.Phosphorylated protein revelation and data acquisition..............95
9.Membrane stripping and re-probing to detect total protein of
JAK/STAT and PI3K/mTOR/Akt pathway.................................96
10.Human colony forming unit (CFU) assay of hematopoietic
stem cells (CD34+)......................................................................99
11.Notes..........................................................................................102
12.Concluding remarks...................................................................105
Disclosures........................................................................................106
References.........................................................................................106
vi Contents
squamous cell carcinoma..........................................63
Priyanka Joshi, Dnyanada S. Ghadi, and
Sanjeev K. Waghmare
1.Introduction..................................................................................64
2.Materials.......................................................................................69
3.Methods........................................................................................70
4.CSCs assays.................................................................................75
5.Precautions to be taken during CSC isolation to avoid
commonly faced problems...........................................................76
6.Concluding remarks.....................................................................77
Disclosures.........................................................................................78
References.........................................................................................78
CHAPTER 5 Immunoblotting-assisted assessment
of JAK/STAT and PI3K/Akt/mTOR signaling
in myeloproliferative neoplasms CD34+ stem
cells............................................................................81
Laura Calabresi, Manjola Balliu, and Niccolo`Bartalucci
1.Introduction..................................................................................82
2.Materials.......................................................................................85
3.Isolation of mononuclear cells (MNCs) from human
peripheral blood by density gradient centrifugation...................89
4.Immunomagnetic separation of hematopoietic stem cells
(CD34+) from MNCs..................................................................90
5.Protein extraction and quantification from CD34+ cells............91
6.Sample preparation and SDS-PAGE Western blotting...............92
7.Membranes probing to detect phosphorylated proteins of
JAK/STAT and PI3K/mTOR/Akt pathway.................................94
8.Phosphorylated protein revelation and data acquisition..............95
9.Membrane stripping and re-probing to detect total protein of
JAK/STAT and PI3K/mTOR/Akt pathway.................................96
10.Human colony forming unit (CFU) assay of hematopoietic
stem cells (CD34+)......................................................................99
11.Notes..........................................................................................102
12.Concluding remarks...................................................................105
Disclosures........................................................................................106
References.........................................................................................106
vi Contents
CHAPTER 6 Isolation of cancer stem cells from cultured
breast cancer cells and xenografted breast
tumors based on aldehyde dehydrogenase
activity......................................................................111
Wasundara Fernando, Derek Rowter, Raj Pranap Arun,
Jaganathan Venkatesh, and Paola Marcato
1.Background................................................................................112
2.The Aldefluor
TM
assay to isolate putative cancer stem cells....113
3.The importance of ALDH1A1 and ALDH1A3 in breast
cancer.........................................................................................113
4.Conclusions................................................................................122
Acknowledgments...........................................................................122
References.......................................................................................123
CHAPTER 7 Intracerebroventricular transplantation of human
iPSC-derived neural stem cells (hiPSC-NSCs)
into neonatal mice...................................................127
Marco Luciani, Chiara Garsia, Elisabeth Mangiameli,
Vasco Meneghini, and Angela Gritti
1.Introduction................................................................................128
2.Materials.....................................................................................130
3.Methods......................................................................................133
4.Notes..........................................................................................140
5.Concluding remarks...................................................................143
Acknowledgments...........................................................................144
References.......................................................................................144
CHAPTER 8 Quantifying epithelial-mesenchymal
heterogeneity and EMT scoring in tumor samples
via tyramide signal amplification (TSA)..................149
Meredith S. Brown, Behnaz Abdollahi, Saeed Hassanpour,
and Diwakar R. Pattabiraman
1.Introduction................................................................................150
2.Materials.....................................................................................153
3.Methods......................................................................................153
4.Imaging and analysis.................................................................156
5.Quantification.............................................................................158
6.Concluding Remarks..................................................................158
7.Limitations.................................................................................158
8.Notes..........................................................................................159
References.......................................................................................159
viiContents
breast cancer cells and xenografted breast
tumors based on aldehyde dehydrogenase
activity......................................................................111
Wasundara Fernando, Derek Rowter, Raj Pranap Arun,
Jaganathan Venkatesh, and Paola Marcato
1.Background................................................................................112
2.The Aldefluor
TM
assay to isolate putative cancer stem cells....113
3.The importance of ALDH1A1 and ALDH1A3 in breast
cancer.........................................................................................113
4.Conclusions................................................................................122
Acknowledgments...........................................................................122
References.......................................................................................123
CHAPTER 7 Intracerebroventricular transplantation of human
iPSC-derived neural stem cells (hiPSC-NSCs)
into neonatal mice...................................................127
Marco Luciani, Chiara Garsia, Elisabeth Mangiameli,
Vasco Meneghini, and Angela Gritti
1.Introduction................................................................................128
2.Materials.....................................................................................130
3.Methods......................................................................................133
4.Notes..........................................................................................140
5.Concluding remarks...................................................................143
Acknowledgments...........................................................................144
References.......................................................................................144
CHAPTER 8 Quantifying epithelial-mesenchymal
heterogeneity and EMT scoring in tumor samples
via tyramide signal amplification (TSA)..................149
Meredith S. Brown, Behnaz Abdollahi, Saeed Hassanpour,
and Diwakar R. Pattabiraman
1.Introduction................................................................................150
2.Materials.....................................................................................153
3.Methods......................................................................................153
4.Imaging and analysis.................................................................156
5.Quantification.............................................................................158
6.Concluding Remarks..................................................................158
7.Limitations.................................................................................158
8.Notes..........................................................................................159
References.......................................................................................159
viiContents
CHAPTER 9 Immunofluorescence staining of colorectal
cancer patient-derived organoids...........................163
Joseph L. Regan
1.Introduction................................................................................164
2.Materials.....................................................................................165
3.Method.......................................................................................166
4.Notes..........................................................................................168
5.Concluding remarks...................................................................169
Disclosures.......................................................................................169
References.......................................................................................169
CHAPTER 10 Isolation of murine bone marrow hematopoietic
stem and progenitor cell populations via flow
cytometry..................................................................173
Weijia Yu, Tessa Schmachtel, Malak Fawaz, and
Michael A. Rieger
1.Introduction................................................................................174
2.Materials.....................................................................................177
3.Methods......................................................................................178
4.Notes..........................................................................................184
5.Concluding remarks...................................................................190
Acknowledgments...........................................................................191
Conflict of interest...........................................................................191
Disclosures.......................................................................................191
References.......................................................................................191
CHAPTER 11 Limiting dilution assay to quantify the self-
renewal potential of cancer stem cells in
hepatocellular carcinoma........................................197
Yingying Lai, Bin Wang, and Xi Zheng
1.Introduction................................................................................198
2.Materials.....................................................................................201
3.Method.......................................................................................202
4.NOTES.......................................................................................209
Funding............................................................................................211
References.......................................................................................211
viiiContents
cancer patient-derived organoids...........................163
Joseph L. Regan
1.Introduction................................................................................164
2.Materials.....................................................................................165
3.Method.......................................................................................166
4.Notes..........................................................................................168
5.Concluding remarks...................................................................169
Disclosures.......................................................................................169
References.......................................................................................169
CHAPTER 10 Isolation of murine bone marrow hematopoietic
stem and progenitor cell populations via flow
cytometry..................................................................173
Weijia Yu, Tessa Schmachtel, Malak Fawaz, and
Michael A. Rieger
1.Introduction................................................................................174
2.Materials.....................................................................................177
3.Methods......................................................................................178
4.Notes..........................................................................................184
5.Concluding remarks...................................................................190
Acknowledgments...........................................................................191
Conflict of interest...........................................................................191
Disclosures.......................................................................................191
References.......................................................................................191
CHAPTER 11 Limiting dilution assay to quantify the self-
renewal potential of cancer stem cells in
hepatocellular carcinoma........................................197
Yingying Lai, Bin Wang, and Xi Zheng
1.Introduction................................................................................198
2.Materials.....................................................................................201
3.Method.......................................................................................202
4.NOTES.......................................................................................209
Funding............................................................................................211
References.......................................................................................211
viiiContents
CHAPTER 12 Methods to isolate adipose tissue-derived stem
cells..........................................................................215
Letizia Ferroni, Francesco De Francesco, Paolo Pinton,
Chiara Gardin, and Barbara Zavan
1.Introduction................................................................................216
2.Stromal vascular fraction (SVF) and adipose tissue-derived
stem cells (ADSCs)....................................................................216
3.Enzymatic isolation of ADSCs: An overview...........................218
4.Non-enzymatic isolation of SVF andADSCs...........................222
5.Novel non-enzymatic systems of ADSCs isolation..................224
6.Conclusions................................................................................225
References.......................................................................................226
CHAPTER 13 Generation of neuronal/glial mixed cultures from
human induced pluripotent stem cells (hiPSCs).....229
Elisabeth Mangiameli, Marta Freschi, Marco Luciani, and
Angela Gritti
1.Introduction................................................................................231
2.Materials.....................................................................................233
3.Methods......................................................................................236
4.Expected results.........................................................................238
5.Notes..........................................................................................241
6.Concluding remarks...................................................................243
Acknowledgments...........................................................................243
References.......................................................................................243
ixContents
cells..........................................................................215
Letizia Ferroni, Francesco De Francesco, Paolo Pinton,
Chiara Gardin, and Barbara Zavan
1.Introduction................................................................................216
2.Stromal vascular fraction (SVF) and adipose tissue-derived
stem cells (ADSCs)....................................................................216
3.Enzymatic isolation of ADSCs: An overview...........................218
4.Non-enzymatic isolation of SVF andADSCs...........................222
5.Novel non-enzymatic systems of ADSCs isolation..................224
6.Conclusions................................................................................225
References.......................................................................................226
CHAPTER 13 Generation of neuronal/glial mixed cultures from
human induced pluripotent stem cells (hiPSCs).....229
Elisabeth Mangiameli, Marta Freschi, Marco Luciani, and
Angela Gritti
1.Introduction................................................................................231
2.Materials.....................................................................................233
3.Methods......................................................................................236
4.Expected results.........................................................................238
5.Notes..........................................................................................241
6.Concluding remarks...................................................................243
Acknowledgments...........................................................................243
References.......................................................................................243
ixContents
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Contributors
Behnaz Abdollahi
Department of Biomedical Data Science, Geisel School of Medicine at Dartmouth,
Hanover, NH, United States
Aysha S. Ansari
Department of Chemical & Materials Engineering, Faculty of Engineering,
University of Alberta, Edmonton, AB, Canada
Raj Pranap Arun
Department of Pathology, Dalhousie University, Halifax, NS, Canada
Manjola Balliu
Center for Research and Innovation for Myeloproliferative Neoplasms,
Department of Experimental and Clinical Medicine, University of Florence,
Florence, Italy
Niccolo`Bartalucci
Center for Research and Innovation for Myeloproliferative Neoplasms,
Department of Experimental and Clinical Medicine, University of Florence,
Florence, Italy
Juan Bayo
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
Meredith S. Brown
Department of Molecular and Systems Biology, Geisel School of Medicine at
Dartmouth, Hanover, NH, United States
Barbara Bueloni
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
Laura Calabresi
Center for Research and Innovation for Myeloproliferative Neoplasms,
Department of Experimental and Clinical Medicine, University of Florence,
Florence, Italy
Francesco De Francesco
Department of Plastic and Reconstructive Surgery-Hand Surgery Unit, Azienda
‘Ospedali Riuniti’ Ancona, Ancona, Italy
Malak Fawaz
Department of Medicine, Hematology/Oncology, Goethe University Hospital,
Frankfurt, Germany
Wasundara Fernando
Department of Pathology, Dalhousie University, Halifax, NS, Canada
xi
Behnaz Abdollahi
Department of Biomedical Data Science, Geisel School of Medicine at Dartmouth,
Hanover, NH, United States
Aysha S. Ansari
Department of Chemical & Materials Engineering, Faculty of Engineering,
University of Alberta, Edmonton, AB, Canada
Raj Pranap Arun
Department of Pathology, Dalhousie University, Halifax, NS, Canada
Manjola Balliu
Center for Research and Innovation for Myeloproliferative Neoplasms,
Department of Experimental and Clinical Medicine, University of Florence,
Florence, Italy
Niccolo`Bartalucci
Center for Research and Innovation for Myeloproliferative Neoplasms,
Department of Experimental and Clinical Medicine, University of Florence,
Florence, Italy
Juan Bayo
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
Meredith S. Brown
Department of Molecular and Systems Biology, Geisel School of Medicine at
Dartmouth, Hanover, NH, United States
Barbara Bueloni
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
Laura Calabresi
Center for Research and Innovation for Myeloproliferative Neoplasms,
Department of Experimental and Clinical Medicine, University of Florence,
Florence, Italy
Francesco De Francesco
Department of Plastic and Reconstructive Surgery-Hand Surgery Unit, Azienda
‘Ospedali Riuniti’ Ancona, Ancona, Italy
Malak Fawaz
Department of Medicine, Hematology/Oncology, Goethe University Hospital,
Frankfurt, Germany
Wasundara Fernando
Department of Pathology, Dalhousie University, Halifax, NS, Canada
xi
Letizia Ferroni
Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy
Esteban Fiore
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
Marta Freschi
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Chiara Gardin
Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy
Chiara Garsia
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Dnyanada S. Ghadi
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced
Centre for Treatment Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai, Maharashtra; Homi Bhabha National
Institute, Mumbai, India
Manuel Gidekel
Universidad Auto´noma de Chile, Providencia, Chile
Angela Gritti
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Saeed Hassanpour
Department of Biomedical Data Science, Geisel School of Medicine at Dartmouth,
Hanover, NH, United States
Xiaoyan Jiang
Terry Fox Laboratory, British Columbia Cancer Research Institute; Department of
Medicine; Department of Medical Genetics, University of British Columbia,
Vancouver, BC, Canada
Priyanka Joshi
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced
Centre for Treatment Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai, Maharashtra; Homi Bhabha National
Institute, Mumbai, India
Yingying Lai
Department of Gastroenterology, Chongqing Hospital of Traditional Chinese
Medicine, Chongqing, PR China
Marco Luciani
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
xiiContributors
Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy
Esteban Fiore
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
Marta Freschi
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Chiara Gardin
Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy
Chiara Garsia
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Dnyanada S. Ghadi
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced
Centre for Treatment Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai, Maharashtra; Homi Bhabha National
Institute, Mumbai, India
Manuel Gidekel
Universidad Auto´noma de Chile, Providencia, Chile
Angela Gritti
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Saeed Hassanpour
Department of Biomedical Data Science, Geisel School of Medicine at Dartmouth,
Hanover, NH, United States
Xiaoyan Jiang
Terry Fox Laboratory, British Columbia Cancer Research Institute; Department of
Medicine; Department of Medical Genetics, University of British Columbia,
Vancouver, BC, Canada
Priyanka Joshi
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced
Centre for Treatment Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai, Maharashtra; Homi Bhabha National
Institute, Mumbai, India
Yingying Lai
Department of Gastroenterology, Chongqing Hospital of Traditional Chinese
Medicine, Chongqing, PR China
Marco Luciani
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
xiiContributors
Valeria Manganelli
Department of Experimental Medicine, "Sapienza" University of Rome, Rome,
Italy
Elisabeth Mangiameli
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Paola Marcato
Department of Pathology; Department of Microbiology & Immunology, Dalhousie
University, Halifax, NS, Canada
Guillermo Mazzolini
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral; Liver Unit, Hospital Universitario Austral, Buenos
Aires, Argentina
Vasco Meneghini
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Diwakar R. Pattabiraman
Department of Molecular and Systems Biology, Geisel School of Medicine at
Dartmouth, Hanover; Dartmouth Cancer Center, Dartmouth-Hitchcock Medical
Center, Lebanon, NH, United States
Paolo Pinton
Department of Medical Sciences, Section of Experimental Medicine, Laboratory
for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara,
Italy
Joseph L. Regan
Charite - Universit€ atsmedizin Berlin, Berlin, Germany
Michael A. Rieger
Department of Medicine, Hematology/Oncology, Goethe University Hospital;
Frankfurt Cancer Institute; Excellence Cluster Cardio-Pulmonary Institute,
Frankfurt; German Cancer Consortium, German Cancer Research Center,
Heidelberg, Germany
Derek Rowter
Flow Cytometry CORES, Dalhousie University, Halifax, NS, Canada
Tessa Schmachtel
Department of Medicine, Hematology/Oncology, Goethe University Hospital,
Frankfurt, Germany
Michele Signore
RPPA Unit, Proteomics Area, Core Facilities, Istituto Superiore di Sanita`, Rome,
Italy
xiiiContributors
Department of Experimental Medicine, "Sapienza" University of Rome, Rome,
Italy
Elisabeth Mangiameli
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Paola Marcato
Department of Pathology; Department of Microbiology & Immunology, Dalhousie
University, Halifax, NS, Canada
Guillermo Mazzolini
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral; Liver Unit, Hospital Universitario Austral, Buenos
Aires, Argentina
Vasco Meneghini
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan, Italy
Diwakar R. Pattabiraman
Department of Molecular and Systems Biology, Geisel School of Medicine at
Dartmouth, Hanover; Dartmouth Cancer Center, Dartmouth-Hitchcock Medical
Center, Lebanon, NH, United States
Paolo Pinton
Department of Medical Sciences, Section of Experimental Medicine, Laboratory
for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara,
Italy
Joseph L. Regan
Charite - Universit€ atsmedizin Berlin, Berlin, Germany
Michael A. Rieger
Department of Medicine, Hematology/Oncology, Goethe University Hospital;
Frankfurt Cancer Institute; Excellence Cluster Cardio-Pulmonary Institute,
Frankfurt; German Cancer Consortium, German Cancer Research Center,
Heidelberg, Germany
Derek Rowter
Flow Cytometry CORES, Dalhousie University, Halifax, NS, Canada
Tessa Schmachtel
Department of Medicine, Hematology/Oncology, Goethe University Hospital,
Frankfurt, Germany
Michele Signore
RPPA Unit, Proteomics Area, Core Facilities, Istituto Superiore di Sanita`, Rome,
Italy
xiiiContributors
Hasan Uludag
Department of Chemical & Materials Engineering, Faculty of Engineering;
Department of Biomedical Engineering, Faculty of Medicine; Faculty of Pharmacy
and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada
Jaganathan Venkatesh
Department of Pathology, Dalhousie University, Halifax, NS, Canada
Sanjeev K. Waghmare
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced
Centre for Treatment Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai, Maharashtra; Homi Bhabha National
Institute, Mumbai, India
Bin Wang
Department of Gastroenterology, Chongqing Key Laboratory of Digestive
Malignancies, Daping Hospital, Army Medical University (Third Military Medical
University), Chongqing, PR China
Andrew Wu
Terry Fox Laboratory, British Columbia Cancer Research Institute; Department of
Medicine, University of British Columbia, Vancouver, BC, Canada
Weijia Yu
Department of Medicine, Hematology/Oncology, Goethe University Hospital,
Frankfurt, Germany
Barbara Zavan
Department of Translational Medicine, University of Ferrara, Ferrara, Italy
Xi Zheng
Department of Gastroenterology, Chongqing University Cancer Hospital,
Chongqing, PR China
xivContributors
Department of Chemical & Materials Engineering, Faculty of Engineering;
Department of Biomedical Engineering, Faculty of Medicine; Faculty of Pharmacy
and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada
Jaganathan Venkatesh
Department of Pathology, Dalhousie University, Halifax, NS, Canada
Sanjeev K. Waghmare
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced
Centre for Treatment Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai, Maharashtra; Homi Bhabha National
Institute, Mumbai, India
Bin Wang
Department of Gastroenterology, Chongqing Key Laboratory of Digestive
Malignancies, Daping Hospital, Army Medical University (Third Military Medical
University), Chongqing, PR China
Andrew Wu
Terry Fox Laboratory, British Columbia Cancer Research Institute; Department of
Medicine, University of British Columbia, Vancouver, BC, Canada
Weijia Yu
Department of Medicine, Hematology/Oncology, Goethe University Hospital,
Frankfurt, Germany
Barbara Zavan
Department of Translational Medicine, University of Ferrara, Ferrara, Italy
Xi Zheng
Department of Gastroenterology, Chongqing University Cancer Hospital,
Chongqing, PR China
xivContributors
CHAPTER
Multiple gene knockdown
strategies for investigating
the properties of human
leukemia stem cells and
exploring new therapies
1
Andrew Wu
a,b
, Aysha S. Ansari
c
, Hasan Uludag
c,d,e
, and Xiaoyan Jiang
a,b,f,
*
a
Terry Fox Laboratory, British Columbia Cancer Research Institute, Vancouver, BC, Canada
b
Department of Medicine, University of British Columbia, Vancouver, BC, Canada
c
Department of Chemical & Materials Engineering, Faculty of Engineering, University of Alberta,
Edmonton, AB, Canada
d
Department of Biomedical Engineering, Faculty of Medicine, University of Alberta, Edmonton,
AB, Canada
e
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada
f
Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
*Corresponding author: e-mail address: [email protected]
Chapter outline
1 Introduction..........................................................................................................2
2 Materials..............................................................................................................6
2.1 Common reagents..................................................................................6
2.1.1 Isolation of CD34
+
or CD34
+
CD38
stem cell-enriched or CD34
+
CD38
+
progenitor populations and cell culture......................................6
2.1.2 FACS staining and sorting of CD34-subpopulations...............................6
2.1.3 Lentiviral-mediated shRNA vector construction.....................................6
2.1.4 Lentiviral-mediated CRISPR-Cas9 vector construction and sgRNA
design................................................................................................6
2.1.5 Lentivirus production...........................................................................8
2.1.6 Selection of transduced CD34
+
stem/progenitor cells.............................8
2.1.7 Nanoparticle-mediated lipopolymer/siRNA preparation and delivery.......8
2.2 Software................................................................................................8
3 Methods...............................................................................................................8
3.1 Isolation of CD34
+
or CD34
+
CD38
stem cell-enriched or CD34
+
CD38
+
progenitor populations and cell culture.....................................................8
Methods in Cell Biology, Volume 171, ISSN 0091-679X,https://doi.org/10.1016/bs.mcb.2022.04.002
Copyright
©2022 Elsevier Inc. All rights reserved.
1
Multiple gene knockdown
strategies for investigating
the properties of human
leukemia stem cells and
exploring new therapies
1
Andrew Wu
a,b
, Aysha S. Ansari
c
, Hasan Uludag
c,d,e
, and Xiaoyan Jiang
a,b,f,
*
a
Terry Fox Laboratory, British Columbia Cancer Research Institute, Vancouver, BC, Canada
b
Department of Medicine, University of British Columbia, Vancouver, BC, Canada
c
Department of Chemical & Materials Engineering, Faculty of Engineering, University of Alberta,
Edmonton, AB, Canada
d
Department of Biomedical Engineering, Faculty of Medicine, University of Alberta, Edmonton,
AB, Canada
e
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada
f
Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
*Corresponding author: e-mail address: [email protected]
Chapter outline
1 Introduction..........................................................................................................2
2 Materials..............................................................................................................6
2.1 Common reagents..................................................................................6
2.1.1 Isolation of CD34
+
or CD34
+
CD38
stem cell-enriched or CD34
+
CD38
+
progenitor populations and cell culture......................................6
2.1.2 FACS staining and sorting of CD34-subpopulations...............................6
2.1.3 Lentiviral-mediated shRNA vector construction.....................................6
2.1.4 Lentiviral-mediated CRISPR-Cas9 vector construction and sgRNA
design................................................................................................6
2.1.5 Lentivirus production...........................................................................8
2.1.6 Selection of transduced CD34
+
stem/progenitor cells.............................8
2.1.7 Nanoparticle-mediated lipopolymer/siRNA preparation and delivery.......8
2.2 Software................................................................................................8
3 Methods...............................................................................................................8
3.1 Isolation of CD34
+
or CD34
+
CD38
stem cell-enriched or CD34
+
CD38
+
progenitor populations and cell culture.....................................................8
Methods in Cell Biology, Volume 171, ISSN 0091-679X,https://doi.org/10.1016/bs.mcb.2022.04.002
Copyright
©2022 Elsevier Inc. All rights reserved.
1
3.2 Lentiviral-mediated shRNA vector construction..........................................9
3.3 Lentiviral-mediated CRISPR-Cas9 vector construction and sgRNA design.......9
3.4 Lentivirus production.................... ........................................................10
3.5 Lentiviral transduction and selection of transduced CD34
+
stem/progenitor cells ...........................................................................10
3.6 Nanoparticle-mediated lipopolymer siRNA preparation and delivery...........12
3.6.1 Preparation of working solutions.........................................................12
3.6.2 Preparation of lipopolymer/siRNA nanoparticles and transfection
of cells.............................................................................................12
3.7 Assessment of gene knockdown.............................................................12
4 Notes.................................................................................................................13
5 Concluding remarks............................................................................................18
Acknowledgments....................................................................................................19
Disclosures.............................................................................................................19
References..............................................................................................................19
Abstract
The past two decades have witnessed significant strides in leukemia therapies through
approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase
activities and key epigenetic and apoptosis regulators. Although these drugs have brought
about complete remission in the majority of patients, many patients face relapse or have
refractory disease. The main factor contributing to relapse is the presence of a small subpop-
ulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leu-
kemia stem cells or LSCs). Thus, overcoming drug resistance and targeting LSCs remain
major challenges for curative treatment of human leukemia. Chronic myeloid leukemia
(CML) is a good example, with rare, propagating LSCs and drug-resistant cells that cannot
be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that
are responsible for disease relapse/progression. Therefore, it is imperative to identify key
players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms,
and their key pathways, so that CML LSCs can be selectively targeted or sensitized to TKIs.
Here, we describe several easily adaptable gene knockdown approaches in CD34
+
CML stem/
progenitor cells that can be used to investigate the biological properties of LSCs and molecular
effects of genes of interest (GOI), which can be further explored as therapeutic modalities
against LSCs in the context of human leukemia.
1Introduction
Chronic myeloid leukemia (CML) is a clonal, myeloproliferative malignancy that
has served as a paradigm for the study of oncogenic signaling pathways and the
development of targeted therapies (Druker et al., 1996;Goldman & Melo, 2003;
Savona & Talpaz, 2008). CML advances through three stages: the chronic phase
2 CHAPTER 1 Multiple gene knockdown strategies
3.3 Lentiviral-mediated CRISPR-Cas9 vector construction and sgRNA design.......9
3.4 Lentivirus production.................... ........................................................10
3.5 Lentiviral transduction and selection of transduced CD34
+
stem/progenitor cells ...........................................................................10
3.6 Nanoparticle-mediated lipopolymer siRNA preparation and delivery...........12
3.6.1 Preparation of working solutions.........................................................12
3.6.2 Preparation of lipopolymer/siRNA nanoparticles and transfection
of cells.............................................................................................12
3.7 Assessment of gene knockdown.............................................................12
4 Notes.................................................................................................................13
5 Concluding remarks............................................................................................18
Acknowledgments....................................................................................................19
Disclosures.............................................................................................................19
References..............................................................................................................19
Abstract
The past two decades have witnessed significant strides in leukemia therapies through
approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase
activities and key epigenetic and apoptosis regulators. Although these drugs have brought
about complete remission in the majority of patients, many patients face relapse or have
refractory disease. The main factor contributing to relapse is the presence of a small subpop-
ulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leu-
kemia stem cells or LSCs). Thus, overcoming drug resistance and targeting LSCs remain
major challenges for curative treatment of human leukemia. Chronic myeloid leukemia
(CML) is a good example, with rare, propagating LSCs and drug-resistant cells that cannot
be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that
are responsible for disease relapse/progression. Therefore, it is imperative to identify key
players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms,
and their key pathways, so that CML LSCs can be selectively targeted or sensitized to TKIs.
Here, we describe several easily adaptable gene knockdown approaches in CD34
+
CML stem/
progenitor cells that can be used to investigate the biological properties of LSCs and molecular
effects of genes of interest (GOI), which can be further explored as therapeutic modalities
against LSCs in the context of human leukemia.
1Introduction
Chronic myeloid leukemia (CML) is a clonal, myeloproliferative malignancy that
has served as a paradigm for the study of oncogenic signaling pathways and the
development of targeted therapies (Druker et al., 1996;Goldman & Melo, 2003;
Savona & Talpaz, 2008). CML advances through three stages: the chronic phase
2 CHAPTER 1 Multiple gene knockdown strategies
(CP), the accelerated phase (AP) and the blast crisis phase (BP). The European
LeukemiaNet (ELN) criteria distinguishes AP from CP by the presence of
15–29% blast cells in peripheral blood (PB) or bone marrow (BM) and>20% baso-
phils in PB (Baccarani et al., 2006, 2013). BP resembles a generally more fatal
acute leukemia and is defined as30% blasts in PB or BM with extramedullary
blast proliferation (Baccarani et al., 2006, 2013). The main defining characteristic
of CML is the occurrence of a t(9;22)(q34;q11.2) chromosomal translocation of
the Abelson murine leukemia viral oncogene homolog 1 (ABL1) on chromosome
9 to the Breakpoint Cluster Region (BCR) on chromosome 22, which gives rise
to the BCR-ABL1 chimeric gene, also known as the ‘Philadelphia chromosome’
(pH) (Rowley, 1973). BCR-ABL1 is considered the primary driver of CML oncogen-
esis and evaluation of BCR-ABL transcript levels have been typically applied to
assess patient post-treatment progress that accurately quantifies minimal residual
disease (MRD) (Baccarani et al., 2006, 2013;Hochhaus et al., 2020).
The standard treatment for CML involves the use of tyrosine kinase inhibitors
(TK
I) to inhibit BCR-ABL1 signaling. These therapeutics include Imatinib Mesylate
(IM) and next-generation inhibitors such as Nilotinib, Dasatinib and Bosutinib.
While these inhibitors have demonstrated efficacy in treating and managing early
stage CML, patients often relapse due to acquired drug-resistance mechanisms.
For example, some TKI-resistant patients have a T315I gate-keeper mutation in
the BCR-ABL1 catalytic pocket which prevents TKI binding (Gorre et al., 2001;
O’Hare, Eide, & Deininger, 2007). To address this, novel allosteric BCR-ABL1
inhibi
tors such as Asciminib are being developed, with positive results in clinical
trials (Hughes et al., 2019). Despite advancements in BCR-ABL1 targeting strate-
gies,
drug-resistance remains a pressing challenge to overcome as some mechanisms
of resistance involve the activation of BCR-ABL1 independent oncogenic signaling
pathways. These mechanisms of resistance are consistently observed in leukemic
stem cells (LSC) which are a key population for sustaining disease (Chu et al.,
2005;Graham et al., 2002;Holtz, Forman, & Bhatia, 2005;Holyoake &
Vetrie,
2017;Jiang et al., 2007;Sloma, Jiang, Eaves, & Eaves, 2010). LSCs are
notorio
usly difficult to target as they often exist in a quiescent state; current therapies
against CML mainly target proliferating cells (Graham et al., 2002;Holtz et al.,
2005;Jiang, Saw, Eaves, & Eaves, 2007;Jiang, Zhao, et al., 2007). Particularly,
LSCs
possess multiple features expected to promote resistance to TKIs, including
a high degree of genetic instability (Holyoake & Vetrie, 2017;Jiang, Saw, et al.,
2007;Jiang, Zhao, et al., 2007). Studies suggest that CML LSCs are not fully depen-
dent
on BCR-ABL activity for survival and therapeutic approaches to kill LSCs or
force them out of their “stem” protective state have not yet proven successful (Corbin
et
al., 2011;Gallipoli et al., 2013;Glauche et al., 2012;Hamilton et al., 2012;Prost
et
al., 2015). Increasing evidence also indicates that the LSCs in CML patients
posse
ss an altered metabolic state and might constitute a targetable vulnerability
(Alvarez-Calderon et al., 2015;Kuntz et al., 2017). These studies emphasize the need
to
identify novel, distinct therapeutic targets/pathways for treatment, to achieve LSC
eradication and disease cure. Furthermore, there have been challenges establishing
31Introduction
LeukemiaNet (ELN) criteria distinguishes AP from CP by the presence of
15–29% blast cells in peripheral blood (PB) or bone marrow (BM) and>20% baso-
phils in PB (Baccarani et al., 2006, 2013). BP resembles a generally more fatal
acute leukemia and is defined as30% blasts in PB or BM with extramedullary
blast proliferation (Baccarani et al., 2006, 2013). The main defining characteristic
of CML is the occurrence of a t(9;22)(q34;q11.2) chromosomal translocation of
the Abelson murine leukemia viral oncogene homolog 1 (ABL1) on chromosome
9 to the Breakpoint Cluster Region (BCR) on chromosome 22, which gives rise
to the BCR-ABL1 chimeric gene, also known as the ‘Philadelphia chromosome’
(pH) (Rowley, 1973). BCR-ABL1 is considered the primary driver of CML oncogen-
esis and evaluation of BCR-ABL transcript levels have been typically applied to
assess patient post-treatment progress that accurately quantifies minimal residual
disease (MRD) (Baccarani et al., 2006, 2013;Hochhaus et al., 2020).
The standard treatment for CML involves the use of tyrosine kinase inhibitors
(TK
I) to inhibit BCR-ABL1 signaling. These therapeutics include Imatinib Mesylate
(IM) and next-generation inhibitors such as Nilotinib, Dasatinib and Bosutinib.
While these inhibitors have demonstrated efficacy in treating and managing early
stage CML, patients often relapse due to acquired drug-resistance mechanisms.
For example, some TKI-resistant patients have a T315I gate-keeper mutation in
the BCR-ABL1 catalytic pocket which prevents TKI binding (Gorre et al., 2001;
O’Hare, Eide, & Deininger, 2007). To address this, novel allosteric BCR-ABL1
inhibi
tors such as Asciminib are being developed, with positive results in clinical
trials (Hughes et al., 2019). Despite advancements in BCR-ABL1 targeting strate-
gies,
drug-resistance remains a pressing challenge to overcome as some mechanisms
of resistance involve the activation of BCR-ABL1 independent oncogenic signaling
pathways. These mechanisms of resistance are consistently observed in leukemic
stem cells (LSC) which are a key population for sustaining disease (Chu et al.,
2005;Graham et al., 2002;Holtz, Forman, & Bhatia, 2005;Holyoake &
Vetrie,
2017;Jiang et al., 2007;Sloma, Jiang, Eaves, & Eaves, 2010). LSCs are
notorio
usly difficult to target as they often exist in a quiescent state; current therapies
against CML mainly target proliferating cells (Graham et al., 2002;Holtz et al.,
2005;Jiang, Saw, Eaves, & Eaves, 2007;Jiang, Zhao, et al., 2007). Particularly,
LSCs
possess multiple features expected to promote resistance to TKIs, including
a high degree of genetic instability (Holyoake & Vetrie, 2017;Jiang, Saw, et al.,
2007;Jiang, Zhao, et al., 2007). Studies suggest that CML LSCs are not fully depen-
dent
on BCR-ABL activity for survival and therapeutic approaches to kill LSCs or
force them out of their “stem” protective state have not yet proven successful (Corbin
et
al., 2011;Gallipoli et al., 2013;Glauche et al., 2012;Hamilton et al., 2012;Prost
et
al., 2015). Increasing evidence also indicates that the LSCs in CML patients
posse
ss an altered metabolic state and might constitute a targetable vulnerability
(Alvarez-Calderon et al., 2015;Kuntz et al., 2017). These studies emphasize the need
to
identify novel, distinct therapeutic targets/pathways for treatment, to achieve LSC
eradication and disease cure. Furthermore, there have been challenges establishing
31Introduction
experimental models to target LSCs in the laboratory; as they are rare, surface
markers that enable direct isolation of CML stem cells are not available and these
primitive cells also undergo rapid differentiation into progenitor and mature cells
(Velten et al., 2021).
To
develop useful approaches to study biological and molecular functions of
LSCs, we isolate the CD34
+
stem/progenitor cell population from primary patient
samples that have been supported with cytokines and growth factors in culture,
for extensivein vitroandin vivostudies, including several long-term assays to
determine phenotypic properties of LSCs and their progeny. Several methods can
be employed to modify the expression of specific genes in these cells. These include
the delivery of short interfering RNAs (siRNA), short hairpin RNA (shRNA) and/or
CRISPR-Cas9-sgRNAs for gene knockdown, as well as delivering gene-encoding
sequences for gene restoration. While CD34
+
leukemic stem/progenitor cells are
known to be difficult to transfect, advanced lentiviral-mediated gene knockdown
or restoration techniques have been successful in delivering targeted constructs in
CD34
+
cells or CD34
+
CD38
γ
stem cell-enriched population. These methods have
allowed us to identify several crucial genes that regulate LSC survival, drug resis-
tance and LSC interaction with the bone marrow niche, including Abelson helper
integration site-1 (AHI-1), ATG4B, microRNA-185 (miR-185), p21-activated
kinase 6 (PAK6), integrin-linked kinase (ILK) and AXL (Chen et al., 2013;Lin
et
al., 2020;Niu et al., 2021;Rothe et al., 2014, 2020). One example is the discovery
that
miR-185 expression is downregulated in CD34
+
CML stem/progenitor cells
compared to healthy normal bone marrow (NBM) samples as well as in TKI-resistant
versus to TKI-sensitive patient cells (Lin et al., 2020). Upon forced miR-185
re-
expression, reduced viability and increased apoptosis were observed in TKI-
resistant CD34
+
stem/progenitor cells and their sensitivity to TKI was restored
in vitro. These results were also demonstrated by colony-forming cell (CFC) and
long-term culture-initiating cell (LTC-IC) assays that measure progenitor and stem
cell functionality (Lin et al., 2020). Most interestingly, restoration of miR-185, com-
bine
d with TKI treatment, effectively eliminated long-term repopulating LSCs, con-
ferring a survival advantage in pre-clinical patient-derived xenotransplantation
(PDX) models (Lin et al., 2020). Following up on the promising results from resto-
rat
ion of miR-185 expression, knockdown of PAK6, a target gene of miRNA-185,
demonstrated similar anti-leukemic effects. Interestingly, genetic silencing of ILK
by lentiviral-mediated transduction in CD34
+
stem/progenitor cells also impaired
the survival of TKI-nonresponder patient cells, sensitizing them to TKIs even in
the presence of protective niche cells (Rothe et al., 2020). Additionally, long-term
ste
m cell maintenance and self-renewal were significantly reduced following ILK
knockdown in CD34
+
TKI-nonresponder cells and injection of these cells into pri-
mary recipients, with further evaluation in secondary recipients using PDX models.
Immunocompromised mouse strains used for these studies wereNOD-Rag1
γ/γ
IL2Rγc
γ/γ
(NRG) or NRG mice producing human IL-3, GM-CSF and Steel factor
constitutively (NRG-3GS mice) as these have been found to promote better expansion
4 CHAPTER 1 Multiple gene knockdown strategies
markers that enable direct isolation of CML stem cells are not available and these
primitive cells also undergo rapid differentiation into progenitor and mature cells
(Velten et al., 2021).
To
develop useful approaches to study biological and molecular functions of
LSCs, we isolate the CD34
+
stem/progenitor cell population from primary patient
samples that have been supported with cytokines and growth factors in culture,
for extensivein vitroandin vivostudies, including several long-term assays to
determine phenotypic properties of LSCs and their progeny. Several methods can
be employed to modify the expression of specific genes in these cells. These include
the delivery of short interfering RNAs (siRNA), short hairpin RNA (shRNA) and/or
CRISPR-Cas9-sgRNAs for gene knockdown, as well as delivering gene-encoding
sequences for gene restoration. While CD34
+
leukemic stem/progenitor cells are
known to be difficult to transfect, advanced lentiviral-mediated gene knockdown
or restoration techniques have been successful in delivering targeted constructs in
CD34
+
cells or CD34
+
CD38
γ
stem cell-enriched population. These methods have
allowed us to identify several crucial genes that regulate LSC survival, drug resis-
tance and LSC interaction with the bone marrow niche, including Abelson helper
integration site-1 (AHI-1), ATG4B, microRNA-185 (miR-185), p21-activated
kinase 6 (PAK6), integrin-linked kinase (ILK) and AXL (Chen et al., 2013;Lin
et
al., 2020;Niu et al., 2021;Rothe et al., 2014, 2020). One example is the discovery
that
miR-185 expression is downregulated in CD34
+
CML stem/progenitor cells
compared to healthy normal bone marrow (NBM) samples as well as in TKI-resistant
versus to TKI-sensitive patient cells (Lin et al., 2020). Upon forced miR-185
re-
expression, reduced viability and increased apoptosis were observed in TKI-
resistant CD34
+
stem/progenitor cells and their sensitivity to TKI was restored
in vitro. These results were also demonstrated by colony-forming cell (CFC) and
long-term culture-initiating cell (LTC-IC) assays that measure progenitor and stem
cell functionality (Lin et al., 2020). Most interestingly, restoration of miR-185, com-
bine
d with TKI treatment, effectively eliminated long-term repopulating LSCs, con-
ferring a survival advantage in pre-clinical patient-derived xenotransplantation
(PDX) models (Lin et al., 2020). Following up on the promising results from resto-
rat
ion of miR-185 expression, knockdown of PAK6, a target gene of miRNA-185,
demonstrated similar anti-leukemic effects. Interestingly, genetic silencing of ILK
by lentiviral-mediated transduction in CD34
+
stem/progenitor cells also impaired
the survival of TKI-nonresponder patient cells, sensitizing them to TKIs even in
the presence of protective niche cells (Rothe et al., 2020). Additionally, long-term
ste
m cell maintenance and self-renewal were significantly reduced following ILK
knockdown in CD34
+
TKI-nonresponder cells and injection of these cells into pri-
mary recipients, with further evaluation in secondary recipients using PDX models.
Immunocompromised mouse strains used for these studies wereNOD-Rag1
γ/γ
IL2Rγc
γ/γ
(NRG) or NRG mice producing human IL-3, GM-CSF and Steel factor
constitutively (NRG-3GS mice) as these have been found to promote better expansion
4 CHAPTER 1 Multiple gene knockdown strategies
of human AML cells (Rothe et al., 2020;Wunderlich et al., 2010). Importantly, inhi-
bi
tion of ILK kinase targets quiescent LSCs from TKI-nonresponders, which is asso-
ciated with effects on mitochondrial oxidative phosphorylation and the generation of
reactive oxygen species (Rothe et al., 2020). We have also demonstrated that these
ge
ne expression knockdown strategies (shRNA or CRISPR-Cas9-sgRNA) can be
effectively applied in acute myeloid leukemia (AML) cells; knockdown of AXL, a
member of the TYRO3, AXL, MER (TAM) family of receptor tyrosine kinases, sen-
sitized AML LSCs and progenitor cells to venetoclax, a selective BCL-2 inhibitor
(Carter et al., 2016;Roberts et al., 2016) against AML in vitro and in PDX models
(Niu et al., 2021). Thus, the use of lentiviral-mediated gene knockdown or restoration
ap
proaches successfully enabled investigation of the properties of LSCs and their pro-
genitors as well as potential molecular mechanisms bothin vitroand in PDX models.
Notably, advances in RNA interference (RNAi) technology have resulted in a
short interfering RNA (siRNA)-based therapeutic platform as an alternative to
knockdown of genes of interest (GOI) and potential molecular therapies. Since
the mechanism of action of siRNA is sequence driven, it offers an advantage over
small molecule and antibody-based drugs, which require a highly specific structural
conformation for spatial recognition and binding of target proteins (Landry et al.,
2015). Furthermore, siRNA allows for specific targeting of malignant cells, while
spar
ing healthy cells, in cases where the leukemia-driving oncogene mutations are
exclusively present in leukemia cells (for example, BCR-ABL fusion gene in
CML and FLT3-ITD mutation in AML), presenting an advantage over conventional
broad-spectrum chemotherapy drugs. Numerous techniques and biomaterials have
been explored to deliver siRNA to leukemia cells, in addition to viral-mediated
techniques. Electroporation is the most popular technique in leukemia clinical trials,
after viral strategies (Ansari, Santerre, & Uludag, 2017). However, electroporation
cannot
be directly implementedin vivo, which is a crucial step in the pre-clinical
evaluation of any therapeutic modality (Ansari et al., 2017). Nanoparticle-mediated
gene
knockdown of leukemogenesis drivers presents a more viable option that has
immense potential to be directly implemented clinically. The relatively facile chem-
istry of the biomaterials that form nanoparticles allows for incorporation of targeting
moieties to enhance targeting specificity to cells of interest (Freeley & Long, 2013;
Rothdiener et al., 2010;Yang et al., 2014;Zhang et al., 2016). Low molecular weight
polye
thyleneimine polymers modified with lipids to yield lipopolymers have been
shown to successfully deliver siRNA and knockdown therapeutically relevant
oncogenes in CML and AML CD34
+
stem/progenitor cells (Gul-Uludag et al.,
2014;Landry et al., 2016;Valencia-Serna et al., 2019).
Here,
we outline the protocols for isolating CD34
+
stem/progenitor cells,
culturing them, and transfecting/transducing them with lentiviral-mediated vectors
(shRNA or CRISPR-Cas9-sgRNA) or lipopolymer/siRNA nanoparticlesin vitro,
to knockdown relevant genes. While we have predominantly implemented this
protocol in CML and AML CD34
+
cells, it can be easily adapted to other types
of leukemia with minimal variations.
51Introduction
bi
tion of ILK kinase targets quiescent LSCs from TKI-nonresponders, which is asso-
ciated with effects on mitochondrial oxidative phosphorylation and the generation of
reactive oxygen species (Rothe et al., 2020). We have also demonstrated that these
ge
ne expression knockdown strategies (shRNA or CRISPR-Cas9-sgRNA) can be
effectively applied in acute myeloid leukemia (AML) cells; knockdown of AXL, a
member of the TYRO3, AXL, MER (TAM) family of receptor tyrosine kinases, sen-
sitized AML LSCs and progenitor cells to venetoclax, a selective BCL-2 inhibitor
(Carter et al., 2016;Roberts et al., 2016) against AML in vitro and in PDX models
(Niu et al., 2021). Thus, the use of lentiviral-mediated gene knockdown or restoration
ap
proaches successfully enabled investigation of the properties of LSCs and their pro-
genitors as well as potential molecular mechanisms bothin vitroand in PDX models.
Notably, advances in RNA interference (RNAi) technology have resulted in a
short interfering RNA (siRNA)-based therapeutic platform as an alternative to
knockdown of genes of interest (GOI) and potential molecular therapies. Since
the mechanism of action of siRNA is sequence driven, it offers an advantage over
small molecule and antibody-based drugs, which require a highly specific structural
conformation for spatial recognition and binding of target proteins (Landry et al.,
2015). Furthermore, siRNA allows for specific targeting of malignant cells, while
spar
ing healthy cells, in cases where the leukemia-driving oncogene mutations are
exclusively present in leukemia cells (for example, BCR-ABL fusion gene in
CML and FLT3-ITD mutation in AML), presenting an advantage over conventional
broad-spectrum chemotherapy drugs. Numerous techniques and biomaterials have
been explored to deliver siRNA to leukemia cells, in addition to viral-mediated
techniques. Electroporation is the most popular technique in leukemia clinical trials,
after viral strategies (Ansari, Santerre, & Uludag, 2017). However, electroporation
cannot
be directly implementedin vivo, which is a crucial step in the pre-clinical
evaluation of any therapeutic modality (Ansari et al., 2017). Nanoparticle-mediated
gene
knockdown of leukemogenesis drivers presents a more viable option that has
immense potential to be directly implemented clinically. The relatively facile chem-
istry of the biomaterials that form nanoparticles allows for incorporation of targeting
moieties to enhance targeting specificity to cells of interest (Freeley & Long, 2013;
Rothdiener et al., 2010;Yang et al., 2014;Zhang et al., 2016). Low molecular weight
polye
thyleneimine polymers modified with lipids to yield lipopolymers have been
shown to successfully deliver siRNA and knockdown therapeutically relevant
oncogenes in CML and AML CD34
+
stem/progenitor cells (Gul-Uludag et al.,
2014;Landry et al., 2016;Valencia-Serna et al., 2019).
Here,
we outline the protocols for isolating CD34
+
stem/progenitor cells,
culturing them, and transfecting/transducing them with lentiviral-mediated vectors
(shRNA or CRISPR-Cas9-sgRNA) or lipopolymer/siRNA nanoparticlesin vitro,
to knockdown relevant genes. While we have predominantly implemented this
protocol in CML and AML CD34
+
cells, it can be easily adapted to other types
of leukemia with minimal variations.
51Introduction
2Materials
2.1Common reagents
2.1.1 Isolation of CD34
+
or CD34
+
CD38
2
stem cell-enriched
or CD34
+
CD38
+
progenitor populations and cell culture
EasySep
®
CD34 Positive Selection Kit (Cat# 18056, STEMCELL Technologies)
(seeNote 1)
Isocove’s Modified Dulbecco’s Medium (IMDM) (Cat# 36150, STEMCELL
Technol
ogies) (seeNote 1)
Complete Cell Culture Media (seeNote 2):
∘BIT (Cat#09500, STEMCELL Technologies)
∘IL-3, human recombinant (Cat# 78040, STEMCELL Technologies)
∘IL-6, human recombinant (Cat# 78050, STEMCELL Technologies)
∘Flt3/Flk-2 Ligand, human recombinant (Cat# 78009, STEMCELL
Technologies)
∘G-CSF, human recombinant (Cat# 78012, STEMCELL Technologies)
Hanks’ Balanced Salt Solution (HBSS) (Cat# 37150, STEMCELL Technologies)
(seeNote 1)
Fetal Bovine Serum (FBS) (Cat# F1051 Invitrogen) (seeNote 1)
2.1.2FACS staining and sorting of CD34-subpopulations
BD FACSAria™Fusion Flow Cytometer (Cat# 34295, BD Biosciences)
(seeNote 1)
Anti-human CD34-APC. Clone 4H11 (Cat# 17–0349-4
2; RRID:AB_2016672,
Thermo
Fisher Scientific)
Anti-human CD38-BV711. Clone HIT2 (Cat# 303527; RRID:AB 11218990,
BioLege
nd)
Anti-human CD38-PE. Clone HIT2 (Cat# 560981; RRID:AB 10563932, BD
Biosciences)
2.1.3Lentiviral-mediated shRNA vector construction
Mission
®
shRNA Control Plasmid, TRC1.5 pLKO.1-puro (Cat# SHC002,
MilliporeSigma) (seeNote 3 andFig. 1A)
PureLink™HiPure
Plasmid Filter Maxiprep Kit (Cat# K210006, Thermo Fisher
Scientific) (seeNotes 1andNote 4)
2.1.4Lentiviral-mediated CRISPR-Cas9 vector construction and sgRNA
design
LentiCRISPRv2GFP (Cat# 82416, Addgene) (seeNote 5andFig. 1B)
PureLink™HiPure
Plasmid Filter Maxiprep Kit (Cat# K210006, Thermo Fisher
Scientific) (seeNotes 1andNote 4)
One Shot
®
Stbl3™Chemically CompetentE. coli(Cat# C737303, Thermo
Fisher Scientific) (seeNote 1)
6 CHAPTER 1 Multiple gene knockdown strategies
2.1Common reagents
2.1.1 Isolation of CD34
+
or CD34
+
CD38
2
stem cell-enriched
or CD34
+
CD38
+
progenitor populations and cell culture
EasySep
®
CD34 Positive Selection Kit (Cat# 18056, STEMCELL Technologies)
(seeNote 1)
Isocove’s Modified Dulbecco’s Medium (IMDM) (Cat# 36150, STEMCELL
Technol
ogies) (seeNote 1)
Complete Cell Culture Media (seeNote 2):
∘BIT (Cat#09500, STEMCELL Technologies)
∘IL-3, human recombinant (Cat# 78040, STEMCELL Technologies)
∘IL-6, human recombinant (Cat# 78050, STEMCELL Technologies)
∘Flt3/Flk-2 Ligand, human recombinant (Cat# 78009, STEMCELL
Technologies)
∘G-CSF, human recombinant (Cat# 78012, STEMCELL Technologies)
Hanks’ Balanced Salt Solution (HBSS) (Cat# 37150, STEMCELL Technologies)
(seeNote 1)
Fetal Bovine Serum (FBS) (Cat# F1051 Invitrogen) (seeNote 1)
2.1.2FACS staining and sorting of CD34-subpopulations
BD FACSAria™Fusion Flow Cytometer (Cat# 34295, BD Biosciences)
(seeNote 1)
Anti-human CD34-APC. Clone 4H11 (Cat# 17–0349-4
2; RRID:AB_2016672,
Thermo
Fisher Scientific)
Anti-human CD38-BV711. Clone HIT2 (Cat# 303527; RRID:AB 11218990,
BioLege
nd)
Anti-human CD38-PE. Clone HIT2 (Cat# 560981; RRID:AB 10563932, BD
Biosciences)
2.1.3Lentiviral-mediated shRNA vector construction
Mission
®
shRNA Control Plasmid, TRC1.5 pLKO.1-puro (Cat# SHC002,
MilliporeSigma) (seeNote 3 andFig. 1A)
PureLink™HiPure
Plasmid Filter Maxiprep Kit (Cat# K210006, Thermo Fisher
Scientific) (seeNotes 1andNote 4)
2.1.4Lentiviral-mediated CRISPR-Cas9 vector construction and sgRNA
design
LentiCRISPRv2GFP (Cat# 82416, Addgene) (seeNote 5andFig. 1B)
PureLink™HiPure
Plasmid Filter Maxiprep Kit (Cat# K210006, Thermo Fisher
Scientific) (seeNotes 1andNote 4)
One Shot
®
Stbl3™Chemically CompetentE. coli(Cat# C737303, Thermo
Fisher Scientific) (seeNote 1)
6 CHAPTER 1 Multiple gene knockdown strategies
FIG. 1
Vector maps for lentiviral-mediated knockdown systems. (A) The pLKO.1-puro shRNA vector (MilliporeSigma) contains an U6 promoter to drive
stable shRNA expression as well as a puromycin resistance gene, for selection. (B) The LentiCRISPRv2GFP vector also contains an U6
promoter to drive the sgRNA and an EF-1 αpromoter to drive Cas9 and GFP expression. Alternatively, the GFP selection marker can be substituted
for a puromycin resistant marker.
Vector maps for lentiviral-mediated knockdown systems. (A) The pLKO.1-puro shRNA vector (MilliporeSigma) contains an U6 promoter to drive
stable shRNA expression as well as a puromycin resistance gene, for selection. (B) The LentiCRISPRv2GFP vector also contains an U6
promoter to drive the sgRNA and an EF-1 αpromoter to drive Cas9 and GFP expression. Alternatively, the GFP selection marker can be substituted
for a puromycin resistant marker.
2.1.5Lentivirus production
293T Cells (Cat# CRL-3216; RRID:CVCL_0063, ATCC)
Dulbecco’s Modified Eagle Medium (DMEM) (Cat# 36350, STEMCELL
Technol
ogies) (seeNote 1)
Fetal Bovine Serum (FBS) (Cat# F1051, Invitrogen) (seeNote 1)
Packaging and Envelope Constructs (seeNotes 6 and 7)
Opti-MEM Reduced Serum Medium (Cat# 31985062, Thermo Fisher Scientific)
(seeNote 1)
2.1.6Selection of transduced CD34
+
stem/progenitor cells
Puromycin Dihydrochloride (Cat# A1113802, Thermo Fisher Scientific)
(seeNote 1)
2.1.7Nanoparticle-mediated lipopolymer/siRNA preparation and delivery
Target GOI siRNA(s) Stock Solution at 100μM(seeNotes 8 and 9)
Negative Control (NC) siRNA Stock Solution at 100μM(seeNotes
8 and 9)
Lipopolymer Stock Solution at 20mg/mL (seeNotes 1 and 10)
Serum-free Cell Culture Media (seeNote 11)
DNase/RNase-free Distilled Water (Cat# 10977015, ThermoFisher Scientific)
(seeNote 1)
Complete Cell Culture Media (seeNotes 2 and 12)
0.4% Trypan Blue Solution (Cat# 15250061, ThermoFisher Scientific)
(seeNote 1)
TRIzol (Cat# 15596026, ThermoFisher Scientific) (seeNote 1)
2.2Software
Benchling Alignment Tool
Synthego CRISPR Design Tool
CRISPOR CRISPR Design Tool
GeneWiz Sanger Sequencing
FlowJo
®
3Methods
3.1Isolation of CD34
+
or CD34
+
CD38
2
stem cell-enriched or
CD34
+
CD38
+
progenitor populations and cell culture
1.Thaw frozen CD34
+
cells and add 0.5mL of DNase for every 1 mL of cell
suspension.
2.Mix
μL EasySep
®
Positive Selection Cocktail into cell suspension and
incubate for 15min at room temperature (RT).
8 CHAPTER 1 Multiple gene knockdown strategies
293T Cells (Cat# CRL-3216; RRID:CVCL_0063, ATCC)
Dulbecco’s Modified Eagle Medium (DMEM) (Cat# 36350, STEMCELL
Technol
ogies) (seeNote 1)
Fetal Bovine Serum (FBS) (Cat# F1051, Invitrogen) (seeNote 1)
Packaging and Envelope Constructs (seeNotes 6 and 7)
Opti-MEM Reduced Serum Medium (Cat# 31985062, Thermo Fisher Scientific)
(seeNote 1)
2.1.6Selection of transduced CD34
+
stem/progenitor cells
Puromycin Dihydrochloride (Cat# A1113802, Thermo Fisher Scientific)
(seeNote 1)
2.1.7Nanoparticle-mediated lipopolymer/siRNA preparation and delivery
Target GOI siRNA(s) Stock Solution at 100μM(seeNotes 8 and 9)
Negative Control (NC) siRNA Stock Solution at 100μM(seeNotes
8 and 9)
Lipopolymer Stock Solution at 20mg/mL (seeNotes 1 and 10)
Serum-free Cell Culture Media (seeNote 11)
DNase/RNase-free Distilled Water (Cat# 10977015, ThermoFisher Scientific)
(seeNote 1)
Complete Cell Culture Media (seeNotes 2 and 12)
0.4% Trypan Blue Solution (Cat# 15250061, ThermoFisher Scientific)
(seeNote 1)
TRIzol (Cat# 15596026, ThermoFisher Scientific) (seeNote 1)
2.2Software
Benchling Alignment Tool
Synthego CRISPR Design Tool
CRISPOR CRISPR Design Tool
GeneWiz Sanger Sequencing
FlowJo
®
3Methods
3.1Isolation of CD34
+
or CD34
+
CD38
2
stem cell-enriched or
CD34
+
CD38
+
progenitor populations and cell culture
1.Thaw frozen CD34
+
cells and add 0.5mL of DNase for every 1 mL of cell
suspension.
2.Mix
μL EasySep
®
Positive Selection Cocktail into cell suspension and
incubate for 15min at room temperature (RT).
8 CHAPTER 1 Multiple gene knockdown strategies
3.Add EasySep
®
Magnetic Nanoparticles (50μL/mL cells) into solution and mix
by pipetting multiple times (seeNote 13). Incubate for 10min.
4.Add EasySep
®
Buffer to increase total volume of cell suspension to 2.5mL and
mix by gentle pipetting.
5.Place the tube containing the cell suspension (without the cap) into the
EasySep
®
Magnet and set aside for 10min.
6.Invert the magnet chamber with the tube inside in one continuous motion to
discard the supernatant. The magnetically labeled cells will remain in the tube.
7.Remove the tube from the EasySep
®
Magnet and mix in 2.5mL EasySep
®
Buffer by pipetting.
8.Place the tube back into the EasySep
®
Magnet and set aside for 5min.
9.Repeat steps 6–8 for a total of 4 washes.
10.Discard the supernatant and remove the tube from the EasySep
®
Magnet before
resuspending cells in 1.2mL of EasySep
®
Buffer. These cells are the purified
CD34
+
cells.
11.For further purity assessment, mix 50μL of CD34
+
cells with 50μL of EasySep
®
Buffer and add 1μL of anti-human CD34-APC (1:100) antibody (seeNote 14).
12.Incubate on ice in the dark for 30min.
13.Wash cells with 1mL EasySep
®
Buffer with 1μL of propidium iodide (PI).
14.Discard the supernatant and resuspend cell pellet in 200μL EasySep
®
Buffer
before FACS analysis using the BD FACSAria™Fusion Flow Cytometer
and FlowJo
®
software (seeNote 1).
15.CD34
+
cells can then be sorted into subpopulations by suspending cells in
HBSS plus 2% FBS and stained with anti-human CD38-PE (1:50) or
anti-human CD38-BV711 (1:50).
16.Culture isolated CD34
+
cells in complete cell culture media at 37°C and 5%
CO
2for lentiviral transduction or other assays (seeNote 2)
3.2Lentiviral-mediated shRNA vector construction
Predesigned MISSION
®
TRC human shRNA vectors targeting the 3
0
untranslated regions (UTR) or coding domain regions (CDS) of genes of
interest may be purchased at MilliporeSigma (https://bit.ly/2YAaApE)
(seeFig. 1A) (seeNotes 3, 15 and 16).
Plasmid DNA can then be isolated using the PureLink™HiPure
Plasmid Filter
Maxiprep Kit (ThermoFisher) or other related product (seeNotes 1 and 4).
3.3Lentiviral-mediated CRISPR-Cas9 vector construction
and sgRNA design
The lentiCRISPRv2 vector was manufactured by the Zhang lab (Broad Institute)
and can be purchased from Addgene with either GFP or puromycin-resistance
selection markers cloned into the backbone (seeFig. 1B). Guide RNAs (sgRNA)
93Methods
®
Magnetic Nanoparticles (50μL/mL cells) into solution and mix
by pipetting multiple times (seeNote 13). Incubate for 10min.
4.Add EasySep
®
Buffer to increase total volume of cell suspension to 2.5mL and
mix by gentle pipetting.
5.Place the tube containing the cell suspension (without the cap) into the
EasySep
®
Magnet and set aside for 10min.
6.Invert the magnet chamber with the tube inside in one continuous motion to
discard the supernatant. The magnetically labeled cells will remain in the tube.
7.Remove the tube from the EasySep
®
Magnet and mix in 2.5mL EasySep
®
Buffer by pipetting.
8.Place the tube back into the EasySep
®
Magnet and set aside for 5min.
9.Repeat steps 6–8 for a total of 4 washes.
10.Discard the supernatant and remove the tube from the EasySep
®
Magnet before
resuspending cells in 1.2mL of EasySep
®
Buffer. These cells are the purified
CD34
+
cells.
11.For further purity assessment, mix 50μL of CD34
+
cells with 50μL of EasySep
®
Buffer and add 1μL of anti-human CD34-APC (1:100) antibody (seeNote 14).
12.Incubate on ice in the dark for 30min.
13.Wash cells with 1mL EasySep
®
Buffer with 1μL of propidium iodide (PI).
14.Discard the supernatant and resuspend cell pellet in 200μL EasySep
®
Buffer
before FACS analysis using the BD FACSAria™Fusion Flow Cytometer
and FlowJo
®
software (seeNote 1).
15.CD34
+
cells can then be sorted into subpopulations by suspending cells in
HBSS plus 2% FBS and stained with anti-human CD38-PE (1:50) or
anti-human CD38-BV711 (1:50).
16.Culture isolated CD34
+
cells in complete cell culture media at 37°C and 5%
CO
2for lentiviral transduction or other assays (seeNote 2)
3.2Lentiviral-mediated shRNA vector construction
Predesigned MISSION
®
TRC human shRNA vectors targeting the 3
0
untranslated regions (UTR) or coding domain regions (CDS) of genes of
interest may be purchased at MilliporeSigma (https://bit.ly/2YAaApE)
(seeFig. 1A) (seeNotes 3, 15 and 16).
Plasmid DNA can then be isolated using the PureLink™HiPure
Plasmid Filter
Maxiprep Kit (ThermoFisher) or other related product (seeNotes 1 and 4).
3.3Lentiviral-mediated CRISPR-Cas9 vector construction
and sgRNA design
The lentiCRISPRv2 vector was manufactured by the Zhang lab (Broad Institute)
and can be purchased from Addgene with either GFP or puromycin-resistance
selection markers cloned into the backbone (seeFig. 1B). Guide RNAs (sgRNA)
93Methods
for CRISPR-Cas9 may be designed and validated using predictive alignment
tools such as Synthego (https://design.synthego.com/#/), CRISPOR (http://
crispor.tefor.net), or other related software ( seeNote 17).
sgRNAs can be cloned into the lentiCRISPRv2GFP vector with One Shot
®
Stbl3™Chemically CompetentE. coliand transformation can be validated by
Sanger Sequencing (GeneWiz) (seeNote 1).
Plasmid DNA can then be isolated using the PureLink™HiPure Plasmid Filter
Maxiprep Kit (ThermoFisher) or other related product (seeNotes 1 and 4).
3.4Lentivirus production(seeFig. 2A)
1.Seed 6Δ10
6
293T cells in 10cm Falcon tissue culture dishes in 7mL DMEM
with 10% FBS for 24h before transfection. Plate 7 dishes per construct
(seeNote 18).
2.The
–4h before
transfection.
3.In sterile tube A, prepare the DNA mixture. For each construct of 7 plates,
prepare: 6μg of plasmid DNA vector, 3.9μgofΔR and 1.5μg of REV packaging
constructs, and 2.1μg of vesicular stomatitis virus glycoprotein (VSV-G)
envelope construct for a total volume of 250μL in Opti-MEM (seeNotes 6,
7 and 19).
4.In sterile tube B, prepare the packaging and envelope plasmids. For each
construct of 7 plates, prepare: 40μg of PEI mixed with 210μL of Opti-MEM
(seeNotes 6, 7 and 19).
5.Add DNA mixture from tube A dropwise into tube B and incubate for 20min at
RT (seeNote 19).
6.Add the combined mixture dropwise to 293T cells and place in an incubator at
37°C and 5% CO
2for 48h (seeNote 19).
7.Collect viral supernatant and filter through a 0.45μm low protein-binding
filter (MilliporeSigma) and spin down by ultracentrifugation for 90min at
25,000RPM and 4°C(seeNotes 19 and 20).
8.Discard the supernatant and resuspend viral pellet in serum-free Isocove’s
Modified Dulbecco’s Medium with 5% DNAse under gentle agitation for at least
1h at RT before aliquoting and storing virus atα80°C until use (seeNote 19).
3.5Lentiviral transduction and selection of transduced CD34
+
stem/progenitor cells(seeFig. 2B)
Plate 2Δ10
5
primary cells in complete cell culture media and incubate for 16h
(seeNotes 2 and 21).
Add 5μL
of protamine sulfate and 5μL of lentiviral suspension per well and
incubate for 6h at 37°C and 5% CO
2(seeNote 19).
10 CHAPTER 1 Multiple gene knockdown strategies
tools such as Synthego (https://design.synthego.com/#/), CRISPOR (http://
crispor.tefor.net), or other related software ( seeNote 17).
sgRNAs can be cloned into the lentiCRISPRv2GFP vector with One Shot
®
Stbl3™Chemically CompetentE. coliand transformation can be validated by
Sanger Sequencing (GeneWiz) (seeNote 1).
Plasmid DNA can then be isolated using the PureLink™HiPure Plasmid Filter
Maxiprep Kit (ThermoFisher) or other related product (seeNotes 1 and 4).
3.4Lentivirus production(seeFig. 2A)
1.Seed 6Δ10
6
293T cells in 10cm Falcon tissue culture dishes in 7mL DMEM
with 10% FBS for 24h before transfection. Plate 7 dishes per construct
(seeNote 18).
2.The
–4h before
transfection.
3.In sterile tube A, prepare the DNA mixture. For each construct of 7 plates,
prepare: 6μg of plasmid DNA vector, 3.9μgofΔR and 1.5μg of REV packaging
constructs, and 2.1μg of vesicular stomatitis virus glycoprotein (VSV-G)
envelope construct for a total volume of 250μL in Opti-MEM (seeNotes 6,
7 and 19).
4.In sterile tube B, prepare the packaging and envelope plasmids. For each
construct of 7 plates, prepare: 40μg of PEI mixed with 210μL of Opti-MEM
(seeNotes 6, 7 and 19).
5.Add DNA mixture from tube A dropwise into tube B and incubate for 20min at
RT (seeNote 19).
6.Add the combined mixture dropwise to 293T cells and place in an incubator at
37°C and 5% CO
2for 48h (seeNote 19).
7.Collect viral supernatant and filter through a 0.45μm low protein-binding
filter (MilliporeSigma) and spin down by ultracentrifugation for 90min at
25,000RPM and 4°C(seeNotes 19 and 20).
8.Discard the supernatant and resuspend viral pellet in serum-free Isocove’s
Modified Dulbecco’s Medium with 5% DNAse under gentle agitation for at least
1h at RT before aliquoting and storing virus atα80°C until use (seeNote 19).
3.5Lentiviral transduction and selection of transduced CD34
+
stem/progenitor cells(seeFig. 2B)
Plate 2Δ10
5
primary cells in complete cell culture media and incubate for 16h
(seeNotes 2 and 21).
Add 5μL
of protamine sulfate and 5μL of lentiviral suspension per well and
incubate for 6h at 37°C and 5% CO
2(seeNote 19).
10 CHAPTER 1 Multiple gene knockdown strategies
FIG. 2
Schematic for lentivirus production and transduction. (A) For lentivirus production, 7 plates of 293T cells are required per construct. For shRNA
constructs, prepare for each plate (1) 6 μg DNA, 3.9μgΔR, 2.1μg VSVG and 1.5μg REV in 250μL of serum-free Opti-MEM and (2) 40μg
PEI in 210μL of serum-free Opti-MEM. For lentiCRISPRv2 constructs, prepare for each plate (1) 4.8 μgΔpsPAX2 packaging construct and 1.6μg
of pVSVG envelope construct in 250μL of serum-free Opti-MEM and (2) 40μg PEI in 210μL of serum-free Opti-MEM. (B) For lentiviral
transduction, CD34
+
cells are first recovered for 16 h in complete cell culture media supplemented with growth factors before being brought to the
viral vector facility. 5 μL of protamine sulfate (P/S) and 5μL of virus are then added to each well before incubation for 6 h. Cells are then
washed and replated to recover for 48 h before selection.
Schematic for lentivirus production and transduction. (A) For lentivirus production, 7 plates of 293T cells are required per construct. For shRNA
constructs, prepare for each plate (1) 6 μg DNA, 3.9μgΔR, 2.1μg VSVG and 1.5μg REV in 250μL of serum-free Opti-MEM and (2) 40μg
PEI in 210μL of serum-free Opti-MEM. For lentiCRISPRv2 constructs, prepare for each plate (1) 4.8 μgΔpsPAX2 packaging construct and 1.6μg
of pVSVG envelope construct in 250μL of serum-free Opti-MEM and (2) 40μg PEI in 210μL of serum-free Opti-MEM. (B) For lentiviral
transduction, CD34
+
cells are first recovered for 16 h in complete cell culture media supplemented with growth factors before being brought to the
viral vector facility. 5 μL of protamine sulfate (P/S) and 5μL of virus are then added to each well before incubation for 6 h. Cells are then
washed and replated to recover for 48 h before selection.
Wash cells 3 times with HBSS plus 2% FBS and replate cells in 350μL of fresh
complete cell culture media (seeNote 2, 19 and 22). Incubate recovered cells for
48h at 37°C and 5% CO
2.
Apply selection mechanism depending on the selection marker cloned in the
lentiviral construct backbone (seeNote 23).
3.6Nanoparticle-mediated lipopolymer siRNA preparation and
delivery
3.6.1 Preparation of working solutions
1.The GOI and NC siRNAs are diluted to 10μM solutions in RNase-free distilled
water (seeNotes 24, 26 and 27).
2.The
concentration of 1mg/mL (seeNote 25).
3.Live cell counts are obtained from a hemocytometer by staining a 20μL aliquot of
cells in suspension with 0.4% trypan blue solution (seeNote 28).
4.Cells are diluted to the desired concentration in complete cell culture medium
in a 50mL conical-bottom centrifuge tube and kept at 37°C under 5% CO
2
(seeNote 29).
3.6.2Preparation of lipopolymer/siRNA nanoparticles and transfection
of
cells
1.Nanoparticles are prepared in triplicate (biological replicates) for each
experimental group (seeNote 30).
2.The
serum-free media in a microcentrifuge tube, followed by the addition of
lipopolymer corresponding to the desired lipopolymer:siRNA (weight/weight)
ratio (seeNote 31).
3.The solution is then vortexed briefly and incubated for 30min at room
temperature, to allow nanoparticle assembly (seeNotes 32 and 33).
4.In each well of a 24-well plate, 200μL of nanoparticles are dispensed gently,
ensuring the solution covers the entire surface of the well (seeNotes 34 and 35).
5.Gently invert the tube containing cells at the desired concentration a couple of
times to ensure a uniform suspension of cells.
6.A volume of 600μL of cell suspension is gently added to each well on top of the
nanoparticles for reverse transfection, following which plates are incubated at
37°C under 5% CO
2(seeNotes 34 and 36).
3.7Assessment of gene knockdown
Direct Evaluation of Gene Knockdown—The level of gene knockdown is
determined by quantifying GOI transcript and protein amounts. These can be
assessed by performing:
12 CHAPTER 1 Multiple gene knockdown strategies
complete cell culture media (seeNote 2, 19 and 22). Incubate recovered cells for
48h at 37°C and 5% CO
2.
Apply selection mechanism depending on the selection marker cloned in the
lentiviral construct backbone (seeNote 23).
3.6Nanoparticle-mediated lipopolymer siRNA preparation and
delivery
3.6.1 Preparation of working solutions
1.The GOI and NC siRNAs are diluted to 10μM solutions in RNase-free distilled
water (seeNotes 24, 26 and 27).
2.The
concentration of 1mg/mL (seeNote 25).
3.Live cell counts are obtained from a hemocytometer by staining a 20μL aliquot of
cells in suspension with 0.4% trypan blue solution (seeNote 28).
4.Cells are diluted to the desired concentration in complete cell culture medium
in a 50mL conical-bottom centrifuge tube and kept at 37°C under 5% CO
2
(seeNote 29).
3.6.2Preparation of lipopolymer/siRNA nanoparticles and transfection
of
cells
1.Nanoparticles are prepared in triplicate (biological replicates) for each
experimental group (seeNote 30).
2.The
serum-free media in a microcentrifuge tube, followed by the addition of
lipopolymer corresponding to the desired lipopolymer:siRNA (weight/weight)
ratio (seeNote 31).
3.The solution is then vortexed briefly and incubated for 30min at room
temperature, to allow nanoparticle assembly (seeNotes 32 and 33).
4.In each well of a 24-well plate, 200μL of nanoparticles are dispensed gently,
ensuring the solution covers the entire surface of the well (seeNotes 34 and 35).
5.Gently invert the tube containing cells at the desired concentration a couple of
times to ensure a uniform suspension of cells.
6.A volume of 600μL of cell suspension is gently added to each well on top of the
nanoparticles for reverse transfection, following which plates are incubated at
37°C under 5% CO
2(seeNotes 34 and 36).
3.7Assessment of gene knockdown
Direct Evaluation of Gene Knockdown—The level of gene knockdown is
determined by quantifying GOI transcript and protein amounts. These can be
assessed by performing:
12 CHAPTER 1 Multiple gene knockdown strategies
∘Real time-quantitative polymerase chain reaction (RT-qPCR), for which
treated cells are washed and resuspended in TRIzol for extraction of total RNA
to subsequently run PCR (seeNotes 22, 37 and 40).
∘Western blot—to measure changes in expression of protein coded by the GOI
(seeNotes 40 and 41).
∘Leukemic cell genotyping in primary samples—to assess gene expression
variations between LSCs with and without GOI knockdown.
Indirect Evaluation of Gene Knockdown—Different functional assays can be
conducted to look at the physiological effects on cells as a consequence of gene
knockdown. Some routinely performed assays are as follows:
∘LTC-IC assay—to measure the capacity of primitive hematopoietic stem cells
to produce progenitors for at least 6weeks.
∘CFC assay—to assess the ability of hematopoietic stem/progenitor cells to
proliferate and differentiate (seeNote 23).
∘Apoptosis assay—to determine if knocking down the GOI leads to apoptosis
and to quantify the extent of apoptosis (seeNotes 40 and 41).
∘Cell growth curves—to assess changes in cell proliferation rate (seeNote 42).
∘Xenotransplantation and secondary transplantations—to assess the long-term
survival and self-maintenance capacity of LSCsin vivoafter GOI
knockdown (seeNotes 43 and 44).
∘Western blot—to assess gene expression of direct downstream targets of the
GOI (seeNotes 40 and 41).
These proposed functional assays to evaluate gene knockdown in LSCs may also be
applied to normal HSCs as well to identify LSC-specific targets that do not adversely
affect normal HSCs.
4Notes
1.Catalog number and supplier are provided as a reference, but an equivalent
product can be obtained from alternative vendors at a similar cost.
2.For
+
cells obtained from leukemia patients, either IMDM
supplemented with 15% BIT serum substitute, 100ng/mL FLT3 ligand
(FLT3-L), 20 ng/mL IL-3, 20ng/mL G-CSF 20ng/mL IL-6, 10
∘4
M
2-mercaptoethanol, or StemSpan SFEM supplemented with 1ΔCD34
+
Expansion Supplement, 500nM SR1, and 1μM UM729, is recommended.
Culture conditions are suitable for both CML and AML primary samples.
3.Mission
®
shRNA plasmids may be purchased as a bacterial glycerol stock and
kept frozen at∘80°C until use.
4.It is recommended to aliquot purified DNA as working stock solutions and
preserve at∘20°C.
5.Alternatively, the GFP selection marker may be substituted for a puromycin
resistance marker. LentiCRISPRv2 plasmids may be purchased as bacteria in
stab culture format, and it is recommended to create glycerol stocks from these
and store at∘80°C until use.
134Notes
treated cells are washed and resuspended in TRIzol for extraction of total RNA
to subsequently run PCR (seeNotes 22, 37 and 40).
∘Western blot—to measure changes in expression of protein coded by the GOI
(seeNotes 40 and 41).
∘Leukemic cell genotyping in primary samples—to assess gene expression
variations between LSCs with and without GOI knockdown.
Indirect Evaluation of Gene Knockdown—Different functional assays can be
conducted to look at the physiological effects on cells as a consequence of gene
knockdown. Some routinely performed assays are as follows:
∘LTC-IC assay—to measure the capacity of primitive hematopoietic stem cells
to produce progenitors for at least 6weeks.
∘CFC assay—to assess the ability of hematopoietic stem/progenitor cells to
proliferate and differentiate (seeNote 23).
∘Apoptosis assay—to determine if knocking down the GOI leads to apoptosis
and to quantify the extent of apoptosis (seeNotes 40 and 41).
∘Cell growth curves—to assess changes in cell proliferation rate (seeNote 42).
∘Xenotransplantation and secondary transplantations—to assess the long-term
survival and self-maintenance capacity of LSCsin vivoafter GOI
knockdown (seeNotes 43 and 44).
∘Western blot—to assess gene expression of direct downstream targets of the
GOI (seeNotes 40 and 41).
These proposed functional assays to evaluate gene knockdown in LSCs may also be
applied to normal HSCs as well to identify LSC-specific targets that do not adversely
affect normal HSCs.
4Notes
1.Catalog number and supplier are provided as a reference, but an equivalent
product can be obtained from alternative vendors at a similar cost.
2.For
+
cells obtained from leukemia patients, either IMDM
supplemented with 15% BIT serum substitute, 100ng/mL FLT3 ligand
(FLT3-L), 20 ng/mL IL-3, 20ng/mL G-CSF 20ng/mL IL-6, 10
∘4
M
2-mercaptoethanol, or StemSpan SFEM supplemented with 1ΔCD34
+
Expansion Supplement, 500nM SR1, and 1μM UM729, is recommended.
Culture conditions are suitable for both CML and AML primary samples.
3.Mission
®
shRNA plasmids may be purchased as a bacterial glycerol stock and
kept frozen at∘80°C until use.
4.It is recommended to aliquot purified DNA as working stock solutions and
preserve at∘20°C.
5.Alternatively, the GFP selection marker may be substituted for a puromycin
resistance marker. LentiCRISPRv2 plasmids may be purchased as bacteria in
stab culture format, and it is recommended to create glycerol stocks from these
and store at∘80°C until use.
134Notes
6.For lentiviral-mediated shRNA packaging, 3.9μgofΔR and 1.5μg of REV
packaging constructs, 2.1μg of vesicular stomatitis virus glycoprotein (VSVG)
envelope construct and 40μg of polyethyenimine (PEI) (Cat# 23966,
Polysciences Inc.seeNote 1) is recommended.
7.For lentiviral-mediated sgRNA packaging, 4.8μgofΔpsPAX2 packaging
construct, 1.6μg of pVSVG envelope construct and 40μg PEI is recommended.
8.Lyophilized siRNA is reconstituted either in TE buffer at pH 7.5 or HPLC-grade
DNase/RNase-free sterile water at pH 7.0 and stored atα20°C.
9.Avoid siRNA reconstitution in DEPC-treated water or water from deionizing
systems for long-term storage as these are often acidic in nature and can degrade
RNA over time.
10.Lyophilized lipopolymer is commercially available from RJH Biosciences Inc.
(Edmonton, Alberta) and is resuspended in DNase/RNase-free sterile water at
pH 7.0 and stored atα20°C. The synthesis and characterization of the
transfection reagents have been described before for CML cells and AML cells
as well as the optimization of transfection conditions for maximal results
(Landry et al., 2016;Valencia-Serna et al., 2019).
11.RPM
I 1640 medium with
L-glutamine is recommended for cell lines, while
Iscove’s Modified Dulbecco’s Medium (IMDM) or StemSpan Serum-Free
Expansion Medium (SFEM) is recommended for use with primary CD34
+
cells
obtained from the bone marrow or peripheral blood of leukemia patients.
12.Complete RPMI 1640 medium is prepared by supplementing it with 10–20%
fetal bovine serum, penicillin (100U/mL) and streptomycin (100μg/mL), and is
used for maintaining cell lines. For some AML cell lines, 5-10ng/mL
of GM-CSF may also be supplemented.
13.Vortexing is not recommended.
14.It is recommended to prepare 4 tubes: 100μL of initial cell suspension, 100μL
of the wash with no CD34 staining, as a negative control, 100μL of wash with
CD34 staining and 50μL of CD34
+
cells with 50μL of EasySep
®
Buffer.
15.Several different shRNA constructs targeting both the UTR and CDS should be
tested for optimal knockdown efficiency.
16.shRNA sequences may be validated using alignment tools such as Benchling or
other related software.
17.sgRNAs designed with these tools will be assigned a predicted efficiency and
off-target score and will automatically account for the Cas9 NGG PAM
motif preference. It is recommended to several sgRNAs which target different
coding domains for optimal knockdown efficiency.
18.Cells may be plated at a density ranging from 5Δ10
6
to 6.5Δ10
6
per plate.
19.Procedure should be performed in a viral vector facility.
20.It is recommended to combine the supernatant of all 7 plates per construct into a
30mL syringe to press through the 0.45μm low protein-binding filter. Filter
may need to be changed if it becomes blocked by debris.
21.Cells may be plated at a density ranging from 2Δ10
5
to 4Δ10
5
per well in
300μL of complete cell culture media in a 24-well culture plate.
14 CHAPTER 1 Multiple gene knockdown strategies
packaging constructs, 2.1μg of vesicular stomatitis virus glycoprotein (VSVG)
envelope construct and 40μg of polyethyenimine (PEI) (Cat# 23966,
Polysciences Inc.seeNote 1) is recommended.
7.For lentiviral-mediated sgRNA packaging, 4.8μgofΔpsPAX2 packaging
construct, 1.6μg of pVSVG envelope construct and 40μg PEI is recommended.
8.Lyophilized siRNA is reconstituted either in TE buffer at pH 7.5 or HPLC-grade
DNase/RNase-free sterile water at pH 7.0 and stored atα20°C.
9.Avoid siRNA reconstitution in DEPC-treated water or water from deionizing
systems for long-term storage as these are often acidic in nature and can degrade
RNA over time.
10.Lyophilized lipopolymer is commercially available from RJH Biosciences Inc.
(Edmonton, Alberta) and is resuspended in DNase/RNase-free sterile water at
pH 7.0 and stored atα20°C. The synthesis and characterization of the
transfection reagents have been described before for CML cells and AML cells
as well as the optimization of transfection conditions for maximal results
(Landry et al., 2016;Valencia-Serna et al., 2019).
11.RPM
I 1640 medium with
L-glutamine is recommended for cell lines, while
Iscove’s Modified Dulbecco’s Medium (IMDM) or StemSpan Serum-Free
Expansion Medium (SFEM) is recommended for use with primary CD34
+
cells
obtained from the bone marrow or peripheral blood of leukemia patients.
12.Complete RPMI 1640 medium is prepared by supplementing it with 10–20%
fetal bovine serum, penicillin (100U/mL) and streptomycin (100μg/mL), and is
used for maintaining cell lines. For some AML cell lines, 5-10ng/mL
of GM-CSF may also be supplemented.
13.Vortexing is not recommended.
14.It is recommended to prepare 4 tubes: 100μL of initial cell suspension, 100μL
of the wash with no CD34 staining, as a negative control, 100μL of wash with
CD34 staining and 50μL of CD34
+
cells with 50μL of EasySep
®
Buffer.
15.Several different shRNA constructs targeting both the UTR and CDS should be
tested for optimal knockdown efficiency.
16.shRNA sequences may be validated using alignment tools such as Benchling or
other related software.
17.sgRNAs designed with these tools will be assigned a predicted efficiency and
off-target score and will automatically account for the Cas9 NGG PAM
motif preference. It is recommended to several sgRNAs which target different
coding domains for optimal knockdown efficiency.
18.Cells may be plated at a density ranging from 5Δ10
6
to 6.5Δ10
6
per plate.
19.Procedure should be performed in a viral vector facility.
20.It is recommended to combine the supernatant of all 7 plates per construct into a
30mL syringe to press through the 0.45μm low protein-binding filter. Filter
may need to be changed if it becomes blocked by debris.
21.Cells may be plated at a density ranging from 2Δ10
5
to 4Δ10
5
per well in
300μL of complete cell culture media in a 24-well culture plate.
14 CHAPTER 1 Multiple gene knockdown strategies
22.Cell lines are washed by transferring samples into microcentrifuge tubes and
centrifuging them at 1600RPM for 5min, while CD34
+
cells obtained from
leukemia patients are centrifuged at 500Δgfor 10min at room temperature,
followed by careful aspiration of cell culture media without disturbing the
cell pellet.
23.For puromycin-resistant vectors, add 2–2.5μg of puromycin and select for
3–5days before knockdown assessment. GFP vectors may be sorted to obtain a
GFP
+
population and may be sorted further into single cell clones for optimal
knockdown efficiency.
24.It is recommended to prepare 50–100μL aliquots of the 10μM siRNA working
stock solution (store atα20°C) to avoid the siRNA undergoing repeated
freeze-thaw cycles.
25.Prepare 100–200μL aliquots of the 1 mg/mL lipopolymer working stock
solution (store atα20°C) to minimize the number of freeze-thaw cycles.
26.The NC siRNA is a nonsense/scrambled sequence that does not target any gene.
It is a good practice to screen 2–3 different NC siRNA sequences to ensure
that it does not have unintended effects on GOI transcript levels (as assessed
by RT-qPCR) and is not cytotoxic.
27.The GOI siRNA can either be a pool of different sequences targeting the same
gene or be an individual sequence targeting a specific region of the GOI. It is
recommended to screen at least 3 sequences targeting different regions of the
GOI, to identify the most potent sequence.
28.Alternatively, an automated cell counter can be used in place of a
hemocytometer.
29.The number of cells/well and volume of cells/well to be used is contingent on
the tissue culture plate used for the experiment (Table 1).
30.While
it is standard practice to run studies with three biological replicates,
nanoparticle preparation can be easily scaled up or down for a user-defined
number of replicates for each experimental group (Table 2).
31.A
range of siRNA doses and lipopolymer:siRNA (weight/weight) ratios should
be tested to identify the most optimal conditions, albeit a good starting range is
30–60nM siRNA and a lipopolymer:siRNA (weight:weight) ratio ranging
from 5 to 12.
32.It is crucial to allow 30min incubation to ensure complete assembly of
nanoparticles before addition to the cells; this process may otherwise not be
effective.
33.The nanoparticles may be dispensed into the tissue culture plates a few minutes
before the 30min incubation period, so that the cells are exposed to the
particles as close to the 30min mark as possible.
34.Although the protocol presented here is based on a 24-well plate, the volume of
nanoparticles and cells can be easily scaled up or down based on the tissue
culture plate utilized for the experiment (Table 1).
35.For
the “no treatment” group, 200μL of serum-free media is added to the wells
in lieu of the nanoparticles.
154Notes
centrifuging them at 1600RPM for 5min, while CD34
+
cells obtained from
leukemia patients are centrifuged at 500Δgfor 10min at room temperature,
followed by careful aspiration of cell culture media without disturbing the
cell pellet.
23.For puromycin-resistant vectors, add 2–2.5μg of puromycin and select for
3–5days before knockdown assessment. GFP vectors may be sorted to obtain a
GFP
+
population and may be sorted further into single cell clones for optimal
knockdown efficiency.
24.It is recommended to prepare 50–100μL aliquots of the 10μM siRNA working
stock solution (store atα20°C) to avoid the siRNA undergoing repeated
freeze-thaw cycles.
25.Prepare 100–200μL aliquots of the 1 mg/mL lipopolymer working stock
solution (store atα20°C) to minimize the number of freeze-thaw cycles.
26.The NC siRNA is a nonsense/scrambled sequence that does not target any gene.
It is a good practice to screen 2–3 different NC siRNA sequences to ensure
that it does not have unintended effects on GOI transcript levels (as assessed
by RT-qPCR) and is not cytotoxic.
27.The GOI siRNA can either be a pool of different sequences targeting the same
gene or be an individual sequence targeting a specific region of the GOI. It is
recommended to screen at least 3 sequences targeting different regions of the
GOI, to identify the most potent sequence.
28.Alternatively, an automated cell counter can be used in place of a
hemocytometer.
29.The number of cells/well and volume of cells/well to be used is contingent on
the tissue culture plate used for the experiment (Table 1).
30.While
it is standard practice to run studies with three biological replicates,
nanoparticle preparation can be easily scaled up or down for a user-defined
number of replicates for each experimental group (Table 2).
31.A
range of siRNA doses and lipopolymer:siRNA (weight/weight) ratios should
be tested to identify the most optimal conditions, albeit a good starting range is
30–60nM siRNA and a lipopolymer:siRNA (weight:weight) ratio ranging
from 5 to 12.
32.It is crucial to allow 30min incubation to ensure complete assembly of
nanoparticles before addition to the cells; this process may otherwise not be
effective.
33.The nanoparticles may be dispensed into the tissue culture plates a few minutes
before the 30min incubation period, so that the cells are exposed to the
particles as close to the 30min mark as possible.
34.Although the protocol presented here is based on a 24-well plate, the volume of
nanoparticles and cells can be easily scaled up or down based on the tissue
culture plate utilized for the experiment (Table 1).
35.For
the “no treatment” group, 200μL of serum-free media is added to the wells
in lieu of the nanoparticles.
154Notes
36.Based on several studies conducted by the authors’ group, reverse transfection
(cells added after the addition of nanoparticles) has been seen to be more
effective than forward transfection (nanoparticles added after the addition of
cells) in knocking down genes in leukemia cells.
37.Besides the TRIzol based organic extraction technique, other methods such as
silica-membrane based spin column technology and paramagnetic particle
technology can be employed for total RNA isolation.
38.For the LTC-IC assay, 3days post puromycin selection, 2Δ10
4
CD34
+
primary
cells are plated onto 6Δ10
4
mixed feeder cells per well of a 24-well plate for
6weeks. Half-volume LTC medium changes with 10
α4
M hydrocortisone are
performed weekly (Rothe et al., 2020).
39.For
the CFC assay, 24h after nanoparticle treatment, 200–400 CD34
+
cell lines
or 1Δ10
4
–2Δ10
4
primary CD34
+
cells are seeded in 400μL of methylcellulose
media per well of a 24-well plate.
40.The duration of nanoparticle treatment has an impact on the results of assays
conducted to evaluate gene knockdown, and hence it is important to explore
effects 1–4days after treatment.
41.The level of gene knockdown in a lentiviral-mediated system is dependent on
the GOI and optimal knockdown may be seen anywhere between 3 and 7days
post-selection.
42.This assay is suitable for cell lines as they proliferate well, but CD34
+
patient
cells grow relatively slowly.
43.For xenotransplantation and secondary transplantation experiments, 2Δ10
6
GOI knockdown CD34
+
CML cells are injected into 10–12-week-old,
sub-lethally irradiated NRG or NRG-3GS mice. Peripheral blood (PB) and bone
marrow (BM) samples are collected at timepoints ranging from 3 to 20weeks
and labeled for FACS analysis (Rothe et al., 2020).
44.For
secondary transplantation experiments, human cells from primary mice BM
can be obtained using an EasySep
®
Mouse/Human Chimera Isolation Kit
(STEMCLL Technologies) and injected into secondary, sub-lethally irradiated
mice. BM samples are collected at serial timepoints and labeled for FACS
analysis (Rothe et al., 2020).
Table 1Recommended volumes and cell numbers for different tissue culture
plates.
Tissue
culture plate
Volume of
nanoparticles
(μL)
Volume of cell
suspension
(μL)
Number of
cells (cell line)
Number of cells
(primary)
12-well plate 400 1200 1.5 Δ10
5
to
2Δ10
5
3Δ10
6
to 5Δ10
6
24-well plate 200 600 72,000–90,000 1Δ10
6
to 2Δ10
6
48-well plate 100 300 35,000–45,000 4Δ10
5
to 8Δ10
5
96-well plate 20 80 12,000–18,000 1Δ10
5
to 2Δ10
5
16 CHAPTER 1 Multiple gene knockdown strategies
(cells added after the addition of nanoparticles) has been seen to be more
effective than forward transfection (nanoparticles added after the addition of
cells) in knocking down genes in leukemia cells.
37.Besides the TRIzol based organic extraction technique, other methods such as
silica-membrane based spin column technology and paramagnetic particle
technology can be employed for total RNA isolation.
38.For the LTC-IC assay, 3days post puromycin selection, 2Δ10
4
CD34
+
primary
cells are plated onto 6Δ10
4
mixed feeder cells per well of a 24-well plate for
6weeks. Half-volume LTC medium changes with 10
α4
M hydrocortisone are
performed weekly (Rothe et al., 2020).
39.For
the CFC assay, 24h after nanoparticle treatment, 200–400 CD34
+
cell lines
or 1Δ10
4
–2Δ10
4
primary CD34
+
cells are seeded in 400μL of methylcellulose
media per well of a 24-well plate.
40.The duration of nanoparticle treatment has an impact on the results of assays
conducted to evaluate gene knockdown, and hence it is important to explore
effects 1–4days after treatment.
41.The level of gene knockdown in a lentiviral-mediated system is dependent on
the GOI and optimal knockdown may be seen anywhere between 3 and 7days
post-selection.
42.This assay is suitable for cell lines as they proliferate well, but CD34
+
patient
cells grow relatively slowly.
43.For xenotransplantation and secondary transplantation experiments, 2Δ10
6
GOI knockdown CD34
+
CML cells are injected into 10–12-week-old,
sub-lethally irradiated NRG or NRG-3GS mice. Peripheral blood (PB) and bone
marrow (BM) samples are collected at timepoints ranging from 3 to 20weeks
and labeled for FACS analysis (Rothe et al., 2020).
44.For
secondary transplantation experiments, human cells from primary mice BM
can be obtained using an EasySep
®
Mouse/Human Chimera Isolation Kit
(STEMCLL Technologies) and injected into secondary, sub-lethally irradiated
mice. BM samples are collected at serial timepoints and labeled for FACS
analysis (Rothe et al., 2020).
Table 1Recommended volumes and cell numbers for different tissue culture
plates.
Tissue
culture plate
Volume of
nanoparticles
(μL)
Volume of cell
suspension
(μL)
Number of
cells (cell line)
Number of cells
(primary)
12-well plate 400 1200 1.5 Δ10
5
to
2Δ10
5
3Δ10
6
to 5Δ10
6
24-well plate 200 600 72,000–90,000 1Δ10
6
to 2Δ10
6
48-well plate 100 300 35,000–45,000 4Δ10
5
to 8Δ10
5
96-well plate 20 80 12,000–18,000 1Δ10
5
to 2Δ10
5
16 CHAPTER 1 Multiple gene knockdown strategies
Table 2Representative experimental set up for lipopolymer/siRNA nanoparticle preparation.
a
Groups
Lipopolymer:
siRNA ratio
b
siRNA
dose (nM)
Lipopolymer
(μg)
siRNA
(μg)
Volume of serum-
free media (μL)
Volume of
lipopolymer (μL)
Volume of
siRNA (μL)
Lipopolymer/
GOI siRNA
126013.101.092 305.0713.106.83
Lipopolymer/
NC siRNA
126013.101.092 305.0713.106.83
Lipopolymer/
GOI siRNA
9609.831.092 308.359.836.83
Lipopolymer/
NC siRNA
9609.831.092 308.359.836.83
Lipopolymer/
GOI siRNA
6606.551.092 311.626.556.83
Lipopolymer/
NC siRNA
6606.551.092 311.626.556.83
No treatment325
Abbreviations: GOI, gene of interest; NC, negative control. a
For cell transfection in 24-well plates with triplicates per group. A total volume of 325 μL of nanoparticles is prepared (with a little excess to account for pipetting
losses), and 100μL is dispensed in each well.
b
Weight/weight ratio.
a
Groups
Lipopolymer:
siRNA ratio
b
siRNA
dose (nM)
Lipopolymer
(μg)
siRNA
(μg)
Volume of serum-
free media (μL)
Volume of
lipopolymer (μL)
Volume of
siRNA (μL)
Lipopolymer/
GOI siRNA
126013.101.092 305.0713.106.83
Lipopolymer/
NC siRNA
126013.101.092 305.0713.106.83
Lipopolymer/
GOI siRNA
9609.831.092 308.359.836.83
Lipopolymer/
NC siRNA
9609.831.092 308.359.836.83
Lipopolymer/
GOI siRNA
6606.551.092 311.626.556.83
Lipopolymer/
NC siRNA
6606.551.092 311.626.556.83
No treatment325
Abbreviations: GOI, gene of interest; NC, negative control. a
For cell transfection in 24-well plates with triplicates per group. A total volume of 325 μL of nanoparticles is prepared (with a little excess to account for pipetting
losses), and 100μL is dispensed in each well.
b
Weight/weight ratio.
5Concluding remarks
The gene knockdown and modification approaches described here have allowed us to
genetically manipulate expression of critical leukemia drivers in hard-to-transfect
CD34
+
stem/progenitor cells from drug-responders and nonresponder patients and
to understand their molecular and cellular actions in regulating LSCs and their pro-
genitor cells, which contribute to drug resistance and leukemia progression. These
studies have also uncovered promising therapeutic targets and enabled exploration
of potential therapeutic modalities. In particular, the CRISPR/Cas9 system has
recently been used to achieve individual gene knockdown on a genome-wide scale
and often results in stronger gene knockdown and phenotypic changes in cells than
that observed when the same genes are knocked down using shRNA. Lipopolymer/
siRNA nanoparticles also offer a simple yet effective approach to performing gene
knockdown in these patient cells. Functional assays to confirm knockdown effi-
ciency in LSCs may also be extended to observe effects of knockdown in normal
HSCs and to identify LSC-specific targets.
While these lentiviral-mediated and lipopolymer/siRNA nanoparticle-mediated
syst
ems are cost-efficient, easily adaptable and generate reliable gene expression
modification efficiencies, there are also a few limitations to consider. Firstly, human
LSCs are rare, without direct surface markers to allow for their isolation, and even
with stem cell-enrichment, there are often not enough materials to perform gene
knockdown in these cells directly. Thus, most gene knockdown experiments can only
be performed with CD34
+
stem/progenitor cells, followed by several long-term bi-
ological assays (LTC-IC and serial engraftment experiments, etc.) to reflect the bi-
ological properties of true LSCs after genetic manipulation (Liu, Miller, & Eaves,
20
13;Thomas & Majeti, 2017). Secondly, the CD34
+
stem/progenitor cell popula-
tion is heterogeneous and undergoes rapid differentiation, and therefore gene knock-
down and transduction efficiency could be different from individual patient samples;
optimization of culture conditions and concentrations of certain reagents may be
needed for effective gene knockdown. Another caveat is that the puromycin selection
method for puro-resistant vectors described in this chapter does not generate single
cell clones, which may be desirable for certain experimental models. Instead, the
CD34
+
cells that have undergone selection are still part of a heterogenous bulk pop-
ulation that may have varying degrees of knockdown efficiencies. The same limita-
tion applies when using the lentiCRISPRv2GFP vectors and sorting for a GFP
+
population. The GFP marker is attached to the Cas9 transcript and while GFP
+
status
is a good marker for vector uptake, it is not necessarily indicative of sgRNA align-
ment nor successful gene editing. Thus, follow-up validation sequencing steps
should be taken to confirm mutagenesis such as leukemic cell genotyping, especially
if single cell knockout clones are desired. Alternative CRISPR-Cas9 knockdown
mechanisms could also be explored, such as ribonucleoprotein (RNP) delivery,
where fluorophores can be directly attached to tracrRNA and isolated through
FACS-sorting (Nasri et al., 2019). However, we have not tested this method in
CD34
+
cells and reports confirming generation ofin vivomouse models using this
18 CHAPTER 1 Multiple gene knockdown strategies
The gene knockdown and modification approaches described here have allowed us to
genetically manipulate expression of critical leukemia drivers in hard-to-transfect
CD34
+
stem/progenitor cells from drug-responders and nonresponder patients and
to understand their molecular and cellular actions in regulating LSCs and their pro-
genitor cells, which contribute to drug resistance and leukemia progression. These
studies have also uncovered promising therapeutic targets and enabled exploration
of potential therapeutic modalities. In particular, the CRISPR/Cas9 system has
recently been used to achieve individual gene knockdown on a genome-wide scale
and often results in stronger gene knockdown and phenotypic changes in cells than
that observed when the same genes are knocked down using shRNA. Lipopolymer/
siRNA nanoparticles also offer a simple yet effective approach to performing gene
knockdown in these patient cells. Functional assays to confirm knockdown effi-
ciency in LSCs may also be extended to observe effects of knockdown in normal
HSCs and to identify LSC-specific targets.
While these lentiviral-mediated and lipopolymer/siRNA nanoparticle-mediated
syst
ems are cost-efficient, easily adaptable and generate reliable gene expression
modification efficiencies, there are also a few limitations to consider. Firstly, human
LSCs are rare, without direct surface markers to allow for their isolation, and even
with stem cell-enrichment, there are often not enough materials to perform gene
knockdown in these cells directly. Thus, most gene knockdown experiments can only
be performed with CD34
+
stem/progenitor cells, followed by several long-term bi-
ological assays (LTC-IC and serial engraftment experiments, etc.) to reflect the bi-
ological properties of true LSCs after genetic manipulation (Liu, Miller, & Eaves,
20
13;Thomas & Majeti, 2017). Secondly, the CD34
+
stem/progenitor cell popula-
tion is heterogeneous and undergoes rapid differentiation, and therefore gene knock-
down and transduction efficiency could be different from individual patient samples;
optimization of culture conditions and concentrations of certain reagents may be
needed for effective gene knockdown. Another caveat is that the puromycin selection
method for puro-resistant vectors described in this chapter does not generate single
cell clones, which may be desirable for certain experimental models. Instead, the
CD34
+
cells that have undergone selection are still part of a heterogenous bulk pop-
ulation that may have varying degrees of knockdown efficiencies. The same limita-
tion applies when using the lentiCRISPRv2GFP vectors and sorting for a GFP
+
population. The GFP marker is attached to the Cas9 transcript and while GFP
+
status
is a good marker for vector uptake, it is not necessarily indicative of sgRNA align-
ment nor successful gene editing. Thus, follow-up validation sequencing steps
should be taken to confirm mutagenesis such as leukemic cell genotyping, especially
if single cell knockout clones are desired. Alternative CRISPR-Cas9 knockdown
mechanisms could also be explored, such as ribonucleoprotein (RNP) delivery,
where fluorophores can be directly attached to tracrRNA and isolated through
FACS-sorting (Nasri et al., 2019). However, we have not tested this method in
CD34
+
cells and reports confirming generation ofin vivomouse models using this
18 CHAPTER 1 Multiple gene knockdown strategies
mechanism are limited. Despite these limitations, bulk sorting of transduced CD34
+
cells may suffice, particularly in cases where the GOI is essential for cell viability
and single cell populations cannot be generated. Lastly, an ideal workflow encom-
passes screening GOI siRNA sequences as well as libraries of lipopolymers to iden-
tify the most potent combination of siRNA and lipopolymer. Such comprehensive
testing is most suitable in cell lines, so that a narrow range of parameters can be iden-
tified for further testing in more precious CD34
+
patient cells; however, knockdown
efficiency observed in cell lines may not necessarily translate exactly to primary
CD34
+
cells (Gul-Uludag et al., 2014;Landry et al., 2016;Valencia-Serna et al.,
2019). Altogether, the methodologies described in this chapter offer relatively
strai
ghtforward approaches to testing the effects of gene knockdowns in LSCs, to
uncover novel survival and proliferation pathways and to identify new targets and
models for developing curative therapies in stem cell-driven cancers.
Acknowledgments
A.W. received a MITACS Accelerate Fellowship and A.S.A. is supported by the Natural
Sciences and Engineering Research Council of Canada (NSERC). H.U. is supported by
NSERC, Canadian Institutes of Health Research (CIHR), RJH Biosciences, MITACS, New
Frontiers Research Fund and Alex’s Lemonade Stand Foundation. X.J. is supported by the
CIHR, the Canadian Cancer Society, the Leukemia & Lymphoma Society of Canada and
the Collings Stevens Chronic Leukemia Research Fund. Figures were generated using
BioRender.
Disclosures
A.W., A.S.A. and X.J. have no conflicts of interest to disclose. H.U. is a founding shareholder
in R.J.H. Biosciences Inc., which commercially develops the described lipopolymers.
References
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19References
+
cells may suffice, particularly in cases where the GOI is essential for cell viability
and single cell populations cannot be generated. Lastly, an ideal workflow encom-
passes screening GOI siRNA sequences as well as libraries of lipopolymers to iden-
tify the most potent combination of siRNA and lipopolymer. Such comprehensive
testing is most suitable in cell lines, so that a narrow range of parameters can be iden-
tified for further testing in more precious CD34
+
patient cells; however, knockdown
efficiency observed in cell lines may not necessarily translate exactly to primary
CD34
+
cells (Gul-Uludag et al., 2014;Landry et al., 2016;Valencia-Serna et al.,
2019). Altogether, the methodologies described in this chapter offer relatively
strai
ghtforward approaches to testing the effects of gene knockdowns in LSCs, to
uncover novel survival and proliferation pathways and to identify new targets and
models for developing curative therapies in stem cell-driven cancers.
Acknowledgments
A.W. received a MITACS Accelerate Fellowship and A.S.A. is supported by the Natural
Sciences and Engineering Research Council of Canada (NSERC). H.U. is supported by
NSERC, Canadian Institutes of Health Research (CIHR), RJH Biosciences, MITACS, New
Frontiers Research Fund and Alex’s Lemonade Stand Foundation. X.J. is supported by the
CIHR, the Canadian Cancer Society, the Leukemia & Lymphoma Society of Canada and
the Collings Stevens Chronic Leukemia Research Fund. Figures were generated using
BioRender.
Disclosures
A.W., A.S.A. and X.J. have no conflicts of interest to disclose. H.U. is a founding shareholder
in R.J.H. Biosciences Inc., which commercially develops the described lipopolymers.
References
Alvarez-Calderon, F., Gregory, M. A., Pham-Danis, C., DeRyckere, D., Stevens, B. M.,
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metabolic state sensitive to mitochondrial perturbations.Clinical Cancer Research,21(6),
1360–1372.https://doi.org/10.1158/1078-0432.ccr-14-2146.
Ansari, A. S., Santerre, P. J., & Uludag, H. (2017). Biomaterials for polynucleotide delivery to
anchorage-independent
cells.Journal of Materials Chemistry B, 5(35), 7238–7261.
https://doi.org/10.1039/c7tb01833a.
Baccarani,
M., Deininger, M. W., Rosti, G., Hochhaus, A., Soverini, S., Apperley, J. F., et al.
(2013). European LeukemiaNet recommendations for the management of chronic myeloid
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Baccarani,
M., Saglio, G., Goldman, J., Hochhaus, A., Simonsson, B., Appelbaum, F., et al.
(2006). Evolving concepts in the management of chronic myeloid leukemia: Recommen-
dations from an expert panel on behalf of the European LeukemiaNet.Blood,108(6),
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20 CHAPTER 1 Multiple gene knockdown strategies
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stem cells.Science Translational Medicine,8(355), 355ra117.https://doi.org/10.1126/sci-
translmed.aag1180.
Chen,
M., Gallipoli, P., DeGeer, D., Sloma, I., Forrest, D. L., Chan, M., et al. (2013). Targeting
primitive chronic myeloid leukemia cells by effective inhibition of a new AHI-1-
BCR-ABL-JAK2 complex.Journal of the National Cancer Institute,105(6), 405–423.
https://doi.org/10.1093/jnci/djt006.
Chu,
S., Xu, H., Shah, N. P., Snyder, D. S., Forman, S. J., Sawyers, C. L., et al. (2005).
Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous
leukemia patients in complete cytogenetic remission on imatinib mesylate treatment.
Blood,105(5), 2093–2098.https://doi.org/10.1182/blood-2004-03-1114.
Corbin,
A. S., Agarwal, A., Loriaux, M., Cortes, J., Deininger, M. W., & Druker, B. J. (2011).
Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition
of BCR-ABL activity.The Journal of Clinical Investigation,121(1), 396–409.https://doi.
org/10.1172/JCI35721.
Druker,
B. J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G. M., Fanning, S., et al. (1996).
Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive
cells.Nature Medicine,2(5), 561–566. Retrieved fromhttp://www.ncbi.nlm.nih.gov/
pubmed/8616716.
Freeley,
M., & Long, A. (2013). Advances in siRNA delivery to T-cells: Potential clinical ap-
plications for inflammatory disease, cancer and infection.Biochemical Journal,455(2),
133–147.https://doi.org/10.1042/BJ20130950.
Gallipoli,
P., Stobo, J., Heaney, N., Nicolini, F. E., Clark, R., Wilson, G., et al. (2013). Safety
and efficacy of pulsed imatinib with or without G-CSF versus continuous imatinib in
chronic phase chronic myeloid leukaemia patients at 5 years follow-up.British Journal
of Haematology,163(5), 674–676.https://doi.org/10.1111/bjh.12532.
Glauche,
I., Horn, K., Horn, M., Thielecke, L., Essers, M. A., Trumpp, A., et al. (2012).
Therapy of chronic myeloid leukaemia can benefit from the activation of stem cells: Sim-
ulation studies of different treatment combinations.British Journal of Cancer,106(11),
1742–1752.https://doi.org/10.1038/bjc.2012.142.
Goldman,
J. M., & Melo, J. V. (2003). Chronic myeloid leukemia—Advances in biology and
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https://doi.org/10.1056/NEJMra020777.
Gorre,
M. E., Mohammed, M., Ellwood, K., Hsu, N., Paquette, R., Rao, P. N., et al. (2001).
Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or
amplification.Science,293(5531), 876–880.https://doi.org/10.1126/science.1062538.
Graham,
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326843.
20 CHAPTER 1 Multiple gene knockdown strategies
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(2020). European LeukemiaNet 2020 recommendations for treating chronic myeloid
leukemia.Leukemia,34(4), 966–984.https://doi.org/10.1038/s41375-020-0776-2.
Holtz,
M. S., Forman, S. J., & Bhatia, R. (2005). Nonproliferating CML CD34+ progenitors
are resistant to apoptosis induced by a wide range of proapoptotic stimuli.Leukemia,
19(6), 1034–1041.https://doi.org/10.1038/sj.leu.2403724.
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L., Story, B. A., Hernandez-Malmierca, P., Raffel, S., Leonce, D. R., Milbank, J.,
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of acute myeloid leukemia.Nanoscale,6(16), 9742–9751.https://doi.org/10.1039/
c4nr01510j.
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22 CHAPTER 1 Multiple gene knockdown strategies
(2016). Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia.
The New England Journal of Medicine,374(4), 311–322.https://doi.org/10.1056/
NEJMoa1513257.
Rothdiener,
M., Muller, D., Castro, P. G., Scholz, A., Schwemmlein, M., Fey, G., et al.
(2010). Targeted delivery of SiRNA to CD33-positive tumor cells with liposomal carrier
systems.Journal of Controlled Release,144(2), 251–258.https://doi.org/10.1016/j.
jconrel.2010.02.020.
Rothe,
K., Babaian, A., Nakamichi, N., Chen, M., Chafe, S. C., Watanabe, A., et al. (2020).
Integrin-linked kinase mediates therapeutic resistance of quiescent CML stem cells to
tyrosine kinase inhibitors.Cell Stem Cell,27(1), 110–124 e119.https://doi.org/10.1016/
j.stem.2020.04.005.
Rothe,
K., Lin, H., Lin, K. B., Leung, A., Wang, H. M., Malekesmaeili, M., et al. (2014). The
core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML
stem/progenitor cells.Blood,123(23), 3622–3634.https://doi.org/10.1182/blood-2013-
07-516807.
Rowley,
J. D. (1973). Letter: A new consistent chromosomal abnormality in chronic myelog-
enous leukaemia identified by quinacrine fluorescence and Giemsa staining.Nature,
243(5405), 290–293. Retrieved fromhttp://www.ncbi.nlm.nih.gov/pubmed/4126434.
Savona,
M., & Talpaz, M. (2008). Getting to the stem of chronic myeloid leukaemia.Nature
Reviews. Cancer,8(5), 341–350.https://doi.org/10.1038/nrc2368.
Sloma,
I., Jiang, X., Eaves, A. C., & Eaves, C. J. (2010). Insights into the stem cells of chronic
myeloid leukemia.Leukemia,24(11), 1823–1833.https://doi.org/10.1038/leu.2010.159.
Thomas,
D., & Majeti, R. (2017). Biology and relevance of human acute myeloid leukemia
stem cells.Blood,129(12), 1577–1585.https://doi.org/10.1182/blood-2016-10-696054.
Valencia-Serna,
J., Kucharski, C., Chen, M., Kc, R., Jiang, X., Brandwein, J., et al. (2019).
siRNA-mediated BCR-ABL silencing in primary chronic myeloid leukemia cells using
lipopolymers.Journal of Controlled Release,310, 141–154.https://doi.org/10.1016/j.
jconrel.2019.08.018.
Velten,
L., Story, B. A., Hernandez-Malmierca, P., Raffel, S., Leonce, D. R., Milbank, J.,
et al. (2021). Identification of leukemic and pre-leukemic stem cells by clonal tracking
from single-cell transcriptomics.Nature Communications,12(1), 1366.https://doi.org/
10.1038/s41467-021-21650-1.
Wunderlich,
M., Chou, F. S., Link, K. A., Mizukawa, B., Perry, R. L., Carroll, M., et al. (2010).
AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitu-
tively expressing human SCF, GM-CSF and IL-3.Leukemia,24(10), 1785–1788.https://
doi.org/10.1038/leu.2010.158.
Yang,
Z., Yu, B., Zhu, J., Huang, X., Xie, J., Xu, S., et al. (2014). A microfluidic method
to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy
of acute myeloid leukemia.Nanoscale,6(16), 9742–9751.https://doi.org/10.1039/
c4nr01510j.
Zhang,
W., Muller, K., Kessel, E., Reinhard, S., He, D., Klein, P. M., et al. (2016). Targeted
siRNA delivery using a lipo-oligoaminoamide nanocore with an influenza peptide and
transferrin shell.Advanced Healthcare Materials,5(12), 1493–1504.https://doi.org/
10.1002/adhm.201600057.
22 CHAPTER 1 Multiple gene knockdown strategies
CHAPTER
Evaluation of cancer stem
cells markers expression
in HCC trough real-time
polymerasechainreaction
2
Barbara Bueloni
a
, Esteban Fiore
a
, Manuel Gidekel
b
, Juan Bayo
a,∗
,
and Guillermo Mazzolini
a,c,∗
a
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
b
Universidad Auto´noma de Chile, Providencia, Chile
c
Liver Unit, Hospital Universitario Austral, Buenos Aires, Argentina
∗
Corresponding authors: e-mail address: [email protected]; [email protected]
Chapter outline
1 Introduction and rationale....................................................................................24
2 Materials............................................................................................................25
2.1 Common reagents................................................................................25
2.2 Common solutions................................................................................25
2.3 Common equipment.............................................................................25
3 Methods.............................................................................................................25
3.1 RNA isolation from cells ortissues.........................................................25
3.2 Removal of genomic DNA contamination................................................26
3.3 cDNA synthesis....................................................................................27
3.4 Designing primers for qRT-PCR.............................................................27
3.5 Primer preparation...............................................................................28
3.6 qRT-PCR set up...................................................................................28
3.7 qRT-PCR thermal settings.....................................................................30
3.8 qRT-PCR analysis.................................................................................30
4 Results and discussion........................................................................................31
Disclosures.............................................................................................................31
References..............................................................................................................32
Methods in Cell Biology, Volume 171, ISSN 0091-679X,
https://doi.org/10.1016/bs.mcb.2022.04.003
Copyright ©2022 Elsevier Inc. All rights reserved.
23
Evaluation of cancer stem
cells markers expression
in HCC trough real-time
polymerasechainreaction
2
Barbara Bueloni
a
, Esteban Fiore
a
, Manuel Gidekel
b
, Juan Bayo
a,∗
,
and Guillermo Mazzolini
a,c,∗
a
Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional,
CONICET- Universidad Austral, Buenos Aires, Argentina
b
Universidad Auto´noma de Chile, Providencia, Chile
c
Liver Unit, Hospital Universitario Austral, Buenos Aires, Argentina
∗
Corresponding authors: e-mail address: [email protected]; [email protected]
Chapter outline
1 Introduction and rationale....................................................................................24
2 Materials............................................................................................................25
2.1 Common reagents................................................................................25
2.2 Common solutions................................................................................25
2.3 Common equipment.............................................................................25
3 Methods.............................................................................................................25
3.1 RNA isolation from cells ortissues.........................................................25
3.2 Removal of genomic DNA contamination................................................26
3.3 cDNA synthesis....................................................................................27
3.4 Designing primers for qRT-PCR.............................................................27
3.5 Primer preparation...............................................................................28
3.6 qRT-PCR set up...................................................................................28
3.7 qRT-PCR thermal settings.....................................................................30
3.8 qRT-PCR analysis.................................................................................30
4 Results and discussion........................................................................................31
Disclosures.............................................................................................................31
References..............................................................................................................32
Methods in Cell Biology, Volume 171, ISSN 0091-679X,
https://doi.org/10.1016/bs.mcb.2022.04.003
Copyright ©2022 Elsevier Inc. All rights reserved.
23
Abstract
Quantitative real-time polymerase chain reaction (qRT-PCR) flexibility, robustness and repro-
ducibility have rapidly extended the scope of the method. Cancer stem cells are gaining
increasing importance since their role in cancer initiation, treatment resistance and recurrence
give rise to a wide range of potential diagnostic and therapeutic applications. The expression
of several characteristic markers is proven a reliable method to assess stem-like-phenotype of
cancer cells. Here, we provided a thorough protocol for the study of cancer stem cells in
hepatocellular carcinoma mouse models and cell cultures using qRT-PCR.
1
Introduction and rationale
Basic principles of qRT-PCR allow its application on quantitative and semi-
quantitative assays, turning it into a widely used technique for gene expression pro-
filing. Therefore, the use of qRT-PCR to measure stem-like features in HCC cells is
an invaluable tool for everyday work in a laboratory.
Despite representing a small subset of tumor cells, CSCs bring together a set of
tumorigenic, metastatic, and chemotherapy-resistant properties that have positioned
their study at the forefront of cancer research (
Liu, Yeh, & Lin, 2020). Among these
properties, self-renewal, high proliferative capability, and maintenance of primary
tumor and metastases can be highlighted (
Liu et al., 2020).
In HCC, the fourth cause of cancer-related death worldwide, genetic and epige-
netic alterations are common events in human HCC, leading to an aggressive gene
expression program and poor clinical prognosis (Bayo et al., 2019). Although a num-
ber of biological therapies have demonstrated success in primary and metastatic liver
tumors in experimental animal models (
Bayo et al., 2014;Inarrairaegui, Melero, &
Sangro, 2018;Ochoa et al., 2013), it was not until very recently that the combination
of atezolizumab and bevacizumab was shown to be effective for advanced HCC in
first line (
Finn et al., 2020). In addition, the presence of several surface markers
including EpCAM (epithelial cell adhesion molecule), CD133, CD44, CD47, and
OCT-4 has been correlated with phenotypic characteristics of liver cancer stem cells
such as tumor-initiating, colony-forming, and immune-evasion capabilities (Liu
et al., 2020;Xiang, Yang, Pang, Zhu, & Liu, 2016). Similarly, the chemoresistance
of hepatic CSCs have been related with DLK1, EpCAM, CD133, CD44, CD24 and
CD13 cell markers (
Chen et al., 2012;Haraguchi et al., 2010;Xiang et al., 2016;Xu
et al., 2012;Zhu et al., 2010). Therefore, comparative RT-PCR measurement of
CSCs markers mRNA levels of cancer cells grown in culture or form tumoral tissues
provides insight into drug effects on the cancer stem cells phenotype (
Rodriguez
et al., 2018;Rodriguez et al., 2021). Here, we report successful implementation
of this technique to evaluate stem-like characteristics of HCC cells and provide a
detailed protocol encompassing critical considerations that should be considered.
24 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
Quantitative real-time polymerase chain reaction (qRT-PCR) flexibility, robustness and repro-
ducibility have rapidly extended the scope of the method. Cancer stem cells are gaining
increasing importance since their role in cancer initiation, treatment resistance and recurrence
give rise to a wide range of potential diagnostic and therapeutic applications. The expression
of several characteristic markers is proven a reliable method to assess stem-like-phenotype of
cancer cells. Here, we provided a thorough protocol for the study of cancer stem cells in
hepatocellular carcinoma mouse models and cell cultures using qRT-PCR.
1
Introduction and rationale
Basic principles of qRT-PCR allow its application on quantitative and semi-
quantitative assays, turning it into a widely used technique for gene expression pro-
filing. Therefore, the use of qRT-PCR to measure stem-like features in HCC cells is
an invaluable tool for everyday work in a laboratory.
Despite representing a small subset of tumor cells, CSCs bring together a set of
tumorigenic, metastatic, and chemotherapy-resistant properties that have positioned
their study at the forefront of cancer research (
Liu, Yeh, & Lin, 2020). Among these
properties, self-renewal, high proliferative capability, and maintenance of primary
tumor and metastases can be highlighted (
Liu et al., 2020).
In HCC, the fourth cause of cancer-related death worldwide, genetic and epige-
netic alterations are common events in human HCC, leading to an aggressive gene
expression program and poor clinical prognosis (Bayo et al., 2019). Although a num-
ber of biological therapies have demonstrated success in primary and metastatic liver
tumors in experimental animal models (
Bayo et al., 2014;Inarrairaegui, Melero, &
Sangro, 2018;Ochoa et al., 2013), it was not until very recently that the combination
of atezolizumab and bevacizumab was shown to be effective for advanced HCC in
first line (
Finn et al., 2020). In addition, the presence of several surface markers
including EpCAM (epithelial cell adhesion molecule), CD133, CD44, CD47, and
OCT-4 has been correlated with phenotypic characteristics of liver cancer stem cells
such as tumor-initiating, colony-forming, and immune-evasion capabilities (Liu
et al., 2020;Xiang, Yang, Pang, Zhu, & Liu, 2016). Similarly, the chemoresistance
of hepatic CSCs have been related with DLK1, EpCAM, CD133, CD44, CD24 and
CD13 cell markers (
Chen et al., 2012;Haraguchi et al., 2010;Xiang et al., 2016;Xu
et al., 2012;Zhu et al., 2010). Therefore, comparative RT-PCR measurement of
CSCs markers mRNA levels of cancer cells grown in culture or form tumoral tissues
provides insight into drug effects on the cancer stem cells phenotype (
Rodriguez
et al., 2018;Rodriguez et al., 2021). Here, we report successful implementation
of this technique to evaluate stem-like characteristics of HCC cells and provide a
detailed protocol encompassing critical considerations that should be considered.
24 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
2Materials
2.1Common reagents
1.TRIzol (Sigma-Aldrich)
2.Carrier (e.g., glycogen or GlycoBlue)
3.DNAse I (RNase-Free) (ThermoFisher Scientific)
4.SuperScript III First-Strand Synthesis SuperMix (ThermoFisher Scientific)
5.SYBR Select Master Mix (ThermoFisher Scientific)
2.2Common solutions
1.Sterile phosphate-buffered saline (PBS)
2.Chloroform
3.Isopropanol
4.Absolute ethanol
5.DEPC treated water
2.3Common equipment
1.T 10 basic ULTRA-TURRAX (IKA)
2.Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies)
3.qRT-PCR an Stratagene Mx3005p (Stratagene)
3Methods
3.1RNA isolation from cells or tissues
A high yield of good-quality RNA can be obtained using TRIzol (Sigma-Aldrich),
taking standard precautions to avoid RNase contamination.
1.For tissues, homogenize samples (fragment size<1mm
2
) in an RNase-free
microcentrifuge tube containing 1mL of TRIzol reagentper 50–100mg of tissue.
Homogenization with a high-speed homogenizer (e.g., IKA T 10 basic ULTRA-
TURRAX) is recommended for a proper tissue disruption.For cells grown in
monolayer, use at least 10
6
cells previously washed with phosphate-buffered
saline (PBS). Add 1mL per 5–10μ10
6
cells of pre-warmed TRIzol directly into
the plate. Remove the TRIzol, passing the cell lysate repeatedly through a pipette.
Transfer to a 1.5mL Eppendorf tube.For suspension cell cultures, pellets cells by
centrifuging at 200μgfor 5min. Discard supernatant and resuspend pellet in
100μL PBS. Centrifuge for 5min at 200μg.Add 1mL TRIzol per 5–10μ10
6
cells and enhance cell lysis by repetitive pipetting
2.Leave sample at room temperature for 5min to allow complete cell lysis and
dissociation of nucleoprotein complexes. Centrifuge tissue homogenates at
12,000μgfor 10 min at 4°C to remove debris, and transfer supernatant to a fresh
1.5mL tube
253Methods
2.1Common reagents
1.TRIzol (Sigma-Aldrich)
2.Carrier (e.g., glycogen or GlycoBlue)
3.DNAse I (RNase-Free) (ThermoFisher Scientific)
4.SuperScript III First-Strand Synthesis SuperMix (ThermoFisher Scientific)
5.SYBR Select Master Mix (ThermoFisher Scientific)
2.2Common solutions
1.Sterile phosphate-buffered saline (PBS)
2.Chloroform
3.Isopropanol
4.Absolute ethanol
5.DEPC treated water
2.3Common equipment
1.T 10 basic ULTRA-TURRAX (IKA)
2.Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies)
3.qRT-PCR an Stratagene Mx3005p (Stratagene)
3Methods
3.1RNA isolation from cells or tissues
A high yield of good-quality RNA can be obtained using TRIzol (Sigma-Aldrich),
taking standard precautions to avoid RNase contamination.
1.For tissues, homogenize samples (fragment size<1mm
2
) in an RNase-free
microcentrifuge tube containing 1mL of TRIzol reagentper 50–100mg of tissue.
Homogenization with a high-speed homogenizer (e.g., IKA T 10 basic ULTRA-
TURRAX) is recommended for a proper tissue disruption.For cells grown in
monolayer, use at least 10
6
cells previously washed with phosphate-buffered
saline (PBS). Add 1mL per 5–10μ10
6
cells of pre-warmed TRIzol directly into
the plate. Remove the TRIzol, passing the cell lysate repeatedly through a pipette.
Transfer to a 1.5mL Eppendorf tube.For suspension cell cultures, pellets cells by
centrifuging at 200μgfor 5min. Discard supernatant and resuspend pellet in
100μL PBS. Centrifuge for 5min at 200μg.Add 1mL TRIzol per 5–10μ10
6
cells and enhance cell lysis by repetitive pipetting
2.Leave sample at room temperature for 5min to allow complete cell lysis and
dissociation of nucleoprotein complexes. Centrifuge tissue homogenates at
12,000μgfor 10 min at 4°C to remove debris, and transfer supernatant to a fresh
1.5mL tube
253Methods
3.Add 200μL of chloroform per mL of TRIzol, cap tubes securely and vortex
vigorously for 15s. Incubate at room temperature for 3min. Centrifuge samples
at 12,000μgfor 15min at 4°C to accelerate phase separation. A two-phase
system, divided by a mucous interphase containing DNA will be obtained.
Carefully transfer upper aqueous, clear phase (Δ500μL), which exclusively
contains RNA, to a new 1.5 mL tube. Extreme caution should be taken to avoid
contamination with the middle interphase layer. It is suggested to sacrifice
aqueous material rather than drawing DNA or organic phase.
(Optional) If the expected RNA concentration is10μg/mL, add a carrier
(e.g., glycogen or GlycoBlue) to the upper phase before proceeding to step 5.
Addition of 1μL of a 20mg/mL glycogen stock will facilitate visualization of the
pellet formed in Step 4.
4.Precipitate RNA from aqueous phase by adding 500μL of isopropanol and gently
mixing by inversion. Let stand for 10min at room temperature. Centrifuge at
12,000μgfor 10min at 4°C to pellet the RNA and remove supernatant
completely.A barely visible pellet should form on the base of the tube.
(Optional) If a low yield is expected, centrifuge for 30min.
5.Wash RNA pellet with 1mL of 75% ethanol (in DEPC treated water). Mix gently.
Centrifuge at no more than 7500μgfor 5min at 4°C.
(Optional) Repeat washing procedure to decrease potential salt
contamination.
6.Remove all traces of ethanol and air-dry RNA pellet at room temperature
until only a meniscus of solution covers the pellet. Pellet should not dry too much
since RNA crystallization difficult solubilization.
7.Resuspend RNA pellet in 20–100μL DEPC treated water by passing the solution
a few times through a pipette tip. Incubate for 10min at 55°C to complete
resuspension.
8.Determine RNA concentration with a spectrophotometer (e.g., Nanodrop
ND-1000 spectrophotometer, NanoDrop Technologies).
Observation: A260nM/A230nM<1.7 indicates salt contamination and
A260nM/A280nM<1.7 indicates protein contamination.
9.Assess RNA integrity by gel electrophoresis
3.2Removal of genomic DNA contamination
The removal of potential genomic DNA contamination can be achieved by treating
RNA samples with DNase I, RNase-free.
Observation: Special cautions must be taken to maintain RNA integrity.
1.Add the following into a sterile microcentrifuge tube, in the indicated order:
2μg of total RNA
1μLof10μDNA buffer
1μL of DNAse I (RNase-free, 1U/μL, Thermo Scientific)
Nuclease free water to final volume of 10μL
2.Incubate at 37°C for 30min
26 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
vigorously for 15s. Incubate at room temperature for 3min. Centrifuge samples
at 12,000μgfor 15min at 4°C to accelerate phase separation. A two-phase
system, divided by a mucous interphase containing DNA will be obtained.
Carefully transfer upper aqueous, clear phase (Δ500μL), which exclusively
contains RNA, to a new 1.5 mL tube. Extreme caution should be taken to avoid
contamination with the middle interphase layer. It is suggested to sacrifice
aqueous material rather than drawing DNA or organic phase.
(Optional) If the expected RNA concentration is10μg/mL, add a carrier
(e.g., glycogen or GlycoBlue) to the upper phase before proceeding to step 5.
Addition of 1μL of a 20mg/mL glycogen stock will facilitate visualization of the
pellet formed in Step 4.
4.Precipitate RNA from aqueous phase by adding 500μL of isopropanol and gently
mixing by inversion. Let stand for 10min at room temperature. Centrifuge at
12,000μgfor 10min at 4°C to pellet the RNA and remove supernatant
completely.A barely visible pellet should form on the base of the tube.
(Optional) If a low yield is expected, centrifuge for 30min.
5.Wash RNA pellet with 1mL of 75% ethanol (in DEPC treated water). Mix gently.
Centrifuge at no more than 7500μgfor 5min at 4°C.
(Optional) Repeat washing procedure to decrease potential salt
contamination.
6.Remove all traces of ethanol and air-dry RNA pellet at room temperature
until only a meniscus of solution covers the pellet. Pellet should not dry too much
since RNA crystallization difficult solubilization.
7.Resuspend RNA pellet in 20–100μL DEPC treated water by passing the solution
a few times through a pipette tip. Incubate for 10min at 55°C to complete
resuspension.
8.Determine RNA concentration with a spectrophotometer (e.g., Nanodrop
ND-1000 spectrophotometer, NanoDrop Technologies).
Observation: A260nM/A230nM<1.7 indicates salt contamination and
A260nM/A280nM<1.7 indicates protein contamination.
9.Assess RNA integrity by gel electrophoresis
3.2Removal of genomic DNA contamination
The removal of potential genomic DNA contamination can be achieved by treating
RNA samples with DNase I, RNase-free.
Observation: Special cautions must be taken to maintain RNA integrity.
1.Add the following into a sterile microcentrifuge tube, in the indicated order:
2μg of total RNA
1μLof10μDNA buffer
1μL of DNAse I (RNase-free, 1U/μL, Thermo Scientific)
Nuclease free water to final volume of 10μL
2.Incubate at 37°C for 30min
26 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
3.Add 1μL of EDTA 50mM (final volume¼12μL) to end the DNase I digestion
4.Incubate at 65°C for 10min
Optional: to ensure RNA purity, add 190μL of nuclease free water, 1μL of a carrier
(e.g., glycogen or GlycoBlue), 20μL of 3M Sodium Acetate (pH 5.2) and 600μLof
ice cold 96% Ethanol. Incubate at80°C for at least 30min. Centrifuge at
12,000μg for 30min at 4°C. Wash precipitate with 1 mL of 75% ethanol (in DEPC
treated water). Centrifuge at no more than 7500μg for 5min at 4°C. Then, air dry
the pellet and resuspend in 11μL of Nuclease free water.
3.3cDNA synthesis
Global cDNA obtention from total isolated mRNA can be achieved through Reverse
Transcription (RT). This procedure leads to the synthesis of double-stranded cDNA,
which can be stored at20°C for future downstream applications, including the
study of multiple targets through RT-qPCR. SuperScript III Reverse Transcriptase
(ThermoFisher Scientific), a genetically engineered MMLV RT, was selected due
to its reduced RNase H activity and its increased thermal stability. Incubations
are carried out in a thermal cycler with a heated lid.
1.Add to prior mix 2μL of oligo(dT) (final volume¼13μL)
2.Incubate at 65°C for 5min. Then place immediately on ice to avoid formation of
secondary structures
3.Add the following in the indicated order:
3.5μLof5μReaction Buffer
0.5μL of RiboLock RNase Inhibitor (20U/μL)
2μL of 10 mM dNTP Mix
1μL of RevertAid M-MuLV RT (200U/μL)
4.Incubate at 42°C for 60min
5.End the reaction by heating at 70°C for 5 min
6.Store at20°C
Observation: to perform qRT-PCR reactions with cDNA synthetized using 50 ng of
RNA dilute the sample adding Nuclease free water to a final volume of 200μL.
3.4Designing primers for qRT-PCR
qRT-PCR success and quality rely on a great extent on primers selection. Multiple
primer design software (e.g., NIH primer blast, Integrated DNA Technologies primer
blast) support this task, facilitating the selection of primer pairs that meet the criteria
that ensures their accuracy and reliability (Rodriguez, Rodriguez, Cordoba, &
Andrade, 2015). Once potential primers that match the target sequences are found,
amplicon and primer length, melting temperature (Tm), GC content, specificity of
the primer pair, primer-dimer and hairpin formation and self-complementarity
should be checked. By doing so, it is aimed to avoid or reduce the formation of
primer-dimers and other non-specific products. In this protocol, SYBRGreen
273Methods
4.Incubate at 65°C for 10min
Optional: to ensure RNA purity, add 190μL of nuclease free water, 1μL of a carrier
(e.g., glycogen or GlycoBlue), 20μL of 3M Sodium Acetate (pH 5.2) and 600μLof
ice cold 96% Ethanol. Incubate at80°C for at least 30min. Centrifuge at
12,000μg for 30min at 4°C. Wash precipitate with 1 mL of 75% ethanol (in DEPC
treated water). Centrifuge at no more than 7500μg for 5min at 4°C. Then, air dry
the pellet and resuspend in 11μL of Nuclease free water.
3.3cDNA synthesis
Global cDNA obtention from total isolated mRNA can be achieved through Reverse
Transcription (RT). This procedure leads to the synthesis of double-stranded cDNA,
which can be stored at20°C for future downstream applications, including the
study of multiple targets through RT-qPCR. SuperScript III Reverse Transcriptase
(ThermoFisher Scientific), a genetically engineered MMLV RT, was selected due
to its reduced RNase H activity and its increased thermal stability. Incubations
are carried out in a thermal cycler with a heated lid.
1.Add to prior mix 2μL of oligo(dT) (final volume¼13μL)
2.Incubate at 65°C for 5min. Then place immediately on ice to avoid formation of
secondary structures
3.Add the following in the indicated order:
3.5μLof5μReaction Buffer
0.5μL of RiboLock RNase Inhibitor (20U/μL)
2μL of 10 mM dNTP Mix
1μL of RevertAid M-MuLV RT (200U/μL)
4.Incubate at 42°C for 60min
5.End the reaction by heating at 70°C for 5 min
6.Store at20°C
Observation: to perform qRT-PCR reactions with cDNA synthetized using 50 ng of
RNA dilute the sample adding Nuclease free water to a final volume of 200μL.
3.4Designing primers for qRT-PCR
qRT-PCR success and quality rely on a great extent on primers selection. Multiple
primer design software (e.g., NIH primer blast, Integrated DNA Technologies primer
blast) support this task, facilitating the selection of primer pairs that meet the criteria
that ensures their accuracy and reliability (Rodriguez, Rodriguez, Cordoba, &
Andrade, 2015). Once potential primers that match the target sequences are found,
amplicon and primer length, melting temperature (Tm), GC content, specificity of
the primer pair, primer-dimer and hairpin formation and self-complementarity
should be checked. By doing so, it is aimed to avoid or reduce the formation of
primer-dimers and other non-specific products. In this protocol, SYBRGreen
273Methods
(Invitrogen, Carlsbad, CA, USA) is employed as fluorescent probe for cDNA ampli-
fication. This underscores the importance of proper primer design, given SYBR-
Green unspecificity and ability to intercalate into double-stranded DNA from
both specific and non-specific products (Rodriguez et al., 2015).
1.Primer length should be restricted to 15–30bp to prevent the formation of
secondary structures. If possible, avoid the selection of GC rich target sequences
since this hinders amplification.
2.Amplicon length should be between70 and 200bp(when working with SYBR
Green I) to guarantee optimal signal detection. Shorter products amplificate
more efficiently than longer ones, being that they are more likely to denaturalize
during the extension step and allow primers binding (
Rodriguez et al., 2015).
Besides, the formation of secondary structures within them is less plausible.
3.Tm consist of the temperature at which half of the primers dissociate from their
complement. Primers with Tm ranging from 55°Cto60°C lead to better results.
4.Aim for a 40–60% GC content.
Observation: after a proper design, a precise validation of the primers is necessary.
3.5Primer preparation
To reduce the probabilities of contaminating the primary source for the primer, as
well as the number of freeze/thaw cycles that it goes through, the preparation of a
master 100μM stock (for each primer) and its further dilution to a 10μM working
stock is highly recommended.
1.Spin down the tubes to ensure that lyophilized primers are spun down to the
bottom before opening the vial.
2.Prepare a stock solution (100μM), by adding the appropriate volume of DNase-
free water. This volume can be easily obtained by multiplying the number of
nmol of primer in the tube by 10.
3.Prepare a work solution (10μM), by diluting the stock solution in a sterile
microcentrifuge tube 1:10 with molecular grade water.
4.Store at20°C.
3.6qRT-PCR set up
To determine the CSCs markers CD133, EpCAM, CD44, CD47, and OCT-4 mRNA
levels the SYBR Green qRT-PCR assay followed by the comparative CT method
(ΔΔCT) analysis was selected. Relative quantification of the aforementioned genes
is attained by normalizing their values to 18S ribosomal RNA (RNA 18s) or Glyc-
eraldehyde 3-phosphate dehydrogenase (GADPH) transcripts as housekeeping
genes, and to their levels in untreated cells (used as reference sample). A non-
template controls (NTC) were run in every assay to verify possible contaminations
or formation of primer dimers. In theTable 1the oligonucleotides used in this study
are listed.
28 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
fication. This underscores the importance of proper primer design, given SYBR-
Green unspecificity and ability to intercalate into double-stranded DNA from
both specific and non-specific products (Rodriguez et al., 2015).
1.Primer length should be restricted to 15–30bp to prevent the formation of
secondary structures. If possible, avoid the selection of GC rich target sequences
since this hinders amplification.
2.Amplicon length should be between70 and 200bp(when working with SYBR
Green I) to guarantee optimal signal detection. Shorter products amplificate
more efficiently than longer ones, being that they are more likely to denaturalize
during the extension step and allow primers binding (
Rodriguez et al., 2015).
Besides, the formation of secondary structures within them is less plausible.
3.Tm consist of the temperature at which half of the primers dissociate from their
complement. Primers with Tm ranging from 55°Cto60°C lead to better results.
4.Aim for a 40–60% GC content.
Observation: after a proper design, a precise validation of the primers is necessary.
3.5Primer preparation
To reduce the probabilities of contaminating the primary source for the primer, as
well as the number of freeze/thaw cycles that it goes through, the preparation of a
master 100μM stock (for each primer) and its further dilution to a 10μM working
stock is highly recommended.
1.Spin down the tubes to ensure that lyophilized primers are spun down to the
bottom before opening the vial.
2.Prepare a stock solution (100μM), by adding the appropriate volume of DNase-
free water. This volume can be easily obtained by multiplying the number of
nmol of primer in the tube by 10.
3.Prepare a work solution (10μM), by diluting the stock solution in a sterile
microcentrifuge tube 1:10 with molecular grade water.
4.Store at20°C.
3.6qRT-PCR set up
To determine the CSCs markers CD133, EpCAM, CD44, CD47, and OCT-4 mRNA
levels the SYBR Green qRT-PCR assay followed by the comparative CT method
(ΔΔCT) analysis was selected. Relative quantification of the aforementioned genes
is attained by normalizing their values to 18S ribosomal RNA (RNA 18s) or Glyc-
eraldehyde 3-phosphate dehydrogenase (GADPH) transcripts as housekeeping
genes, and to their levels in untreated cells (used as reference sample). A non-
template controls (NTC) were run in every assay to verify possible contaminations
or formation of primer dimers. In theTable 1the oligonucleotides used in this study
are listed.
28 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
Table 1Oligonucleotides for quantitative real time pcr used in this protocol. Primers Gene name Species Sequence (5
0–3
0
) Start Stop Exon Amplicon length (bp)
mCD133 PROM1 Mouse Fw TCG GCA TAG GGA AAG CCA CG 2609 2628 22 119
Rv GGG CAC AGT CTC AAC ATC GTC 2727 2707 24
hCD133 PROM1 Human Fw AAA CAG TTT GCC CCC AGG AA 2232 2251 18/19 155
Rv ACA ATC CAT TCC CTG TGC GT 2386 2367 20
mCD44 CD44 Mouse Fw CAC CTT GGC CAC CAC TCC TA 979 998 5/6 112
Rv GGA GTC TTC ACT TGG GGT AGG G 1090 1069 6
hCD44 CD44 Human Fw GCG CAG ATC GAT TTG AAT ATA A 191 212 1/2 198
Rv GTG CCC TTC TAT GAA CCC A 388 370 3
mOCT-4 Pou5f1 Mouse Fw CCT TTC CCT CTG TTC CCG TC 713 732 5 103
Rv GTC TAC CTC CCT TGC CTT GG 815 796 5
mCD47 CD47 Mouse Fw ATG CTT CTG GAC TTG GCC TC 737 756 5 148
Rv CCG ACC AAA GCA AGG ACG TA 884 865 6
EpCAM EpCAM Mouse Fw CGG AGT CCG AAG AAC CGA CA 821 840 4 118
Rv GCT CTG ATG GTC GTA GGG GC 938 919 5
18S RNA18S1 Mouse Fw ACC GCA GCT AGG AAT AAT GGA 850 870 – 63
Rv GCC TCA GTT CCG AAA ACC A 912 894 –
RNA18SN1 Human Fw ACC GCA GCT AGG AAT AAT GGA 849 869 – 63
Rv GCC TCA GTT CCG AAA ACC A 911 893 –
GAPDH GAPDH Mouse Fw GGG GCT GCC CAG AAC ATC AT 687 706 5 194
Rv GCC TGC TTC ACC ACC TTC TTG 880 860 6
GAPDH Human Fw GGG GCT GCC CAG AAC ATC AT 677 696 8 194
Rv GCC TGC TTC ACC ACC TTC TTG 870 850 8
0–3
0
) Start Stop Exon Amplicon length (bp)
mCD133 PROM1 Mouse Fw TCG GCA TAG GGA AAG CCA CG 2609 2628 22 119
Rv GGG CAC AGT CTC AAC ATC GTC 2727 2707 24
hCD133 PROM1 Human Fw AAA CAG TTT GCC CCC AGG AA 2232 2251 18/19 155
Rv ACA ATC CAT TCC CTG TGC GT 2386 2367 20
mCD44 CD44 Mouse Fw CAC CTT GGC CAC CAC TCC TA 979 998 5/6 112
Rv GGA GTC TTC ACT TGG GGT AGG G 1090 1069 6
hCD44 CD44 Human Fw GCG CAG ATC GAT TTG AAT ATA A 191 212 1/2 198
Rv GTG CCC TTC TAT GAA CCC A 388 370 3
mOCT-4 Pou5f1 Mouse Fw CCT TTC CCT CTG TTC CCG TC 713 732 5 103
Rv GTC TAC CTC CCT TGC CTT GG 815 796 5
mCD47 CD47 Mouse Fw ATG CTT CTG GAC TTG GCC TC 737 756 5 148
Rv CCG ACC AAA GCA AGG ACG TA 884 865 6
EpCAM EpCAM Mouse Fw CGG AGT CCG AAG AAC CGA CA 821 840 4 118
Rv GCT CTG ATG GTC GTA GGG GC 938 919 5
18S RNA18S1 Mouse Fw ACC GCA GCT AGG AAT AAT GGA 850 870 – 63
Rv GCC TCA GTT CCG AAA ACC A 912 894 –
RNA18SN1 Human Fw ACC GCA GCT AGG AAT AAT GGA 849 869 – 63
Rv GCC TCA GTT CCG AAA ACC A 911 893 –
GAPDH GAPDH Mouse Fw GGG GCT GCC CAG AAC ATC AT 687 706 5 194
Rv GCC TGC TTC ACC ACC TTC TTG 880 860 6
GAPDH Human Fw GGG GCT GCC CAG AAC ATC AT 677 696 8 194
Rv GCC TGC TTC ACC ACC TTC TTG 870 850 8
All qPCR reactions were performed as triplicates for each sample.
1.To prepare PCR reaction mixture add:
10μL of SYBR Select Master Mix
0.45μL of forward primer
0.45μL of reverse primer
4.1μL of Nuclease free water
Observation: since multiple qRT-PCR experiment are needed it is recommended to
assemble a cocktail containing the reagents common to all reactions into a 1.8mL
microcentrifuge tube. For instance, to prepare 7 reactions (2 samples by triplicate
+ 1 for the pipetting error), then add 70μL(7μ10μL) of SYBR Select Master Mix
+ 3.15μL of each primer (7μ0.45μL) and 28.7μL(7μ4.1μL) of Nuclease free
water. The reagents should be mixed thoroughly by gently pumping the plunger of
a micropipetor up and down about 20 times as described above.
2.Seed the PCR reaction addin the bottom ofa 96-well platewell.
(1)Add 5μL of diluted cDNA
(2)15μL of reaction mixture for each reaction
3.7qRT-PCR thermal settings
1.1cycle of:
50°C for 2min (UDG Activation)
95°C for 2min (AmpliTaq DNA Polymerase, UP Activation)
2.40cycles of:
95°C for 15s (denaturation)
58°C for 30s (annealing)
72°C for 30s (extension)
3.Generation of a dissociation curve by a stepwise increase in temperature from
58°Cto95°C at a rate of 2°C/min, and fluorescence measurement every 15s.
Observation: analysis allows to confirm the amplification of specific RT-qPCR prod-
ucts. Any doubtful dissociation curve should be excluded.
4.Final cooling to 12°C.
3.8qRT-PCR analysis
To determine the relative expression (RE) of the selected genes avoiding the
preparation of standard curves, the comparative CT method (ΔΔCT) was selected.
To determine the fold change (FC) the following equation should be used:
1.ΔCt¼Ct (gene of interest)Ct (housekeeping gene)
2.ΔΔCt¼ΔCt (treated sample)ΔCt (reference sample)
3.Fold Change¼2
–ΔΔCt
.
30 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
1.To prepare PCR reaction mixture add:
10μL of SYBR Select Master Mix
0.45μL of forward primer
0.45μL of reverse primer
4.1μL of Nuclease free water
Observation: since multiple qRT-PCR experiment are needed it is recommended to
assemble a cocktail containing the reagents common to all reactions into a 1.8mL
microcentrifuge tube. For instance, to prepare 7 reactions (2 samples by triplicate
+ 1 for the pipetting error), then add 70μL(7μ10μL) of SYBR Select Master Mix
+ 3.15μL of each primer (7μ0.45μL) and 28.7μL(7μ4.1μL) of Nuclease free
water. The reagents should be mixed thoroughly by gently pumping the plunger of
a micropipetor up and down about 20 times as described above.
2.Seed the PCR reaction addin the bottom ofa 96-well platewell.
(1)Add 5μL of diluted cDNA
(2)15μL of reaction mixture for each reaction
3.7qRT-PCR thermal settings
1.1cycle of:
50°C for 2min (UDG Activation)
95°C for 2min (AmpliTaq DNA Polymerase, UP Activation)
2.40cycles of:
95°C for 15s (denaturation)
58°C for 30s (annealing)
72°C for 30s (extension)
3.Generation of a dissociation curve by a stepwise increase in temperature from
58°Cto95°C at a rate of 2°C/min, and fluorescence measurement every 15s.
Observation: analysis allows to confirm the amplification of specific RT-qPCR prod-
ucts. Any doubtful dissociation curve should be excluded.
4.Final cooling to 12°C.
3.8qRT-PCR analysis
To determine the relative expression (RE) of the selected genes avoiding the
preparation of standard curves, the comparative CT method (ΔΔCT) was selected.
To determine the fold change (FC) the following equation should be used:
1.ΔCt¼Ct (gene of interest)Ct (housekeeping gene)
2.ΔΔCt¼ΔCt (treated sample)ΔCt (reference sample)
3.Fold Change¼2
–ΔΔCt
.
30 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
Observation 1: the threshold cycle (Ct) value represents the cycle number at which
sample fluorescence rises to a statistically significant level above the background.
Observation 2: According with the MIQE guidelines, to consider valid a differ-
ence in the gene expression similar results should be observed using at least two
housekeepings (Bustin et al.,2009).
4Results and discussion
To validate the protocol, we analyzed the mRNA expression of CSCs markers
between cancer stem cells and non-cancer stem cells isolated from the murine
HCC cell line Hepa129 by magnetic sorting using an anti-CD133 antibody. As
expected, CD133
+
Hepa129 cells express higher level of all the CSCs markers stud-
ied highlighting the robustness of our protocol (Fig. 1) in comparison with the non-
cancer stem cell population. Therefore, it can be concluded that the quantification of
CD133, CD44, EpCAM, CD47, and OCT-4 expression through qRT-PCR is a useful
approach to evaluate cancer stem cells features in HCC.
Disclosures
B.B., E.F., M.G., J.B. and G.M. have no conflicts of interest to disclose.
FIG. 1
CSCs Markers expression on HCC cells. CD133
+
and CD133
Hepa 129 cells were isolated
using an anti-CD133 antibody and magnetic sorting. Then, mRNA levels of CD133, EpCAM,
CD44, CD47 and OCT4 were determined by qRT-PCR as detailed in the present protocol.
*P<0.05 vs CD133—by Mann-Whitney.
31Disclosures
sample fluorescence rises to a statistically significant level above the background.
Observation 2: According with the MIQE guidelines, to consider valid a differ-
ence in the gene expression similar results should be observed using at least two
housekeepings (Bustin et al.,2009).
4Results and discussion
To validate the protocol, we analyzed the mRNA expression of CSCs markers
between cancer stem cells and non-cancer stem cells isolated from the murine
HCC cell line Hepa129 by magnetic sorting using an anti-CD133 antibody. As
expected, CD133
+
Hepa129 cells express higher level of all the CSCs markers stud-
ied highlighting the robustness of our protocol (Fig. 1) in comparison with the non-
cancer stem cell population. Therefore, it can be concluded that the quantification of
CD133, CD44, EpCAM, CD47, and OCT-4 expression through qRT-PCR is a useful
approach to evaluate cancer stem cells features in HCC.
Disclosures
B.B., E.F., M.G., J.B. and G.M. have no conflicts of interest to disclose.
FIG. 1
CSCs Markers expression on HCC cells. CD133
+
and CD133
Hepa 129 cells were isolated
using an anti-CD133 antibody and magnetic sorting. Then, mRNA levels of CD133, EpCAM,
CD44, CD47 and OCT4 were determined by qRT-PCR as detailed in the present protocol.
*P<0.05 vs CD133—by Mann-Whitney.
31Disclosures
References
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A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies
potential therapeutic targets.Journal of Hepatology,71,78–90.
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The MIQE guidelines: Minimum information for publication of quantitative real-time
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32 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
Bayo, J., Fiore, E., Aquino, J. B., Malvicini, M., Rizzo, M., Peixoto, E., et al. (2014). Increased
migration of human mesenchymal stromal cells by autocrine motility factor (AMF)
resulted in enhanced recruitment towards hepatocellular carcinoma.PLoS One,9(4),
e95171.https://doi.org/10.1371/journal.pone.0095171.
Bayo, J., Fiore, E. J., Dominguez, L. M., Real, A., Malvicini, M., Rizzo, M., et al. (2019).
A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies
potential therapeutic targets.Journal of Hepatology,71,78–90.
Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., et al. (2009).
The MIQE guidelines: Minimum information for publication of quantitative real-time
PCR experiments.Clinical Chemistry,55, 611–622.
Chen, Y., Yu, D., Zhang, H., He, H., Zhang, C., Zhao, W., et al. (2012). CD133(+)EpCAM(+)
phenotype possesses more characteristics of tumor initiating cells in hepatocellular carci-
noma Huh7 cells.International Journal of Biological Sciences,8, 992–1004.
Finn, R. S., Qin, S., Ikeda, M., Galle, P. R., Ducreux, M., Kim, T. Y., et al. (2020). Atezoli-
zumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma.The New England
Journal of Medicine,382, 1894–1905.
Haraguchi, N., Ishii, H., Mimori, K., Tanaka, F., Ohkuma, M., Kim, H. M., et al. (2010). CD13
is a therapeutic target in human liver cancer stem cells.The Journal of Clinical Investiga-
tion,120, 3326–3339.
Inarrairaegui, M., Melero, I., & Sangro, B. (2018). Immunotherapy of hepatocellular
carcinoma: Facts and hopes.Clinical Cancer Research,24, 1518–1524.
Liu, Y. C., Yeh, C. T., & Lin, K. H. (2020). Cancer stem cell functions in hepatocellular
carcinoma and comprehensive therapeutic strategies.Cell,9.
Ochoa, M. C., Fioravanti, J., Rodriguez, I., Hervas-Stubbs, S., Azpilikueta, A., Mazzolini, G.,
et al. (2013). Antitumor immunotherapeutic and toxic properties of an HDL-conjugated
chimeric IL-15 fusion protein.Cancer Research,73, 139–149.
Rodriguez, M. M., Fiore, E., Bayo, J., Atorrasagasti, C., Garcia, M., Onorato, A., et al. (2018).
4Mu decreases CD47 expression on hepatic cancer stem cells and primes a potent antitu-
mor T cell response induced by Interleukin-12.Molecular Therapy,26, 2738–2750.
Rodriguez, M. M., Onorato, A., Cantero, M. J., Dominguez, L., Bayo, J., Fiore, E., et al.
(2021). 4-methylumbelliferone-mediated polarization of M1 macrophages correlate with
decreased hepatocellular carcinoma aggressiveness in mice.Scientific Reports,11, 6310.
Rodriguez, A., Rodriguez, M., Cordoba, J. J., & Andrade, M. J. (2015). Design of primers and
probes for quantitative real-time PCR methods.Methods in Molecular Biology,1275,
31–56.
Xiang, Y., Yang, T., Pang, B. Y., Zhu, Y., & Liu, Y. N. (2016). The Progress and prospects of
putative biomarkers for liver cancer stem cells in hepatocellular carcinoma.Stem Cells
International,2016, 7614971.
Xu, X., Liu, R. F., Zhang, X., Huang, L. Y., Chen, F., Fei, Q. L., et al. (2012). DLK1 as a
potential target against cancer stem/progenitor cells of hepatocellular carcinoma.Molec-
ular Cancer Therapeutics,11, 629–638.
Zhu, Z., Hao, X., Yan, M., Yao, M., Ge, C., Gu, J., et al. (2010). Cancer stem/progenitor cells
are highly enriched in CD133+CD44+ population in hepatocellular carcinoma.Interna-
tional Journal of Cancer,126, 2067–2078.
32 CHAPTER 2 Evaluation of cancer stem cells markers expression in HCC
CHAPTER
Reverse Phase Protein
Arraysincancerstemcells
3
Michele Signore
a,
*and Valeria Manganelli
b
a
RPPA Unit, Proteomics Area, Core Facilities, Istituto Superiore di Sanita`, Rome, Italy
b
Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
*Corresponding author: e-mail address: [email protected]
Chapter outline
1 Introduction........................................................................................................34
2 Materials............................................................................................................36
3 Methods.............................................................................................................40
3.1 Plating and treating cells......................................................................40
3.2 Sample preparation for printing.............................................................41
3.3 Preliminary control printing...................................................................43
3.4 Sample printing...................................................................................43
3.5 Quantification of total protein content....................................................45
3.6 Immunostaining...................................................................................46
3.7 Scanning and image analysis.................................................................47
4 Notes.................................................................................................................48
5 Concluding remarks............................................................................................55
Disclosures.............................................................................................................56
References..............................................................................................................57
Abstract
The scenario of proteogenomics is rapidly evolving and novel technologies are enabling com-
prehensive molecular exploration down to single cells. Likewise, digital (immuno-)assays are
revolutionizing the field of biomarker detection and have reached the grade for population-
level screenings with single-molecule sensitivity. Nonetheless, cost- and time-effective,
high-throughput targeted phospho-proteomics at a preclinical stage still relies on ad hoc mi-
croarray platforms, such as the Reverse-Phase Protein microArrays (RPPA). Although this
technique requires specific knowledge and equipment and different laboratories worldwide
have implemented alternative methodological strategies, the application of RPPA to
Methods in Cell Biology, Volume 171, ISSN 0091-679X,https://doi.org/10.1016/bs.mcb.2022.04.004
Copyright ©2022 Elsevier Inc. All rights reserved.
33
Reverse Phase Protein
Arraysincancerstemcells
3
Michele Signore
a,
*and Valeria Manganelli
b
a
RPPA Unit, Proteomics Area, Core Facilities, Istituto Superiore di Sanita`, Rome, Italy
b
Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
*Corresponding author: e-mail address: [email protected]
Chapter outline
1 Introduction........................................................................................................34
2 Materials............................................................................................................36
3 Methods.............................................................................................................40
3.1 Plating and treating cells......................................................................40
3.2 Sample preparation for printing.............................................................41
3.3 Preliminary control printing...................................................................43
3.4 Sample printing...................................................................................43
3.5 Quantification of total protein content....................................................45
3.6 Immunostaining...................................................................................46
3.7 Scanning and image analysis.................................................................47
4 Notes.................................................................................................................48
5 Concluding remarks............................................................................................55
Disclosures.............................................................................................................56
References..............................................................................................................57
Abstract
The scenario of proteogenomics is rapidly evolving and novel technologies are enabling com-
prehensive molecular exploration down to single cells. Likewise, digital (immuno-)assays are
revolutionizing the field of biomarker detection and have reached the grade for population-
level screenings with single-molecule sensitivity. Nonetheless, cost- and time-effective,
high-throughput targeted phospho-proteomics at a preclinical stage still relies on ad hoc mi-
croarray platforms, such as the Reverse-Phase Protein microArrays (RPPA). Although this
technique requires specific knowledge and equipment and different laboratories worldwide
have implemented alternative methodological strategies, the application of RPPA to
Methods in Cell Biology, Volume 171, ISSN 0091-679X,https://doi.org/10.1016/bs.mcb.2022.04.004
Copyright ©2022 Elsevier Inc. All rights reserved.
33
biomarker discovery has proven successful on diverse types of samples, including tissues and
biological fluids as well as nanovesicles and in vitro cultured lines. Among these, cancer stem
(-like) cells (CSC) represent an ideal experimental model system for preclinical discovery and
definition of novel drug targets.
The present methodological article provides the basic knowledge and steps on how to de-
ploy an RPPA analysis with specific reference to an ideal experimental setup of drug testing on
CSC.
1Introduction
Technological developments in the latest 15 years have revolutionized mole-
cular phenotyping by enabling simultaneous measurement of RNA or DNA and
(phospho-)proteins in individual cells (Gerlach et al., 2019;Stuart & Satija,
2019). Multi-omics measurements are now accomplished through multiplexed mas-
sive parallel sequencing on single cells isolated from cellular suspensions (Lee,
Hyeon, & Hwang, 2020;Stuart & Satija, 2019) as well as directly on ad hoc-stained
tissue sections (Lewis et al., 2021). Indeed, the still relatively low throughput of such
approaches prevents their application for screening as well as diagnostic and prog-
nostic purposes (Stuart & Satija, 2019). This holds true particularly for the limited
amount of (phospho-)proteomic targets that can be measured in concomitance with
mRNA expression in single-cell measurements but also applies to most advanced
label-free measurements, whereby both the proteome coverage and the minimum
cell number required are far from reaching the standard levels of applicability of
sequencing-based technologies (Kelly, 2020). Next-generation, single-molecule pro-
tein sequencing methods is a brand new area of research that is actively working to
fill the technological gaps in proteomics (Alfaro et al., 2021;Perkel, 2021). Further-
more, the clinical applicability of digital assays with single-molecule sensitivity for
both nucleic acids (Rowlands et al., 2019) and proteins (Abasıyanık et al., 2020;
Norman et al., 2020) is boosting the field of biomarker discovery. Therefore, in a
hopefully near future case scenario, multi-omic reconstruction of biological re-
sponses will be possible at the single-cell resolution with a deep coverage of all
analyzed macro-molecules and research-grade discoveries will be easily converted
to diagnostic-grade tests.
In the mean-while, the excitement for such an envisioned high-tech future of pre-
cision medicine is subdued by the actual need for a robust, high-throughput platforms
for pre-clinical drug and/or biomarker screening. Indeed, fostered by the availability
of comprehensive databases and powerful analytical algorithms, researchers have fo-
cused their attention on previously unexplored areas of the genome in search of ther-
apeutic opportunities (
Gerdes et al., 2021;Munoz, 2017;Oprea et al., 2018). While
mass spectrometry (MS) is frequently used for identification of drug targets and/or
actions in experimental models (Muroi & Osada, 2021;Ruprecht et al., 2020) the
application of extensive, ad hoc data analysis methods and models, prevents its
34 CHAPTER 3 Reverse Phase Protein Arrays in cancer stem cells
biological fluids as well as nanovesicles and in vitro cultured lines. Among these, cancer stem
(-like) cells (CSC) represent an ideal experimental model system for preclinical discovery and
definition of novel drug targets.
The present methodological article provides the basic knowledge and steps on how to de-
ploy an RPPA analysis with specific reference to an ideal experimental setup of drug testing on
CSC.
1Introduction
Technological developments in the latest 15 years have revolutionized mole-
cular phenotyping by enabling simultaneous measurement of RNA or DNA and
(phospho-)proteins in individual cells (Gerlach et al., 2019;Stuart & Satija,
2019). Multi-omics measurements are now accomplished through multiplexed mas-
sive parallel sequencing on single cells isolated from cellular suspensions (Lee,
Hyeon, & Hwang, 2020;Stuart & Satija, 2019) as well as directly on ad hoc-stained
tissue sections (Lewis et al., 2021). Indeed, the still relatively low throughput of such
approaches prevents their application for screening as well as diagnostic and prog-
nostic purposes (Stuart & Satija, 2019). This holds true particularly for the limited
amount of (phospho-)proteomic targets that can be measured in concomitance with
mRNA expression in single-cell measurements but also applies to most advanced
label-free measurements, whereby both the proteome coverage and the minimum
cell number required are far from reaching the standard levels of applicability of
sequencing-based technologies (Kelly, 2020). Next-generation, single-molecule pro-
tein sequencing methods is a brand new area of research that is actively working to
fill the technological gaps in proteomics (Alfaro et al., 2021;Perkel, 2021). Further-
more, the clinical applicability of digital assays with single-molecule sensitivity for
both nucleic acids (Rowlands et al., 2019) and proteins (Abasıyanık et al., 2020;
Norman et al., 2020) is boosting the field of biomarker discovery. Therefore, in a
hopefully near future case scenario, multi-omic reconstruction of biological re-
sponses will be possible at the single-cell resolution with a deep coverage of all
analyzed macro-molecules and research-grade discoveries will be easily converted
to diagnostic-grade tests.
In the mean-while, the excitement for such an envisioned high-tech future of pre-
cision medicine is subdued by the actual need for a robust, high-throughput platforms
for pre-clinical drug and/or biomarker screening. Indeed, fostered by the availability
of comprehensive databases and powerful analytical algorithms, researchers have fo-
cused their attention on previously unexplored areas of the genome in search of ther-
apeutic opportunities (
Gerdes et al., 2021;Munoz, 2017;Oprea et al., 2018). While
mass spectrometry (MS) is frequently used for identification of drug targets and/or
actions in experimental models (Muroi & Osada, 2021;Ruprecht et al., 2020) the
application of extensive, ad hoc data analysis methods and models, prevents its
34 CHAPTER 3 Reverse Phase Protein Arrays in cancer stem cells
broad-range use. High-content screening (HCS) is a powerful, broadly used approach
for image-based phenotyping (Chandrasekaran, Ceulemans, Boyd, & Carpenter,
2021), but its application to three-dimensional cell models such as organoids
(Bock et al., 2021;Jiang et al., 2020;Yan et al., 2018) has proven challenging
(Carragher et al., 2018).
However, antibody as well as functional microarrays represent cost-effective,
high-throughput and high-sensitivity research tools, require low input sample vol-
ume and have broad applicability (Chen, Dodig-Crnkovic, Schwenk, & Tao,
2018;Syu, Dunn, & Zhu, 2020). Among array-based immunoassay platforms, the
Reverse-Phase Protein microArrays (RPPA) have been specifically designed for tar-
geted investigation of signal transduction cascades (
Signore, Manganelli, & Vitale,
2017). The main advantages of RPPA over other sister techniques like antibody (i.e.,
forward-phase) arrays, Western blotting, ELISA or tissue microarrays, are combined
sample and analyte throughput as well as sensitivity. The Cancer Genome Atlas
(TCGA) applied RPPA as the main application of (phospho-)proteomic technology
(
Akbani et al., 2014;Chen, Chandrashekar, Varambally, & Creighton, 2019) and
enabled its use within other broad-scale cancer cell line molecular and functional
characterization initiatives (
Li et al., 2017). The setup of RPPA requires non-
negligible funding and dedicated laboratory training, but its implementation and
deployment as a service within institutional core facilities has proven successful
(
Signore, Manganelli, & Vitale, 2017). Moreover, while the basic immunoassay
principles behind RPPA have been readjusted through custom, alternative proce-
dures by diverse laboratories worldwide, overall the technique has been demon-
strated extremely robust (Byron et al., 2020). Although RPPA is not suited for
single-cell measurements, its sensitivity still allows detection down to as low as
200 cell equivalents (
Grote et al., 2008) and, depending on the antibody used, below
the picogram level (Signore et al., 2021). Therefore RPPA is an ideal technique for
screening of low-abundance biomarker in biological fluids and samples (Boellner &
Becker, 2015;Grote et al., 2008;Sewell et al., 2014;Spreafico et al., 2017) as well as
for biased definition of pharmacodynamic biomarkers and drugs’ mechanisms of
action (MoA) (Manic et al., 2018;Matteoni et al., 2019;Zhao et al., 2021, 2020).
Among diverse biological platforms used in the pre-clinical, experimental phase
of biomarker discovery in cancer, tumor organoids represent the most acknowledged
cellular model to faithfully recapitulate in vitro and in vivo an individual patients’
molecular responses, prospectively enabling precision oncology (
Bleijs, van de
Wetering, Clevers, & Drost, 2019;Bock et al., 2021;Lo, Karlsson, & Kuo, 2020;
Weeber, Ooft, Dijkstra, & Voest, 2017). Of note, tumor organoids have since posed
challenges in terms of reproducibility, standardization of operating procedures and
ease of handling, all of which represent open issues that are being tackled by re-
searchers worldwide (LeSavage, Suhar, Broguiere, Lutolf, & Heilshorn, 2021). In-
deed, patient-derived cancer stem-like cells (CSC) present with similar issues related
to standardization of procedures and the biological rationale as well as the role of
CSC have been questioned and revisited over time (
Batlle & Clevers, 2017;
Garber, 2018;Kreso & Dick, 2014). Nonetheless, the low intrinsic complexity
351Introduction
for image-based phenotyping (Chandrasekaran, Ceulemans, Boyd, & Carpenter,
2021), but its application to three-dimensional cell models such as organoids
(Bock et al., 2021;Jiang et al., 2020;Yan et al., 2018) has proven challenging
(Carragher et al., 2018).
However, antibody as well as functional microarrays represent cost-effective,
high-throughput and high-sensitivity research tools, require low input sample vol-
ume and have broad applicability (Chen, Dodig-Crnkovic, Schwenk, & Tao,
2018;Syu, Dunn, & Zhu, 2020). Among array-based immunoassay platforms, the
Reverse-Phase Protein microArrays (RPPA) have been specifically designed for tar-
geted investigation of signal transduction cascades (
Signore, Manganelli, & Vitale,
2017). The main advantages of RPPA over other sister techniques like antibody (i.e.,
forward-phase) arrays, Western blotting, ELISA or tissue microarrays, are combined
sample and analyte throughput as well as sensitivity. The Cancer Genome Atlas
(TCGA) applied RPPA as the main application of (phospho-)proteomic technology
(
Akbani et al., 2014;Chen, Chandrashekar, Varambally, & Creighton, 2019) and
enabled its use within other broad-scale cancer cell line molecular and functional
characterization initiatives (
Li et al., 2017). The setup of RPPA requires non-
negligible funding and dedicated laboratory training, but its implementation and
deployment as a service within institutional core facilities has proven successful
(
Signore, Manganelli, & Vitale, 2017). Moreover, while the basic immunoassay
principles behind RPPA have been readjusted through custom, alternative proce-
dures by diverse laboratories worldwide, overall the technique has been demon-
strated extremely robust (Byron et al., 2020). Although RPPA is not suited for
single-cell measurements, its sensitivity still allows detection down to as low as
200 cell equivalents (
Grote et al., 2008) and, depending on the antibody used, below
the picogram level (Signore et al., 2021). Therefore RPPA is an ideal technique for
screening of low-abundance biomarker in biological fluids and samples (Boellner &
Becker, 2015;Grote et al., 2008;Sewell et al., 2014;Spreafico et al., 2017) as well as
for biased definition of pharmacodynamic biomarkers and drugs’ mechanisms of
action (MoA) (Manic et al., 2018;Matteoni et al., 2019;Zhao et al., 2021, 2020).
Among diverse biological platforms used in the pre-clinical, experimental phase
of biomarker discovery in cancer, tumor organoids represent the most acknowledged
cellular model to faithfully recapitulate in vitro and in vivo an individual patients’
molecular responses, prospectively enabling precision oncology (
Bleijs, van de
Wetering, Clevers, & Drost, 2019;Bock et al., 2021;Lo, Karlsson, & Kuo, 2020;
Weeber, Ooft, Dijkstra, & Voest, 2017). Of note, tumor organoids have since posed
challenges in terms of reproducibility, standardization of operating procedures and
ease of handling, all of which represent open issues that are being tackled by re-
searchers worldwide (LeSavage, Suhar, Broguiere, Lutolf, & Heilshorn, 2021). In-
deed, patient-derived cancer stem-like cells (CSC) present with similar issues related
to standardization of procedures and the biological rationale as well as the role of
CSC have been questioned and revisited over time (
Batlle & Clevers, 2017;
Garber, 2018;Kreso & Dick, 2014). Nonetheless, the low intrinsic complexity
351Introduction
coupled to a still three-dimensional architecture and improved manipulation as com-
pared to tumor organoids, have granted CSC respect as an accessible and faithful cell
model system in translational oncology (
Buccarelli et al., 2021;Carstens et al., 2015;
Clarke, 2019;Dirkse et al., 2019;Manic et al., 2021;Prager, Xie, Bao, &
Rich, 2019).
The curious reader may find elsewhere further information on the main signaling
pathways driving CSC (Yang et al., 2020) and on the use of CSC-based models for
biomarker discovery in cancer research (Manic et al., 2018). Likewise, other
published protocols are available for RPPA setup (Spurrier, Ramalingam, &
Nishizuka, 2008). Herein, we will describe the experimental steps and materials
required to perform RPPA analysis as per our established protocols. In particular,
we will refer to an example sample set encompassing drug concentration and time
series of CSC derived from colorectal cancer (CRC) and commercially available
CRC lines, confronted with diverse chemotherapeutics.
2Materials
Common disposables
General purpose
• 15 and 50mL centrifuge tubes, polypropylene, conical bottom (e.g.,
#339652, Thermo Fisher Scientific, Waltham, MA, USA)
• 2.0 and 1.5mL microcentrifuge tubes (e.g., Eppendorf Safe-Lock Tubes,
Eppendorf AG, Hamburg, Germany)
• Sterile, plastic serological pipets (2, 5, 10 and 25mL volume sizes, e.g.,
Stripette™Serological Pipets, Polystyrene, Individually Paper/Plastic
Wrapped, Sterile, Corning Inc., NY, USA)
• Graduated beakers (1000 mL), flasks (250mL) and cylinders (50, 100, 1000
and 2000 mL)
• Micropipette tips (P10, P20, P200, and P1000 microliters range), standard,
clear, beveled, graduated, non-sterile (e.g., #LW1136, #LW8503,
#LW6481, Alpha Laboratories, Hampshire, UK)
• 500mL Vacuum Filter/Storage Bottle System, 0.22μm Pore 33.2cm
2
PES
Membrane, Sterile (e.g., #431097, Corning Inc., NY, USA)
• Sterile syringe filters, PES, 0.22μm pour size, 25mm diameter (e.g.,
#CH2225-PES, Thermo Fisher Scientific, Waltham, MA, USA)
• Sterile syringe filters, PES, 0.45μm pour size, 25mm diameter (e.g.,
#CH4525-PES, Thermo Fisher Scientific, Waltham, MA, USA)
• 50mL disposable graduated eccentric luer-slip syringe without needle (e.g.,
#300866 BD Plastipak, Becton, Dickinson and Company, Franklin Lakes,
NJ, USA) (seeNote 3)
• Syringe needles gauge 23, L 1¼
00
(e.g., #Z192430 BD Precisionglide
®
syringe needles, Sigma-Aldrich
®
, Merck KGaA, Darmstadt, Germany)
36 CHAPTER 3 Reverse Phase Protein Arrays in cancer stem cells
pared to tumor organoids, have granted CSC respect as an accessible and faithful cell
model system in translational oncology (
Buccarelli et al., 2021;Carstens et al., 2015;
Clarke, 2019;Dirkse et al., 2019;Manic et al., 2021;Prager, Xie, Bao, &
Rich, 2019).
The curious reader may find elsewhere further information on the main signaling
pathways driving CSC (Yang et al., 2020) and on the use of CSC-based models for
biomarker discovery in cancer research (Manic et al., 2018). Likewise, other
published protocols are available for RPPA setup (Spurrier, Ramalingam, &
Nishizuka, 2008). Herein, we will describe the experimental steps and materials
required to perform RPPA analysis as per our established protocols. In particular,
we will refer to an example sample set encompassing drug concentration and time
series of CSC derived from colorectal cancer (CRC) and commercially available
CRC lines, confronted with diverse chemotherapeutics.
2Materials
Common disposables
General purpose
• 15 and 50mL centrifuge tubes, polypropylene, conical bottom (e.g.,
#339652, Thermo Fisher Scientific, Waltham, MA, USA)
• 2.0 and 1.5mL microcentrifuge tubes (e.g., Eppendorf Safe-Lock Tubes,
Eppendorf AG, Hamburg, Germany)
• Sterile, plastic serological pipets (2, 5, 10 and 25mL volume sizes, e.g.,
Stripette™Serological Pipets, Polystyrene, Individually Paper/Plastic
Wrapped, Sterile, Corning Inc., NY, USA)
• Graduated beakers (1000 mL), flasks (250mL) and cylinders (50, 100, 1000
and 2000 mL)
• Micropipette tips (P10, P20, P200, and P1000 microliters range), standard,
clear, beveled, graduated, non-sterile (e.g., #LW1136, #LW8503,
#LW6481, Alpha Laboratories, Hampshire, UK)
• 500mL Vacuum Filter/Storage Bottle System, 0.22μm Pore 33.2cm
2
PES
Membrane, Sterile (e.g., #431097, Corning Inc., NY, USA)
• Sterile syringe filters, PES, 0.22μm pour size, 25mm diameter (e.g.,
#CH2225-PES, Thermo Fisher Scientific, Waltham, MA, USA)
• Sterile syringe filters, PES, 0.45μm pour size, 25mm diameter (e.g.,
#CH4525-PES, Thermo Fisher Scientific, Waltham, MA, USA)
• 50mL disposable graduated eccentric luer-slip syringe without needle (e.g.,
#300866 BD Plastipak, Becton, Dickinson and Company, Franklin Lakes,
NJ, USA) (seeNote 3)
• Syringe needles gauge 23, L 1¼
00
(e.g., #Z192430 BD Precisionglide
®
syringe needles, Sigma-Aldrich
®
, Merck KGaA, Darmstadt, Germany)
36 CHAPTER 3 Reverse Phase Protein Arrays in cancer stem cells
Cell culture and protein lysates
• Multiwell plates for suspension culture, 12- and 24-well, polystyrene, clear,
lid with condensation rings, sterile, individually packed (#665970 and
#662102, Greiner Bio-One International GmbH, Kremsm€unster, Austria)
• Cell scrapers (e.g., Falcon
®
Cell Scraper with 25cm Handle and 1.8cm
Blade, Sterile, Individually Packaged, #353086, Corning Inc., NY, USA)
(seeNote 23)
• Visible Range Cuvette, Semi-Micro, 1.5mL, 10mm path length
(e.g., #1938, Kartell S.p.A., Milan, Italy)
Slide printing
• 384-Well Low Profile Microplate, round well, V-bottom, sterile, with cover
(#X6004, Molecular Devices, LLC., San Jose, CA) (seeNote 1)
• ONCYTE
®
AVID nitrocellulose-coated slides (#305170, Grace Bio-Labs,
Bend, Oregon, USA) (seeNote 2)
Slide staining
• Kimwipes
®
disposable wipers (#Z188956, Sigma-Aldrich
®
, Merck KGaA,
Darmstadt, Germany)
• DAKO Reagent Bottles, User-Fillable, for Autostainer Link Instruments,
25 bottles, 5mL (#SK20005-2, Agilent Technologies Inc., CA, USA)
• DAKO Reagent Bottles, User-Fillable, for Autostainer Link Instruments,
25 bottles, 12mL (# SK20110-2, Agilent Technologies Inc., CA, USA)
Common reagents
Vehicle and drugs
• DMSO (#41639-500ML, Sigma-Aldrich
®
, Merck KGaA, Darmstadt,
Germany) (seeNote 4)
• 5-Fluorouracil, Oxaliplatin and Irinotecan hydrochloride trihydrate
(#ALX-480-099-G005, #ALX-400-042-M025 and #ALX-430-139-M025,
Enzo Life Sciences, Inc., NY, USA) (seeNote 5)
Protein lysates and RPPA sample preparation
• T-PER™Tissue Protein Extraction Reagent (500mL) (#78510, Thermo
Fisher Scientific, Waltham, MA, USA)
• Sodium chloride (NaCl, #S9888, Sigma-Aldrich
®
, Merck KGaA,
Darmstadt, Germany) (seeNote 6)
• Protease Inhibitor Cocktail, PIC (#P8340-1ML, Sigma-Aldrich
®
, Merck
KGaA, Darmstadt, Germany)
• Phosphatase Inhibitor Cocktail 2, PII (#P5726-1ML, Sigma-Aldrich
®
,
Merck KGaA, Darmstadt, Germany)
• Phosphatase Inhibitor Cocktail 3, PIII (P0044-1ML, Sigma-Aldrich
®
,
Merck KGaA, Darmstadt, Germany)
• Bio-Rad Protein Assay Dye Reagent Concentrate, 450 mL (#5000006,
Bio-Rad Laboratories, Hercules, CA, USA) (seeNote 7)
• Bond-Breaker™TCEP Solution, Neutral pH (5mL) (#77720, Thermo
Fisher Scientific, Waltham, MA, USA)
372Materials
• Multiwell plates for suspension culture, 12- and 24-well, polystyrene, clear,
lid with condensation rings, sterile, individually packed (#665970 and
#662102, Greiner Bio-One International GmbH, Kremsm€unster, Austria)
• Cell scrapers (e.g., Falcon
®
Cell Scraper with 25cm Handle and 1.8cm
Blade, Sterile, Individually Packaged, #353086, Corning Inc., NY, USA)
(seeNote 23)
• Visible Range Cuvette, Semi-Micro, 1.5mL, 10mm path length
(e.g., #1938, Kartell S.p.A., Milan, Italy)
Slide printing
• 384-Well Low Profile Microplate, round well, V-bottom, sterile, with cover
(#X6004, Molecular Devices, LLC., San Jose, CA) (seeNote 1)
• ONCYTE
®
AVID nitrocellulose-coated slides (#305170, Grace Bio-Labs,
Bend, Oregon, USA) (seeNote 2)
Slide staining
• Kimwipes
®
disposable wipers (#Z188956, Sigma-Aldrich
®
, Merck KGaA,
Darmstadt, Germany)
• DAKO Reagent Bottles, User-Fillable, for Autostainer Link Instruments,
25 bottles, 5mL (#SK20005-2, Agilent Technologies Inc., CA, USA)
• DAKO Reagent Bottles, User-Fillable, for Autostainer Link Instruments,
25 bottles, 12mL (# SK20110-2, Agilent Technologies Inc., CA, USA)
Common reagents
Vehicle and drugs
• DMSO (#41639-500ML, Sigma-Aldrich
®
, Merck KGaA, Darmstadt,
Germany) (seeNote 4)
• 5-Fluorouracil, Oxaliplatin and Irinotecan hydrochloride trihydrate
(#ALX-480-099-G005, #ALX-400-042-M025 and #ALX-430-139-M025,
Enzo Life Sciences, Inc., NY, USA) (seeNote 5)
Protein lysates and RPPA sample preparation
• T-PER™Tissue Protein Extraction Reagent (500mL) (#78510, Thermo
Fisher Scientific, Waltham, MA, USA)
• Sodium chloride (NaCl, #S9888, Sigma-Aldrich
®
, Merck KGaA,
Darmstadt, Germany) (seeNote 6)
• Protease Inhibitor Cocktail, PIC (#P8340-1ML, Sigma-Aldrich
®
, Merck
KGaA, Darmstadt, Germany)
• Phosphatase Inhibitor Cocktail 2, PII (#P5726-1ML, Sigma-Aldrich
®
,
Merck KGaA, Darmstadt, Germany)
• Phosphatase Inhibitor Cocktail 3, PIII (P0044-1ML, Sigma-Aldrich
®
,
Merck KGaA, Darmstadt, Germany)
• Bio-Rad Protein Assay Dye Reagent Concentrate, 450 mL (#5000006,
Bio-Rad Laboratories, Hercules, CA, USA) (seeNote 7)
• Bond-Breaker™TCEP Solution, Neutral pH (5mL) (#77720, Thermo
Fisher Scientific, Waltham, MA, USA)
372Materials
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yourself, and it’s necessary for me to be there to look to ’t. We all
have our callin’s and duties, and this is mine. I stick by the Molly and
her captain until both are out of this scrape, or both are condemned.
I know nothing of treason; but if the law wants another victim, I
must take my chance.”
Mulford was surprised at this steadiness of Jack’s, in what he
thought a very bad cause, and he was quite as much surprised that
Rose did not join him, in his endeavors to persuade the steward not
to be so fool-hardy, as to endeavor to go back to the brig. Rose did
not, however; sitting silently eating her dinner the whole time,
though she occasionally cast glances of interest at both the speakers
the while. In this state of things the mate abandoned the attempt,
for the moment, intending to return to the subject, after having had
a private conference with his betrothed.
Notwithstanding the little drawback just related, that was a
happy as well as a delicious repast. The mate did full justice to the
soup, and afterward to the fish with the unpoetical name; and Rose
ate more than she had done in the last three days. The habits of
discipline prevented Jack from taking his seat at table, though
pressed by both Rose and Harry to do so, but he helped himself to
the contents of a bowl, and did full justice to his own art, on one
aside. The little fellow was delighted with the praises that were
bestowed on his dishes; and for the moment, the sea, its dangers,
its tornadoes, wrecks and races, were all forgotten in the security
and pleasures of so savory a repast.
“Folk ashore don’t know how sailors sometimes live,” said Jack,
holding a large spoon filled with the soup ready to plunge into a
tolerably capacious mouth.
as to have no qualms about eating the bread of idleness.”
“Thank ’e, thank ’e, maty,” cried Jack, dashing a tear out of his
eye with the back of his hand, “thank ’e, sir, from the bottom of my
heart. The time may come, but not now. My papers is signed for this
v’y’ge. Stephen Spike has a halter round his neck, as you say
yourself, and it’s necessary for me to be there to look to ’t. We all
have our callin’s and duties, and this is mine. I stick by the Molly and
her captain until both are out of this scrape, or both are condemned.
I know nothing of treason; but if the law wants another victim, I
must take my chance.”
Mulford was surprised at this steadiness of Jack’s, in what he
thought a very bad cause, and he was quite as much surprised that
Rose did not join him, in his endeavors to persuade the steward not
to be so fool-hardy, as to endeavor to go back to the brig. Rose did
not, however; sitting silently eating her dinner the whole time,
though she occasionally cast glances of interest at both the speakers
the while. In this state of things the mate abandoned the attempt,
for the moment, intending to return to the subject, after having had
a private conference with his betrothed.
Notwithstanding the little drawback just related, that was a
happy as well as a delicious repast. The mate did full justice to the
soup, and afterward to the fish with the unpoetical name; and Rose
ate more than she had done in the last three days. The habits of
discipline prevented Jack from taking his seat at table, though
pressed by both Rose and Harry to do so, but he helped himself to
the contents of a bowl, and did full justice to his own art, on one
aside. The little fellow was delighted with the praises that were
bestowed on his dishes; and for the moment, the sea, its dangers,
its tornadoes, wrecks and races, were all forgotten in the security
and pleasures of so savory a repast.
“Folk ashore don’t know how sailors sometimes live,” said Jack,
holding a large spoon filled with the soup ready to plunge into a
tolerably capacious mouth.
“Or how they sometimes starve,” answered Rose. “Remember
our own situation, less than forty-eight hours since!”
“All very true, Miss Rose; yet, you see, turtle-soup brings us up,
a’ter all. Would you choose a glass of wine, maty?”
“Very much indeed, Jack, after so luscious a soup; but wishing
for it will not bring it here.”
“That remains to be seen, sir. I call this a bottle of something
that looks wery much like a wine.”
“Claret, as I live! Why, where should light-house keepers get the
taste for claret?”
“I’ve thought of that myself, Mr. Mulford, and have supposed that
some of Uncle Sam’s officers have brought the liquor to this part of
the world. I understand a party on ’em was here surveyin’ all last
winter. It seems they come in the cool weather, and get their sights
and measure their distances, and go home in the warm weather, and
work out their traverses in the shade, as it might be.”
“This seems likely, Jack; but come, whence it may, it is welcome,
and we will taste it.”
Mulford then drew the cork of this mild and grateful liquor, and
helped his companions and himself. In this age of moral tours de
force, one scarcely dare say any thing favorable of a liquid that even
bears the name of wine, or extol the shape of a bottle. It is truly the
era of exaggeration. Nothing is treated in the old-fashioned, natural,
common sense way. Virtue is no longer virtue, unless it get upon
stilts; and, as for sins being confined to “transgression against the
law of God,” audacious would be the wretch who should presume to
limit the sway of the societies by any dogma so narrow! A man may
be as abstemious as an anchorite and get no credit for it, unless “he
sign the pledge;” or, signing the pledge, he may get fuddled in
corners, and be cited as a miracle of sobriety. The test of morals is
no longer in the abuse of the gifts of Providence, but in their use;
prayers are deserting the closet for the corners of streets, and
charity (not the giving of alms) has got to be so earnest in the
demonstration of its nature, as to be pretty certain to “begin at
our own situation, less than forty-eight hours since!”
“All very true, Miss Rose; yet, you see, turtle-soup brings us up,
a’ter all. Would you choose a glass of wine, maty?”
“Very much indeed, Jack, after so luscious a soup; but wishing
for it will not bring it here.”
“That remains to be seen, sir. I call this a bottle of something
that looks wery much like a wine.”
“Claret, as I live! Why, where should light-house keepers get the
taste for claret?”
“I’ve thought of that myself, Mr. Mulford, and have supposed that
some of Uncle Sam’s officers have brought the liquor to this part of
the world. I understand a party on ’em was here surveyin’ all last
winter. It seems they come in the cool weather, and get their sights
and measure their distances, and go home in the warm weather, and
work out their traverses in the shade, as it might be.”
“This seems likely, Jack; but come, whence it may, it is welcome,
and we will taste it.”
Mulford then drew the cork of this mild and grateful liquor, and
helped his companions and himself. In this age of moral tours de
force, one scarcely dare say any thing favorable of a liquid that even
bears the name of wine, or extol the shape of a bottle. It is truly the
era of exaggeration. Nothing is treated in the old-fashioned, natural,
common sense way. Virtue is no longer virtue, unless it get upon
stilts; and, as for sins being confined to “transgression against the
law of God,” audacious would be the wretch who should presume to
limit the sway of the societies by any dogma so narrow! A man may
be as abstemious as an anchorite and get no credit for it, unless “he
sign the pledge;” or, signing the pledge, he may get fuddled in
corners, and be cited as a miracle of sobriety. The test of morals is
no longer in the abuse of the gifts of Providence, but in their use;
prayers are deserting the closet for the corners of streets, and
charity (not the giving of alms) has got to be so earnest in the
demonstration of its nature, as to be pretty certain to “begin at
home,” and to end where it begins. Even the art of mendacity has
been aroused by the great progress which is making by all around it,
and many manifest the strength of their ambition by telling ten lies
where their fathers would have been satisfied with telling only one.
This art has made an extraordinary progress within the last quarter
of a century, aspiring to an ascendancy that was formerly conceded
only to truth, until he who gains his daily bread by it has some such
contempt for the sneaking wretch who does business on the small
scale, as the slayer of his thousands in the field is known to
entertain for him who kills only a single man in the course of a long
life.
At the risk of damaging the reputations of our hero and heroine,
we shall frankly aver the fact that both Harry and Rose partook of
the vin de Bordeaux, a very respectable bottle of Medoc, by the way,
which had been forgotten by Uncle Sam’s people, in the course of
the preceding winter, agreeably to Jack Tier’s conjecture. One glass
sufficed for Rose, and, contrary as it may be to all modern theory,
she was somewhat the better for it; while the mate and Jack Tier
quite half emptied the bottle, being none the worse. There they sat,
enjoying the security and abundance which had succeeded to their
late danger, happy in that security, happy in themselves, and happy
in the prospects of a bright future. It was just as practicable for
them to remain at the Dry Tortugas, as it was for the family which
ordinarily dwelt at the light. The place was amply supplied with
every thing that would be necessary for their wants, for months to
come, and Harry caused his betrothed to blush, as he whispered to
her, should the chaplain arrive, he should delight in passing the
honey-moon where they then were.
“I could tend the light,” he added, smiling, “which would be not
only an occupation, but a useful occupation; you could read all those
books from beginning to end, and Jack could keep us supplied with
fish. By the way, master steward, are you in the humor for motion,
so soon after your hearty meal?”
“Any thing to be useful,” answered Jack, cheerfully.
been aroused by the great progress which is making by all around it,
and many manifest the strength of their ambition by telling ten lies
where their fathers would have been satisfied with telling only one.
This art has made an extraordinary progress within the last quarter
of a century, aspiring to an ascendancy that was formerly conceded
only to truth, until he who gains his daily bread by it has some such
contempt for the sneaking wretch who does business on the small
scale, as the slayer of his thousands in the field is known to
entertain for him who kills only a single man in the course of a long
life.
At the risk of damaging the reputations of our hero and heroine,
we shall frankly aver the fact that both Harry and Rose partook of
the vin de Bordeaux, a very respectable bottle of Medoc, by the way,
which had been forgotten by Uncle Sam’s people, in the course of
the preceding winter, agreeably to Jack Tier’s conjecture. One glass
sufficed for Rose, and, contrary as it may be to all modern theory,
she was somewhat the better for it; while the mate and Jack Tier
quite half emptied the bottle, being none the worse. There they sat,
enjoying the security and abundance which had succeeded to their
late danger, happy in that security, happy in themselves, and happy
in the prospects of a bright future. It was just as practicable for
them to remain at the Dry Tortugas, as it was for the family which
ordinarily dwelt at the light. The place was amply supplied with
every thing that would be necessary for their wants, for months to
come, and Harry caused his betrothed to blush, as he whispered to
her, should the chaplain arrive, he should delight in passing the
honey-moon where they then were.
“I could tend the light,” he added, smiling, “which would be not
only an occupation, but a useful occupation; you could read all those
books from beginning to end, and Jack could keep us supplied with
fish. By the way, master steward, are you in the humor for motion,
so soon after your hearty meal?”
“Any thing to be useful,” answered Jack, cheerfully.
“Then do me the favor to go up into the lantern of the light-
house, and take a look for the sloop-of-war. If she’s in sight at all,
you’ll find her off here to the northward; and while you are aloft you
may as well make a sweep of the whole horizon. There hangs the
light-house keeper’s glass, which may help your eyes, by stepping
into the gallery outside of the lantern.”
Jack willingly complied, taking the glass and proceeding forthwith
to the other building. Mulford had two objects in view in giving this
commission to the steward. He really wished to ascertain what was
the chance of seeing the Poughkeepsie, in the neighborhood of the
islets, and felt just that indisposition to move himself, that is apt to
come over one who has recently made a very bountiful meal, while
he also desired to have another private conversation with Rose.
A good portion of the time that Jack was gone, and he staid quite
an hour in the lantern, our lovers conversed as lovers are much
inclined to converse; that is to say, of themselves, their feelings, and
their prospects. Mulford told Rose of his hopes and fears, while he
visited at the house of her aunt, previously to sailing, and the
manner in which his suspicions had been first awakened in reference
to the intentions of Spike—intentions, so far as they were connected
with an admiration of his old commander’s niece, and possibly in
connection also with the little fortune she was known to possess, but
not in reference to the bold project to which he had, in fact,
resorted. No distrust of the scheme finally put in practice had ever
crossed the mind of the young mate, until he received the
unexpected order, mentioned in our opening chapter, to prepare the
brig for the reception of Mrs. Budd and her party. Harry confessed
his jealousy of one youth whom he dreaded far more even than he
had ever dreaded Spike, and whose apparent favor with Rose, and
actual favor with her aunt, had given him many a sleepless night.
They next conversed of the future, which to them seemed full of
flowers. Various were the projects started, discussed, and dismissed,
between them, the last almost as soon as proposed. On one thing
they were of a mind, as soon as proposed. Harry was to have a ship
as quick as one could be purchased by Rose’s means, and the
house, and take a look for the sloop-of-war. If she’s in sight at all,
you’ll find her off here to the northward; and while you are aloft you
may as well make a sweep of the whole horizon. There hangs the
light-house keeper’s glass, which may help your eyes, by stepping
into the gallery outside of the lantern.”
Jack willingly complied, taking the glass and proceeding forthwith
to the other building. Mulford had two objects in view in giving this
commission to the steward. He really wished to ascertain what was
the chance of seeing the Poughkeepsie, in the neighborhood of the
islets, and felt just that indisposition to move himself, that is apt to
come over one who has recently made a very bountiful meal, while
he also desired to have another private conversation with Rose.
A good portion of the time that Jack was gone, and he staid quite
an hour in the lantern, our lovers conversed as lovers are much
inclined to converse; that is to say, of themselves, their feelings, and
their prospects. Mulford told Rose of his hopes and fears, while he
visited at the house of her aunt, previously to sailing, and the
manner in which his suspicions had been first awakened in reference
to the intentions of Spike—intentions, so far as they were connected
with an admiration of his old commander’s niece, and possibly in
connection also with the little fortune she was known to possess, but
not in reference to the bold project to which he had, in fact,
resorted. No distrust of the scheme finally put in practice had ever
crossed the mind of the young mate, until he received the
unexpected order, mentioned in our opening chapter, to prepare the
brig for the reception of Mrs. Budd and her party. Harry confessed
his jealousy of one youth whom he dreaded far more even than he
had ever dreaded Spike, and whose apparent favor with Rose, and
actual favor with her aunt, had given him many a sleepless night.
They next conversed of the future, which to them seemed full of
flowers. Various were the projects started, discussed, and dismissed,
between them, the last almost as soon as proposed. On one thing
they were of a mind, as soon as proposed. Harry was to have a ship
as quick as one could be purchased by Rose’s means, and the
promised bride laughingly consented to make one voyage to Europe
along with her husband.
“I wonder, dear Rose, my poverty has never presented any
difficulties in the way of our union,” said Harry, sensibly touched with
the free way his betrothed disposed of her own money in his behalf;
“but neither you nor Mrs. Budd has ever seemed to think of the
difference there is between us in this respect.”
“What is the trifle I possess, Harry, set in the balance against
your worth? My aunt, as you say, has thought I might even be the
gainer by the exchange.”
“I am sure I feel a thousand times indebted to Mrs. Budd —”
“Aunt Budd. You must learn to say, ‘my Aunt Budd,’ Mr. Henry
Mulford, if you mean to live in peace with her unworthy niece.”
“Aunt Budd, then,” returned Harry, laughing, for the laugh came
easily that evening; “Aunt Budd, if you wish it, Rose. I can have no
objection to call any relative of yours, uncle or aunt.”
“I think we are intimate enough, now, to ask you a question or
two, Harry, touching my aunt,” continued Rose, looking stealthily
over her shoulder, as if apprehensive of being overheard. “You know
how fond she is of speaking of the sea, and of indulging in nautical
phrases?”
“Any one must have observed that, Rose,” answered the young
man, gazing up at the wall, in order not to be compelled to look the
beautiful creature before him in the eyes—“Mrs. Budd has very
strong tastes that way.”
“Now tell me, Harry—that is, answer me frankly—I mean—she is
not always right, is she?”
“Why, no; not absolutely so—that is, not absolutely always so—
few persons are always right, you know.”
Rose remained silent and embarrassed for a moment; after which
she pursued the discourse.
“But aunty does not know as much of the sea and of ships as she
thinks she does?”
along with her husband.
“I wonder, dear Rose, my poverty has never presented any
difficulties in the way of our union,” said Harry, sensibly touched with
the free way his betrothed disposed of her own money in his behalf;
“but neither you nor Mrs. Budd has ever seemed to think of the
difference there is between us in this respect.”
“What is the trifle I possess, Harry, set in the balance against
your worth? My aunt, as you say, has thought I might even be the
gainer by the exchange.”
“I am sure I feel a thousand times indebted to Mrs. Budd —”
“Aunt Budd. You must learn to say, ‘my Aunt Budd,’ Mr. Henry
Mulford, if you mean to live in peace with her unworthy niece.”
“Aunt Budd, then,” returned Harry, laughing, for the laugh came
easily that evening; “Aunt Budd, if you wish it, Rose. I can have no
objection to call any relative of yours, uncle or aunt.”
“I think we are intimate enough, now, to ask you a question or
two, Harry, touching my aunt,” continued Rose, looking stealthily
over her shoulder, as if apprehensive of being overheard. “You know
how fond she is of speaking of the sea, and of indulging in nautical
phrases?”
“Any one must have observed that, Rose,” answered the young
man, gazing up at the wall, in order not to be compelled to look the
beautiful creature before him in the eyes—“Mrs. Budd has very
strong tastes that way.”
“Now tell me, Harry—that is, answer me frankly—I mean—she is
not always right, is she?”
“Why, no; not absolutely so—that is, not absolutely always so—
few persons are always right, you know.”
Rose remained silent and embarrassed for a moment; after which
she pursued the discourse.
“But aunty does not know as much of the sea and of ships as she
thinks she does?”
“Perhaps not. We all overrate our own acquirements. I dare say
that even I am not as good a seaman as I fancy myself to be.”
“Even Spike admits that you are what he calls ‘a prime seaman.’
But it is not easy for a woman to get a correct knowledge of the use
of all the strange, and sometimes uncouth, terms that you sailors
use.”
“Certainly not; and for that reason I would rather you should
never attempt it, Rose. We rough sons of the ocean would prefer to
hear our wives make divers pretty blunders, rather than to be
swaggering about like so many ‘old salts.’ ”
“Mr. Mulford! Does Aunt Budd swagger like an old salt?”
“Dearest Rose, I was not thinking of your aunt, but of you. Of
you, as you are, feminine, spirited, lovely alike in form and character,
and of you a graduate of the ocean, and full of its language and
ideas.”
It was probable Rose was not displeased at this allusion to
herself, for a smile struggled around her pretty mouth, and she did
not look at all angry. After another short pause, she resumed the
discourse.
“My aunt did not very clearly comprehend those explanations of
yours about the time of day, and the longitude,” she said, “nor am I
quite certain that I did myself.”
“You understood them far better than Mrs. Budd, Rose. Women
are so little accustomed to think on such subjects at all, that it is not
surprising they sometimes get confused. I do wish, however, that
your aunt could be persuaded to be more cautious in the presence
of strangers, on the subject of terms she does not understand.”
“I feared it might be so, Harry,” answered Rose, in a low voice, as
if unwilling even he should know the full extent of her thoughts on
this subject; “but my aunt’s heart is most excellent, though she may
make mistakes occasionally. I owe her a great deal, if not absolutely
my education, certainly my health and comfort through childhood,
and more prudent, womanly advice than you may suppose, perhaps,
since I have left school. How she became the dupe of Spike, indeed,
that even I am not as good a seaman as I fancy myself to be.”
“Even Spike admits that you are what he calls ‘a prime seaman.’
But it is not easy for a woman to get a correct knowledge of the use
of all the strange, and sometimes uncouth, terms that you sailors
use.”
“Certainly not; and for that reason I would rather you should
never attempt it, Rose. We rough sons of the ocean would prefer to
hear our wives make divers pretty blunders, rather than to be
swaggering about like so many ‘old salts.’ ”
“Mr. Mulford! Does Aunt Budd swagger like an old salt?”
“Dearest Rose, I was not thinking of your aunt, but of you. Of
you, as you are, feminine, spirited, lovely alike in form and character,
and of you a graduate of the ocean, and full of its language and
ideas.”
It was probable Rose was not displeased at this allusion to
herself, for a smile struggled around her pretty mouth, and she did
not look at all angry. After another short pause, she resumed the
discourse.
“My aunt did not very clearly comprehend those explanations of
yours about the time of day, and the longitude,” she said, “nor am I
quite certain that I did myself.”
“You understood them far better than Mrs. Budd, Rose. Women
are so little accustomed to think on such subjects at all, that it is not
surprising they sometimes get confused. I do wish, however, that
your aunt could be persuaded to be more cautious in the presence
of strangers, on the subject of terms she does not understand.”
“I feared it might be so, Harry,” answered Rose, in a low voice, as
if unwilling even he should know the full extent of her thoughts on
this subject; “but my aunt’s heart is most excellent, though she may
make mistakes occasionally. I owe her a great deal, if not absolutely
my education, certainly my health and comfort through childhood,
and more prudent, womanly advice than you may suppose, perhaps,
since I have left school. How she became the dupe of Spike, indeed,
is to me unaccountable; for in all that relates to health, she is, in
general, both acute and skillful.”
“Spike is a man of more art than he appears to be to superficial
observers. On my first acquaintance with him, I mistook him for a
frank, fearless, but well-meaning sailor, who loved hazardous
voyages and desperate speculation—a sort of innocent gambler; but
I have learned to know better. His means are pretty much reduced
to his brig, and she is getting old, and can do but little more service.
His projects are plain enough, now. By getting you into his power, he
hoped to compel a marriage, in which case both your fortune and
your aunt’s would contribute to repair his.”
“He might have killed me, but I never would have married him,”
rejoined Rose, firmly. “Is not that Jack coming down the steps of the
light-house?”
“It is. I find that fellow’s attachment to Spike very extraordinary,
Rose. Can you, in any manner, account for it?”
Rose at first seemed disposed to reply. Her lips parted, as if
about to speak, and closed again, as glancing her eyes toward the
open door, she seemed to expect the appearance of the steward’s
little, rotund form on its threshold, which held her tongue-tied. A
brief interval elapsed, however, ere Jack actually arrived, and Rose,
perceiving that Harry was curiously expecting her answer, said
hurriedly—“it may be hatred, not attachment.”
The next instant Jack Tier entered the room. He had been gone
rather more than an hour, not returning until just as the sun was
about to set in a flame of fire.
“Well, Jack, what news from the Poughkeepsie?” demanded the
mate. “You have been gone long enough to make sure of your
errand. Is it certain that we are not to see the man-of-war’s-men to-
night?”
“Whatever you see, my advice to you is to keep close, and to be
on your guard,” answered Jack, evasively.
“I have little fear of any of Uncle Sam’s craft. A plain story, and
an honest heart, will make all clear to a well-disposed listener. We
general, both acute and skillful.”
“Spike is a man of more art than he appears to be to superficial
observers. On my first acquaintance with him, I mistook him for a
frank, fearless, but well-meaning sailor, who loved hazardous
voyages and desperate speculation—a sort of innocent gambler; but
I have learned to know better. His means are pretty much reduced
to his brig, and she is getting old, and can do but little more service.
His projects are plain enough, now. By getting you into his power, he
hoped to compel a marriage, in which case both your fortune and
your aunt’s would contribute to repair his.”
“He might have killed me, but I never would have married him,”
rejoined Rose, firmly. “Is not that Jack coming down the steps of the
light-house?”
“It is. I find that fellow’s attachment to Spike very extraordinary,
Rose. Can you, in any manner, account for it?”
Rose at first seemed disposed to reply. Her lips parted, as if
about to speak, and closed again, as glancing her eyes toward the
open door, she seemed to expect the appearance of the steward’s
little, rotund form on its threshold, which held her tongue-tied. A
brief interval elapsed, however, ere Jack actually arrived, and Rose,
perceiving that Harry was curiously expecting her answer, said
hurriedly—“it may be hatred, not attachment.”
The next instant Jack Tier entered the room. He had been gone
rather more than an hour, not returning until just as the sun was
about to set in a flame of fire.
“Well, Jack, what news from the Poughkeepsie?” demanded the
mate. “You have been gone long enough to make sure of your
errand. Is it certain that we are not to see the man-of-war’s-men to-
night?”
“Whatever you see, my advice to you is to keep close, and to be
on your guard,” answered Jack, evasively.
“I have little fear of any of Uncle Sam’s craft. A plain story, and
an honest heart, will make all clear to a well-disposed listener. We
have not been accomplices in Spike’s treasons, and cannot be made
to answer for them.”
“Take my advice, maty, and be in no hurry to hail every vessel
you see. Uncle Sam’s fellows may not always be at hand to help you.
Do you not know that this island will be tabooed to seamen for some
time to come?”
“Why so, Jack? The islet has done no harm, though others may
have performed wicked deeds near it.”
“Two of the drowned men lie within a hundred yards of this spot,
and sailors never go near new-made graves, if they can find any
other place to resort to.”
“You deal in enigmas, Jack; and did I not know that you are very
temperate, I might suspect that the time you have been gone has
been passed in the company of a bottle of brandy.”
“That will explain my meaning,” said Jack, laconically, pointing as
he spoke, seemingly at some object that was to be seen without.
The door of the house was wide open, for the admission of air. It
faced the haven of the islets, and just as the mate’s eyes were
turned to it, the end of a flying-jib boom, with the sail down, and
fluttering beneath it, was coming into the view. “The Poughkeepsie!”
exclaimed Mulford, in delight, seeing all his hopes realized, while
Rose blushed to the eyes. A pause succeeded, during which Mulford
drew aside, keeping his betrothed in the back-ground, and as much
out of sight as possible. The vessel was shooting swiftly into view,
and presently all there could see it was the Swash.
[To be continued.
to answer for them.”
“Take my advice, maty, and be in no hurry to hail every vessel
you see. Uncle Sam’s fellows may not always be at hand to help you.
Do you not know that this island will be tabooed to seamen for some
time to come?”
“Why so, Jack? The islet has done no harm, though others may
have performed wicked deeds near it.”
“Two of the drowned men lie within a hundred yards of this spot,
and sailors never go near new-made graves, if they can find any
other place to resort to.”
“You deal in enigmas, Jack; and did I not know that you are very
temperate, I might suspect that the time you have been gone has
been passed in the company of a bottle of brandy.”
“That will explain my meaning,” said Jack, laconically, pointing as
he spoke, seemingly at some object that was to be seen without.
The door of the house was wide open, for the admission of air. It
faced the haven of the islets, and just as the mate’s eyes were
turned to it, the end of a flying-jib boom, with the sail down, and
fluttering beneath it, was coming into the view. “The Poughkeepsie!”
exclaimed Mulford, in delight, seeing all his hopes realized, while
Rose blushed to the eyes. A pause succeeded, during which Mulford
drew aside, keeping his betrothed in the back-ground, and as much
out of sight as possible. The vessel was shooting swiftly into view,
and presently all there could see it was the Swash.
[To be continued.
STOCK-JOBBING IN NEW YORK.
———
BY PETER PENCIL.
———
“Nothing venture, nothing win.”
Theêe are comparatively few people, even in New York, who
know, or have the most remote idea of, the amount of the daily
transactions of various kinds that take place in Wall street. If the
truth could be arrived at, it would appear, I doubt not, that the
operations there, in the course of a year, exceed, in their aggregate
amount, those of all other cities in the United States combined. This
opinion may startle some, but it will not startle those who are in the
practice of visiting that place, and seeing what is going forward
among the countless capitalists, brokers, merchants, and others,
whose vocation draws them to that vicinity. Nor can one who is a
visiter merely, form a conjecture approximating to the truth,
concerning the multiplicity and extent of Wall street affairs, any
more than a man who travels straight through the middle of a state,
can form an idea as to what quantity of corn is growing upon the
whole surface. It would be necessary to penetrate the hundreds of
offices, both great and small, public and private, and to see all that
is done therein, before one could begin, as the boys say, to estimate
the amount of business transacted in that short street, and its
immediate vicinity, in the course of a single day.
The stock operations alone would stagger the credulity even of
the initiated, who should keep an accurate account of the amount
changing hands from day to-day, and sum the whole at the
expiration of the year. Many millions’ worth of this species of
property would be found to have been bought and sold, making
some richer and some poorer, and leaving some, but, doubtless, very
———
BY PETER PENCIL.
———
“Nothing venture, nothing win.”
Theêe are comparatively few people, even in New York, who
know, or have the most remote idea of, the amount of the daily
transactions of various kinds that take place in Wall street. If the
truth could be arrived at, it would appear, I doubt not, that the
operations there, in the course of a year, exceed, in their aggregate
amount, those of all other cities in the United States combined. This
opinion may startle some, but it will not startle those who are in the
practice of visiting that place, and seeing what is going forward
among the countless capitalists, brokers, merchants, and others,
whose vocation draws them to that vicinity. Nor can one who is a
visiter merely, form a conjecture approximating to the truth,
concerning the multiplicity and extent of Wall street affairs, any
more than a man who travels straight through the middle of a state,
can form an idea as to what quantity of corn is growing upon the
whole surface. It would be necessary to penetrate the hundreds of
offices, both great and small, public and private, and to see all that
is done therein, before one could begin, as the boys say, to estimate
the amount of business transacted in that short street, and its
immediate vicinity, in the course of a single day.
The stock operations alone would stagger the credulity even of
the initiated, who should keep an accurate account of the amount
changing hands from day to-day, and sum the whole at the
expiration of the year. Many millions’ worth of this species of
property would be found to have been bought and sold, making
some richer and some poorer, and leaving some, but, doubtless, very
few, about the same in purse at the end of the year, as they were at
the beginning.
If a person, standing on the steps of the exchange, were
endowed with the faculty of reading the heart of every man that
passed him, what numbers of agitated bosoms, what hopes, what
fears, what emotions of vexation, sorrow, anger, and despair, would
come under review; particularly after a panic among the speculators,
and a consequent fall of stock!
There are a few fortunate individuals, who owe to Wall street all
they possess—having speculated and staked high under the benign
influence of Fortune, while that goddess was in a kindly humor; but
there are hundreds, nay, thousands, who have seen their wealth
melt away there, like snow in a sunny nook on an April morn. “Make
or break—neck or no joint,” are the mottoes there; for when a man
once gets into the spirit of speculating, as this species of gambling is
mildly termed, he is not apt to back out till he has made a fortune,
or lost what he possessed—won the horse, or lost the saddle.
The reader will see, in the course of this essay, to which of these
categories I belong; for I, too, have been afflicted with the prevailing
mania for stock-jobbing, and have shared in the hopes and fears, joy
and sorrow, which are produced by the uncertainty of such
operations, and the momentous consequences which often follow in
their train. It is my purpose to give a short sketch of my doings in
that line of business, (now so much in vogue,) for the amusement of
those who never go into Wall street, and the benefit of such
adventurous spirits as may be disposed to try their fortune at the
same table.
It may not be known to the majority of my readers, that the
prices of stocks, in New York, are very much influenced by the
weather; indeed, I have sometimes thought that their value, as a
marketable commodity, depended more on the state of the
atmosphere than on their intrinsic worth. I have known a snow-
storm cause a sudden fall of two to five per cent.; and an April
shower, though it lasted but an hour, more or less, have the same
effect to the extent of one or two per cent. I have myself suffered in
the beginning.
If a person, standing on the steps of the exchange, were
endowed with the faculty of reading the heart of every man that
passed him, what numbers of agitated bosoms, what hopes, what
fears, what emotions of vexation, sorrow, anger, and despair, would
come under review; particularly after a panic among the speculators,
and a consequent fall of stock!
There are a few fortunate individuals, who owe to Wall street all
they possess—having speculated and staked high under the benign
influence of Fortune, while that goddess was in a kindly humor; but
there are hundreds, nay, thousands, who have seen their wealth
melt away there, like snow in a sunny nook on an April morn. “Make
or break—neck or no joint,” are the mottoes there; for when a man
once gets into the spirit of speculating, as this species of gambling is
mildly termed, he is not apt to back out till he has made a fortune,
or lost what he possessed—won the horse, or lost the saddle.
The reader will see, in the course of this essay, to which of these
categories I belong; for I, too, have been afflicted with the prevailing
mania for stock-jobbing, and have shared in the hopes and fears, joy
and sorrow, which are produced by the uncertainty of such
operations, and the momentous consequences which often follow in
their train. It is my purpose to give a short sketch of my doings in
that line of business, (now so much in vogue,) for the amusement of
those who never go into Wall street, and the benefit of such
adventurous spirits as may be disposed to try their fortune at the
same table.
It may not be known to the majority of my readers, that the
prices of stocks, in New York, are very much influenced by the
weather; indeed, I have sometimes thought that their value, as a
marketable commodity, depended more on the state of the
atmosphere than on their intrinsic worth. I have known a snow-
storm cause a sudden fall of two to five per cent.; and an April
shower, though it lasted but an hour, more or less, have the same
effect to the extent of one or two per cent. I have myself suffered in
my speculations by a change of weather; and the only fortunate hit I
ever made, I ascribe entirely to the opportune clearing up of a long
storm.
It is really surprising what effect the weather has upon the minds
of stock-operators. Apparently, those enterprising fellows are as
susceptible to the influence of the atmosphere, as poets; though in
every thing else, it must be confessed, they are as different from the
genus irritabile vatum, as Horace calls them, as the orange-water on
a lady’s toilet is from the plain, unperfumed Croton in which she
laves her hands. On a bright, sunny day their countenances wear a
cheerful expression, their bosoms throb with joyful expectations of
an advance or fall in prices, as may happen to suit their purpose;
and, in a word, they feel richer and better, and are prepared to
renew their operations with increased spirit. Hence the expression so
often seen in the “Money Articles” of our daily papers, “there was a
better feeling at both boards to-day;” and this stereotyped phrase
has become equivalent to the announcement that the weather has
become exceedingly fine.
In cloudy weather, on the contrary, particularly if it rains, their
faces are generally augmented longitudinally to a very considerable
extent; and so true an interpreter is a broker’s face of the state of
the heavens, that one might safely depend on it for information
without looking at the sky. I regard a speculator’s countenance as far
more reliable than a weathercock, because I have known the latter
to deceive me by pointing westward, when, according to the
weather, it should have stood in the opposite stormy quarter. But the
face of a stock-operator of New York was never known to play tricks
of this kind, within the far-reaching memory of that most
respectable, and often referred-to individual, the Oldest Inhabitant.
No man ever saw a smile on his phiz, except when the sun shone.
There are some shrewd men in New York, who perfectly
understand these “skyey influences,” and regulate their speculative
movements accordingly—buying in a storm, especially if it be a long
and severe one, and selling out whenever the succeeding clear
weather has produced a favorable reaction in prices. One rich
ever made, I ascribe entirely to the opportune clearing up of a long
storm.
It is really surprising what effect the weather has upon the minds
of stock-operators. Apparently, those enterprising fellows are as
susceptible to the influence of the atmosphere, as poets; though in
every thing else, it must be confessed, they are as different from the
genus irritabile vatum, as Horace calls them, as the orange-water on
a lady’s toilet is from the plain, unperfumed Croton in which she
laves her hands. On a bright, sunny day their countenances wear a
cheerful expression, their bosoms throb with joyful expectations of
an advance or fall in prices, as may happen to suit their purpose;
and, in a word, they feel richer and better, and are prepared to
renew their operations with increased spirit. Hence the expression so
often seen in the “Money Articles” of our daily papers, “there was a
better feeling at both boards to-day;” and this stereotyped phrase
has become equivalent to the announcement that the weather has
become exceedingly fine.
In cloudy weather, on the contrary, particularly if it rains, their
faces are generally augmented longitudinally to a very considerable
extent; and so true an interpreter is a broker’s face of the state of
the heavens, that one might safely depend on it for information
without looking at the sky. I regard a speculator’s countenance as far
more reliable than a weathercock, because I have known the latter
to deceive me by pointing westward, when, according to the
weather, it should have stood in the opposite stormy quarter. But the
face of a stock-operator of New York was never known to play tricks
of this kind, within the far-reaching memory of that most
respectable, and often referred-to individual, the Oldest Inhabitant.
No man ever saw a smile on his phiz, except when the sun shone.
There are some shrewd men in New York, who perfectly
understand these “skyey influences,” and regulate their speculative
movements accordingly—buying in a storm, especially if it be a long
and severe one, and selling out whenever the succeeding clear
weather has produced a favorable reaction in prices. One rich
individual, living up town, the moment he rises in the morning,
opens his window and looks at the vane on a neighboring steeple—
the only part of the church, by the way, he cares a fig about—and if
the wind happen to blow from a rainy point, he hastens down town,
and orders his broker to dive deep into some of the “fancies.” If,
however, the day be clear, he stays at home, his broker being
already instructed to sell out some previous purchase, as soon as
the weather should warrant.
But the weather, though a most powerful agent in the fluctuation
of prices, is by no means the only cause of those great and sudden
changes in the marketable value of “securities,” which take money
from one pocket, and put it into another. An apprehension, well or ill
founded, (it is the same thing in effect,) of an increased demand for
money; a paragraph in a newspaper, announcing, mysteriously, that
some sort of news, concerning nobody knows what, may be
expected in a few days; wars, and rumors of wars; and reports
about different matters, however trifling and uninteresting to the
majority of the people; all these are sufficient to dash a broker’s
spirits; and produce a panic in the market.
Stepping into the great room of the exchange one day, to see the
doings at the public board of brokers, I, like the rest of the crowd
that stood looking on, became interested in their proceedings, and
was soon seized with a desire to try my luck in speculation. I had
previously heard of this man and that, having realized their
thousands in as many weeks; and as stocks were advancing, and
likely, for aught that appeared, to have an “upward tendency” for
some time to come, I saw no good reason why I, too, might not
increase my little capital in the same rapid manner. “The prospect
before us is cheering,” said I to myself, “the boundary question,
thanks to the great Daniel, is settled; money is plentiful, and as
cheap as dirt; and, in all human probability, Harry Clay, or somebody
equally worthy, will be our next president. It follows, therefore, as a
necessary consequence, that good dividend-paying stocks must
advance.”
opens his window and looks at the vane on a neighboring steeple—
the only part of the church, by the way, he cares a fig about—and if
the wind happen to blow from a rainy point, he hastens down town,
and orders his broker to dive deep into some of the “fancies.” If,
however, the day be clear, he stays at home, his broker being
already instructed to sell out some previous purchase, as soon as
the weather should warrant.
But the weather, though a most powerful agent in the fluctuation
of prices, is by no means the only cause of those great and sudden
changes in the marketable value of “securities,” which take money
from one pocket, and put it into another. An apprehension, well or ill
founded, (it is the same thing in effect,) of an increased demand for
money; a paragraph in a newspaper, announcing, mysteriously, that
some sort of news, concerning nobody knows what, may be
expected in a few days; wars, and rumors of wars; and reports
about different matters, however trifling and uninteresting to the
majority of the people; all these are sufficient to dash a broker’s
spirits; and produce a panic in the market.
Stepping into the great room of the exchange one day, to see the
doings at the public board of brokers, I, like the rest of the crowd
that stood looking on, became interested in their proceedings, and
was soon seized with a desire to try my luck in speculation. I had
previously heard of this man and that, having realized their
thousands in as many weeks; and as stocks were advancing, and
likely, for aught that appeared, to have an “upward tendency” for
some time to come, I saw no good reason why I, too, might not
increase my little capital in the same rapid manner. “The prospect
before us is cheering,” said I to myself, “the boundary question,
thanks to the great Daniel, is settled; money is plentiful, and as
cheap as dirt; and, in all human probability, Harry Clay, or somebody
equally worthy, will be our next president. It follows, therefore, as a
necessary consequence, that good dividend-paying stocks must
advance.”
Now this seemed well reasoned, to say the least, and the
conclusion a just one; but, alas! for human foresight! the good
stocks, in which alone I ventured at first, like a balking horse, stood
still, or if they moved at all, refused to budge an inch in the right
direction. The bad stocks, those not intrinsically worth a fig, were
those which I should have purchased. They went up like a rocket;
but mine, from the moment that I bought it, seemed to have
suddenly acquired one of the properties of lead, for it would go
down, in spite of every effort made to keep it up—and the papers
called it heavy. Heavy enough I found it, heaven knows! But I am
anticipating, and running ahead of my story.
When I entered the exchange, I was the possessor of fifteen
hundred dollars—the savings of many years of industry; but I was
tired of work, and longed to make a fortune rather by the exercise of
intellect, than by the labor of my hands. It promised me a fortune in
a hundredth part of the time that it would take me to accumulate
one in any other way; and then it was so fine, I thought, to be
considered a heavy dealer in stocks, and to be regarded as a great,
bold operator, and a capitalist. How could I, with such lofty ideas in
my head, and with such a consciousness of possessing superior tact
and talent, go back quietly to work! Pah! the very thought of such a
thing sickened me.
I caught the eye of a broker with whom I was acquainted, and,
having beckoned him to me, requested him to buy ten thousand
dollars worth of Ohio sixes, at the market price, which happened, I
remember, (and I shall never forget it the longest day I live,) to be
one hundred and four. The day was pleasant, the room light, and
well filled with cheerful spectators; the brokers were in good spirits,
and disposed to go deep in their favorite game, and, to use a
common expression, the steam was up to the highest point at both
boards, and in the street.
Methinks I hear some one ask how so much stock was paid for
by a man worth but fifteen hundred dollars, all told. Innocent one! I
will tell thee. I borrowed the money, or about ninety per cent. of it
at least, for a few days, and gave the stock itself as security. How
conclusion a just one; but, alas! for human foresight! the good
stocks, in which alone I ventured at first, like a balking horse, stood
still, or if they moved at all, refused to budge an inch in the right
direction. The bad stocks, those not intrinsically worth a fig, were
those which I should have purchased. They went up like a rocket;
but mine, from the moment that I bought it, seemed to have
suddenly acquired one of the properties of lead, for it would go
down, in spite of every effort made to keep it up—and the papers
called it heavy. Heavy enough I found it, heaven knows! But I am
anticipating, and running ahead of my story.
When I entered the exchange, I was the possessor of fifteen
hundred dollars—the savings of many years of industry; but I was
tired of work, and longed to make a fortune rather by the exercise of
intellect, than by the labor of my hands. It promised me a fortune in
a hundredth part of the time that it would take me to accumulate
one in any other way; and then it was so fine, I thought, to be
considered a heavy dealer in stocks, and to be regarded as a great,
bold operator, and a capitalist. How could I, with such lofty ideas in
my head, and with such a consciousness of possessing superior tact
and talent, go back quietly to work! Pah! the very thought of such a
thing sickened me.
I caught the eye of a broker with whom I was acquainted, and,
having beckoned him to me, requested him to buy ten thousand
dollars worth of Ohio sixes, at the market price, which happened, I
remember, (and I shall never forget it the longest day I live,) to be
one hundred and four. The day was pleasant, the room light, and
well filled with cheerful spectators; the brokers were in good spirits,
and disposed to go deep in their favorite game, and, to use a
common expression, the steam was up to the highest point at both
boards, and in the street.
Methinks I hear some one ask how so much stock was paid for
by a man worth but fifteen hundred dollars, all told. Innocent one! I
will tell thee. I borrowed the money, or about ninety per cent. of it
at least, for a few days, and gave the stock itself as security. How
simple! did I hear thee say? Truly the process was exceedingly
simple; natheless I advise thee not to follow my example.
I considered myself uncommonly lucky in thus securing what I
wanted at so low a price, as I then regarded it; for the broker
assured me, and such seemed to be the prevailing opinion among
the knowing ones, that the stock I bought would rise six per cent. at
least within two or three months. I expected, so sanguine is my
temperament, to sell at that advance in less than a fortnight; and
already considered myself as six hundred dollars richer than I was
before. “A nice little sum that,” thought I, “for a beginning, and will
furnish the out-goings for a month, next summer, at Saratoga, and
the disbursements of a trip to Niagara, returning by way of Montreal,
Quebec, and Lake George.”
There is a proverb about counting the young of barn-yard fowls,
before the tender chickens are fairly out of their shells; which
proverb admonishes us never to make such a reckoning till the
hatching is completed, lest we should be disappointed as to the
number. Experience has taught me that this proverb, with some
slight verbal alterations, would apply equally well to the expected
profits from speculation in stocks. One should never count his gains,
nor appropriate them to any specific purpose, until they be realized.
In a day or two I found, much to my chagrin, that the stock I
had so fortunately purchased, instead of being on the high road to
one hundred and ten, began to grow tired of advancing, as though it
were leg-weary, and turning suddenly about, took, like a school-boy
coming home, “cross lots” the shortest possible way back to its old
position on the wrong side of par. I ascribe this sudden change to
two causes; first, I was the owner of some of the stock, which
reason was enough of itself to knock down that or any other
security; as I never in my life touched any thing of the kind that did
not immediately become “heavy,” and of less value than it was
before. Tom Moore complained most beautifully of similar ill-luck,
and said, in his own inimitable way,
simple; natheless I advise thee not to follow my example.
I considered myself uncommonly lucky in thus securing what I
wanted at so low a price, as I then regarded it; for the broker
assured me, and such seemed to be the prevailing opinion among
the knowing ones, that the stock I bought would rise six per cent. at
least within two or three months. I expected, so sanguine is my
temperament, to sell at that advance in less than a fortnight; and
already considered myself as six hundred dollars richer than I was
before. “A nice little sum that,” thought I, “for a beginning, and will
furnish the out-goings for a month, next summer, at Saratoga, and
the disbursements of a trip to Niagara, returning by way of Montreal,
Quebec, and Lake George.”
There is a proverb about counting the young of barn-yard fowls,
before the tender chickens are fairly out of their shells; which
proverb admonishes us never to make such a reckoning till the
hatching is completed, lest we should be disappointed as to the
number. Experience has taught me that this proverb, with some
slight verbal alterations, would apply equally well to the expected
profits from speculation in stocks. One should never count his gains,
nor appropriate them to any specific purpose, until they be realized.
In a day or two I found, much to my chagrin, that the stock I
had so fortunately purchased, instead of being on the high road to
one hundred and ten, began to grow tired of advancing, as though it
were leg-weary, and turning suddenly about, took, like a school-boy
coming home, “cross lots” the shortest possible way back to its old
position on the wrong side of par. I ascribe this sudden change to
two causes; first, I was the owner of some of the stock, which
reason was enough of itself to knock down that or any other
security; as I never in my life touched any thing of the kind that did
not immediately become “heavy,” and of less value than it was
before. Tom Moore complained most beautifully of similar ill-luck,
and said, in his own inimitable way,
“I never nursed a dear gazelle,
To glad me with its soft black eye,
But when it came to know me well,
And love me, it was sure to die.”
And I can and do say with more truth, (for Tom evidently fibbed, or
rather made Hinda do so,) and with equally good rhymes, that
I never bought a single mill
Of stock, in that vile street named Wall,
That rose a peg, or e’en stood still;
Dod rot it!—it was sure to fall.
Secondly, a paragraph appeared in the Herald, saying something
about England and war; and this circumstance, combined with the
fact of my being a holder, was too much for Ohio sixes, and down
they went. Nothing short of a miracle could have sustained them
under such a pressure. But this was not all; for, in the incipient stage
of the panic which followed, the wind suddenly veered round to
north-east, and a storm came on to increase the difficulty. Such a
scene as ensued has rarely been witnessed since Wall street became
a theatre for speculation. Faces became elongated many hundred
feet in the aggregate; eyes opened to their widest capacity, and
seemed to be looking wildly about for that greatest of bug-bears,
the British; and every speculator’s heart, like Macbeth’s, did
————“Knock at the ribs,
Against the use of nature,”
as though some terrible calamity, involving the annihilation of every
thing in the shape of stocks and money, were impending.
If some giant from another globe had come upon the earth, and
suddenly knocked the foundation stones from under that noble
structure, the merchants’ exchange, the crash would hardly have
been greater or more alarming than that which took place, on the
day in question, among the stocks. I stood silently by, and saw my
property vanish, as it were, before my eyes; but I will not attempt to
describe my feelings, for I am sure that I should not be able to
To glad me with its soft black eye,
But when it came to know me well,
And love me, it was sure to die.”
And I can and do say with more truth, (for Tom evidently fibbed, or
rather made Hinda do so,) and with equally good rhymes, that
I never bought a single mill
Of stock, in that vile street named Wall,
That rose a peg, or e’en stood still;
Dod rot it!—it was sure to fall.
Secondly, a paragraph appeared in the Herald, saying something
about England and war; and this circumstance, combined with the
fact of my being a holder, was too much for Ohio sixes, and down
they went. Nothing short of a miracle could have sustained them
under such a pressure. But this was not all; for, in the incipient stage
of the panic which followed, the wind suddenly veered round to
north-east, and a storm came on to increase the difficulty. Such a
scene as ensued has rarely been witnessed since Wall street became
a theatre for speculation. Faces became elongated many hundred
feet in the aggregate; eyes opened to their widest capacity, and
seemed to be looking wildly about for that greatest of bug-bears,
the British; and every speculator’s heart, like Macbeth’s, did
————“Knock at the ribs,
Against the use of nature,”
as though some terrible calamity, involving the annihilation of every
thing in the shape of stocks and money, were impending.
If some giant from another globe had come upon the earth, and
suddenly knocked the foundation stones from under that noble
structure, the merchants’ exchange, the crash would hardly have
been greater or more alarming than that which took place, on the
day in question, among the stocks. I stood silently by, and saw my
property vanish, as it were, before my eyes; but I will not attempt to
describe my feelings, for I am sure that I should not be able to
convey an idea of them to the reader’s mind. Suffice it to say that I
was hurt—cut to the very soul. “Farewell, Niagara, Quebec, and
Montreal,” thought I; “if I can keep out of the almshouse, the way
things are going, I shall be remarkably lucky.”
After consulting with my friend, the broker, who, to do him
justice, it must be confessed, gave me the best advice that his fears
permitted, I concluded to sell out my stock at ninety-eight, while it
was on the descent, and buy again the moment it should reach the
lowest point, which the broker and I thought would be about ninety.
Then, if our expectations should be realized, and the stock again
reach what I had before given, namely, one hundred and four, it is
clear that I should, beside recovering my loss, make eight per cent.
profit.
Here was a most glorious opportunity for a speculation—one of
those that occur about twice in a century. It was a happy thought in
me to sell even at a great loss, with a view of repurchasing on better
terms; and I could not help regarding it as a singularly bold move—
one indicating great genius, and just such a one as Napoleon
himself, under similar circumstances, might have conceived and
made. I became elated at the prospect, and bade my friend sell out
with all possible expedition. He did so at ninety-eight, being a loss to
me of six per cent., or six hundred dollars—a pretty fair clip from the
back of my little capital of fifteen hundred.
I should have been exceedingly annoyed by this docking of my
fortune, had not the certainty which I felt of making good the
deficiency, encouraged me; and but for the most perfect confidence
I entertained in the success of my next adventure, I should, in all
human probability, have retired from Wall street with much the same
feeling that a fox has when he sneaks off to his hole, after parting
with his tail in a trap.
But what short-sighted mortals we are, and how the blindfolded
goddess loves to sport with human calculations!
was hurt—cut to the very soul. “Farewell, Niagara, Quebec, and
Montreal,” thought I; “if I can keep out of the almshouse, the way
things are going, I shall be remarkably lucky.”
After consulting with my friend, the broker, who, to do him
justice, it must be confessed, gave me the best advice that his fears
permitted, I concluded to sell out my stock at ninety-eight, while it
was on the descent, and buy again the moment it should reach the
lowest point, which the broker and I thought would be about ninety.
Then, if our expectations should be realized, and the stock again
reach what I had before given, namely, one hundred and four, it is
clear that I should, beside recovering my loss, make eight per cent.
profit.
Here was a most glorious opportunity for a speculation—one of
those that occur about twice in a century. It was a happy thought in
me to sell even at a great loss, with a view of repurchasing on better
terms; and I could not help regarding it as a singularly bold move—
one indicating great genius, and just such a one as Napoleon
himself, under similar circumstances, might have conceived and
made. I became elated at the prospect, and bade my friend sell out
with all possible expedition. He did so at ninety-eight, being a loss to
me of six per cent., or six hundred dollars—a pretty fair clip from the
back of my little capital of fifteen hundred.
I should have been exceedingly annoyed by this docking of my
fortune, had not the certainty which I felt of making good the
deficiency, encouraged me; and but for the most perfect confidence
I entertained in the success of my next adventure, I should, in all
human probability, have retired from Wall street with much the same
feeling that a fox has when he sneaks off to his hole, after parting
with his tail in a trap.
But what short-sighted mortals we are, and how the blindfolded
goddess loves to sport with human calculations!
————Heu, Fortuna, quis est crudelior in nos
Te Deus?—ut semper gaudes illudere rebus
Humanis!
exclaimed Horace; and depend upon it, if stocks were the subject of
traffic in Rome, he had just been nicked when he wrote that
passage. Most courteous reader, I was doomed to suffer another
grievous disappointment; stocks took a different turn from what I
had expected. The storm cleared away, and the panic abated. The
sun again shone out bright, and smiles reappeared on the brokers’
faces. Prices had reached their lowest point, precisely at the moment
that I sold out mine, and instead of going down to ninety, as they
would have done had I continued to hold, they “rallied,” as the
saying is, and rose to par. I looked and felt blue, and counted over
my money again and again; I ciphered and calculated for half a
morning, in endeavoring to make my loss less than it was. It was of
no use, however, for the result of my counting and my ciphering
were precisely the same, showing a deficiency of six hundred dollars
and the brokerage. “O, if I could but get back my stock,” thought I,
“I would hold it till doomsday, before I would again sell it for a less
sum than it cost me.” That was an idle thought, for the money
having been borrowed, I had not the power to do as I wished.
Well, I found that complaining would do no good, and it was
plain that I could not recover my losses by sitting down and doing
nothing; beside, it was very unlike a bold operator—a Napoleon of
the exchange—to be disheartened by the first reverse or two; so I
determined, as there was now a strong probability of an immediate
advance of prices, to get back my Ohio stock at par. I was too late in
deciding by a day, and was obliged to give one per cent. premium.
That trifling difference, however, I did not regard; for what was one
per cent., more or less, to a man who was sure of making ten of
them?
I now felt certain that I had hit the nail on the head. “Rem tetigi
acu,” said I; and what made me more confident of success was the
fact, that the newspapers, disagreeing upon almost every other
subject, were agreed upon one point, namely, that, in consequence
Te Deus?—ut semper gaudes illudere rebus
Humanis!
exclaimed Horace; and depend upon it, if stocks were the subject of
traffic in Rome, he had just been nicked when he wrote that
passage. Most courteous reader, I was doomed to suffer another
grievous disappointment; stocks took a different turn from what I
had expected. The storm cleared away, and the panic abated. The
sun again shone out bright, and smiles reappeared on the brokers’
faces. Prices had reached their lowest point, precisely at the moment
that I sold out mine, and instead of going down to ninety, as they
would have done had I continued to hold, they “rallied,” as the
saying is, and rose to par. I looked and felt blue, and counted over
my money again and again; I ciphered and calculated for half a
morning, in endeavoring to make my loss less than it was. It was of
no use, however, for the result of my counting and my ciphering
were precisely the same, showing a deficiency of six hundred dollars
and the brokerage. “O, if I could but get back my stock,” thought I,
“I would hold it till doomsday, before I would again sell it for a less
sum than it cost me.” That was an idle thought, for the money
having been borrowed, I had not the power to do as I wished.
Well, I found that complaining would do no good, and it was
plain that I could not recover my losses by sitting down and doing
nothing; beside, it was very unlike a bold operator—a Napoleon of
the exchange—to be disheartened by the first reverse or two; so I
determined, as there was now a strong probability of an immediate
advance of prices, to get back my Ohio stock at par. I was too late in
deciding by a day, and was obliged to give one per cent. premium.
That trifling difference, however, I did not regard; for what was one
per cent., more or less, to a man who was sure of making ten of
them?
I now felt certain that I had hit the nail on the head. “Rem tetigi
acu,” said I; and what made me more confident of success was the
fact, that the newspapers, disagreeing upon almost every other
subject, were agreed upon one point, namely, that, in consequence
of the “better feeling” that prevailed, stocks would certainly rise. I
believed them, having naturally a strong inclination to credit what I
see in print.
A good feeling unquestionably did exist at the time I bought, and
the prices of stocks were likewise very good; but, as usual, when the
time came in which I was compelled to sell, a very different feeling
seemed to be rife, and symptoms of another panic began to make
their appearance simultaneously with the approach of a storm. On
the day I sold out, everything was at sixes and sevens; the rain
came down in floods, the wind blew, and the whole army of brokers,
like a flock of sheep that had lost their shepherd, were again in the
greatest alarm and confusion. My poor stock, like the parting spirit of
Napoleon, went off in a whirlwind, at ninety-eight; and I went home
that day mad, and drenched with rain, (having mislaid my umbrella,)
and a loser of three hundred dollars more. I felt exceedingly bad—I
was disgusted.
The prospect of my going to Niagara was now unpromising; and
I prudently resolved to postpone the visit for another year at least.
Such a thing was not again to be thought of, till, in gambler’s
phrase, I should be on velvet, that is, have some winnings over and
above my capital; but so far from being on velvet, I was on the
sharpest kind of paving-stones, nay, figuratively speaking, I was on
spikes. I was now reduced to the point of struggling, not for victory,
but for safety; and I was like a general who, having abandoned all
hopes of conquest, would be too happy to save his own bacon, and
get safe home. My discouragement, however, was of short duration,
and with my reviving spirits, I resumed the hazardous business.
I made several other operations in what are technically called the
“fancies”—stocks that pay no dividends, and the value of which is
rather imaginary or fanciful, (whence their name, probably,) than
real. I had enough of good stocks—they had well-nigh ruined me;
and I resolved to try my luck among those that are good for nothing,
except to be bought and sold. Ill-fortune still pursued me. What with
stormy weather, increased demand for money, paragraphs
containing bad news from Washington, and flying reports of some
believed them, having naturally a strong inclination to credit what I
see in print.
A good feeling unquestionably did exist at the time I bought, and
the prices of stocks were likewise very good; but, as usual, when the
time came in which I was compelled to sell, a very different feeling
seemed to be rife, and symptoms of another panic began to make
their appearance simultaneously with the approach of a storm. On
the day I sold out, everything was at sixes and sevens; the rain
came down in floods, the wind blew, and the whole army of brokers,
like a flock of sheep that had lost their shepherd, were again in the
greatest alarm and confusion. My poor stock, like the parting spirit of
Napoleon, went off in a whirlwind, at ninety-eight; and I went home
that day mad, and drenched with rain, (having mislaid my umbrella,)
and a loser of three hundred dollars more. I felt exceedingly bad—I
was disgusted.
The prospect of my going to Niagara was now unpromising; and
I prudently resolved to postpone the visit for another year at least.
Such a thing was not again to be thought of, till, in gambler’s
phrase, I should be on velvet, that is, have some winnings over and
above my capital; but so far from being on velvet, I was on the
sharpest kind of paving-stones, nay, figuratively speaking, I was on
spikes. I was now reduced to the point of struggling, not for victory,
but for safety; and I was like a general who, having abandoned all
hopes of conquest, would be too happy to save his own bacon, and
get safe home. My discouragement, however, was of short duration,
and with my reviving spirits, I resumed the hazardous business.
I made several other operations in what are technically called the
“fancies”—stocks that pay no dividends, and the value of which is
rather imaginary or fanciful, (whence their name, probably,) than
real. I had enough of good stocks—they had well-nigh ruined me;
and I resolved to try my luck among those that are good for nothing,
except to be bought and sold. Ill-fortune still pursued me. What with
stormy weather, increased demand for money, paragraphs
containing bad news from Washington, and flying reports of some
diabolical measure contemplated by England, all my adventures
turned out unfavorably, and I was reduced in pocket to a very low
ebb. My little capital was on its last legs.
One day, almost in despair, I took up a newspaper, (it was the
Journal of Commerce,) and my eye alighted on a remark of the
editor’s to the effect that a stock-speculator should be in no hurry
either to buy or sell; but, waiting coolly and patiently for
opportunities, with his feet elevated upon a stove, he should always
buy when stocks are low, and sell out when they are high. I was
struck with amazement at the wisdom displayed in this advice, and
wondered why so obviously correct a course had not occurred to me
in my deep cogitations upon this subject. It was perfectly plain—a
child might see it—that if this recommendation were strictly
followed, success would crown my efforts; and I forthwith
determined to commence another career on this excellent and safe
principle. Failure was impossible. “Buy when they are low,” I
repeated, “and sell when they are high. How wonderful, yet, at the
same time, how simple!” I had all along been pursuing the wrong
track. My practice had been, whatever my intentions were, to buy
when they were high, and sell when they were low; and this had
been the result of a want of patience, and of too much precipitancy
in my purchases and sales. I was now in possession of a grand
secret, and that secret was to WAIT, BUY LOW AND SELL HIGH.
Well, I did wait, and that most patiently, for a fall of stocks—and
a fall at length occurred, a greater one than had been known for a
long time, and prices were depressed below what they had been in
several months. “Now,” thought I, “is the time to take down my feet
from the stove, and walk into the fancies;” whereupon I went into
Wall street, and borrowed a considerable sum for a fortnight,
pledging the stock as security, according to the modus operandi well
understood in that region. Every thing promised well; and I felt
encouraged, deeming it next to impossible that fortune should
always fight against me. I bought the stock very low, comparatively,
and went home to replace my feet upon the stove, and await
patiently another rise.
turned out unfavorably, and I was reduced in pocket to a very low
ebb. My little capital was on its last legs.
One day, almost in despair, I took up a newspaper, (it was the
Journal of Commerce,) and my eye alighted on a remark of the
editor’s to the effect that a stock-speculator should be in no hurry
either to buy or sell; but, waiting coolly and patiently for
opportunities, with his feet elevated upon a stove, he should always
buy when stocks are low, and sell out when they are high. I was
struck with amazement at the wisdom displayed in this advice, and
wondered why so obviously correct a course had not occurred to me
in my deep cogitations upon this subject. It was perfectly plain—a
child might see it—that if this recommendation were strictly
followed, success would crown my efforts; and I forthwith
determined to commence another career on this excellent and safe
principle. Failure was impossible. “Buy when they are low,” I
repeated, “and sell when they are high. How wonderful, yet, at the
same time, how simple!” I had all along been pursuing the wrong
track. My practice had been, whatever my intentions were, to buy
when they were high, and sell when they were low; and this had
been the result of a want of patience, and of too much precipitancy
in my purchases and sales. I was now in possession of a grand
secret, and that secret was to WAIT, BUY LOW AND SELL HIGH.
Well, I did wait, and that most patiently, for a fall of stocks—and
a fall at length occurred, a greater one than had been known for a
long time, and prices were depressed below what they had been in
several months. “Now,” thought I, “is the time to take down my feet
from the stove, and walk into the fancies;” whereupon I went into
Wall street, and borrowed a considerable sum for a fortnight,
pledging the stock as security, according to the modus operandi well
understood in that region. Every thing promised well; and I felt
encouraged, deeming it next to impossible that fortune should
always fight against me. I bought the stock very low, comparatively,
and went home to replace my feet upon the stove, and await
patiently another rise.
No rise, however, occurred within the fortnight that I was able to
hold my new acquisition. Prices moved, it is true, but they moved
the wrong way for me; they “advanced backward.” I thought when I
purchased, that they were low enough in all conscience; but it
appears there were lower depths still to which they were destined to
attain. I did not wait long enough. The principle on which I had
acted was a good one—the fault was in me.
A man falling from the roof of a house, would not reach the
ground more quickly than my stock tumbled to a point five per cent.
below what I had given. A new element had arisen to produce this
sudden, unlooked for, and extraordinary change. The Texas question
came upon the brokers like a thunderbolt, knocking every thing into
a cocked-hat; and the upshot was, that I sold my stock at a loss
which swept away the remainder of my capital, and left me as
penniless as a street-beggar.
This was the last of my operations; and thus the savings of
several years disappeared like dew on a summer’s morning. Nor is
that the worst feature of this unfortunate business; for the
excitement of speculation, the handling of large sums of money, the
high-wrought expectation of realising large profits in a short time,
have totally unfitted me for the labors by which I accumulated what
money I have lost. How can I go to work again on a mere salary,
two-thirds of which I must spend in support of my family, the
remainder being a petty sum only, which any lucky broker would
make on a clear morning at a single throw? I am ready to die
through pure vexation; but I’ll not leave the ground yet. I know a
friend who will lend me five hundred dollars, and by hypothecating
the stock I shall buy, I can borrow of Jack Little five thousand. Yes, I
must have one more chance—one more—and then, if fortune favors
me, as she always does the brave, (so the Latin grammar declares,)
I shall soon be on my feet again; but if she should continue to
frown, and disappoint my hopes, I will abandon speculation forever
—perhaps.
hold my new acquisition. Prices moved, it is true, but they moved
the wrong way for me; they “advanced backward.” I thought when I
purchased, that they were low enough in all conscience; but it
appears there were lower depths still to which they were destined to
attain. I did not wait long enough. The principle on which I had
acted was a good one—the fault was in me.
A man falling from the roof of a house, would not reach the
ground more quickly than my stock tumbled to a point five per cent.
below what I had given. A new element had arisen to produce this
sudden, unlooked for, and extraordinary change. The Texas question
came upon the brokers like a thunderbolt, knocking every thing into
a cocked-hat; and the upshot was, that I sold my stock at a loss
which swept away the remainder of my capital, and left me as
penniless as a street-beggar.
This was the last of my operations; and thus the savings of
several years disappeared like dew on a summer’s morning. Nor is
that the worst feature of this unfortunate business; for the
excitement of speculation, the handling of large sums of money, the
high-wrought expectation of realising large profits in a short time,
have totally unfitted me for the labors by which I accumulated what
money I have lost. How can I go to work again on a mere salary,
two-thirds of which I must spend in support of my family, the
remainder being a petty sum only, which any lucky broker would
make on a clear morning at a single throw? I am ready to die
through pure vexation; but I’ll not leave the ground yet. I know a
friend who will lend me five hundred dollars, and by hypothecating
the stock I shall buy, I can borrow of Jack Little five thousand. Yes, I
must have one more chance—one more—and then, if fortune favors
me, as she always does the brave, (so the Latin grammar declares,)
I shall soon be on my feet again; but if she should continue to
frown, and disappoint my hopes, I will abandon speculation forever
—perhaps.
Salvator Rosa, pinx. A.L. Dick, sc.
JACOB’S DREAM. GEN. XXVIII, 10, 11, 12.
JACOB’S DREAM. GEN. XXVIII, 10, 11, 12.
JACOB’S DREAM.
WITH AN ENGRAVING.
The Patriarch slept—and dreaming there appeared,
In the deep watches of the silent night,
A ladder, high from earth to heaven upreared,
Steadfast and firm, to his astonished sight:
And seraph angels thronged that thoroughfare,
Descending from the glorious realms above,
And thence returning, their bright robes to wear
In the pure presence of the God of love.
The Patriarch listened—and his Maker’s voice
Broke with soft music on his raptured ear.
Quelling his fears and bidding him rejoice
In the abundance of a Father’s care.
Wide as the earth shall Israel’s power extend,
Countless as ocean’s sands his issue be,
While all the nations to his rule shall bend,
And in his seed a rich salvation see.
The Patriarch from his wondrous dream awoke,
And knew that the Almighty Lord was there—
And where the Maker to the creature spoke,
Built him an altar sanctified with prayer.
So, when the Lord with tender care imparts
Unnumbered blessings to us, let us raise,
Like Israel’s Patriarch, in believing hearts,
Altars of love and thankfulness and praise.
WITH AN ENGRAVING.
The Patriarch slept—and dreaming there appeared,
In the deep watches of the silent night,
A ladder, high from earth to heaven upreared,
Steadfast and firm, to his astonished sight:
And seraph angels thronged that thoroughfare,
Descending from the glorious realms above,
And thence returning, their bright robes to wear
In the pure presence of the God of love.
The Patriarch listened—and his Maker’s voice
Broke with soft music on his raptured ear.
Quelling his fears and bidding him rejoice
In the abundance of a Father’s care.
Wide as the earth shall Israel’s power extend,
Countless as ocean’s sands his issue be,
While all the nations to his rule shall bend,
And in his seed a rich salvation see.
The Patriarch from his wondrous dream awoke,
And knew that the Almighty Lord was there—
And where the Maker to the creature spoke,
Built him an altar sanctified with prayer.
So, when the Lord with tender care imparts
Unnumbered blessings to us, let us raise,
Like Israel’s Patriarch, in believing hearts,
Altars of love and thankfulness and praise.
LOLAH LALANDE.
A PACKAGE FROM MY OLD WRITING-DESK.
———
BY ENNA DUVAL.
———
She can show art rules to astonish her.
How like the nimble winds, which play upon
The tender grass, yet press it not, or fly
Over the crystal face of smoothest streams,
Leaving no curl behind them.
She makes
Motion the god of every excellence,
And what the muses would with study find,
She teaches in her dancing——
To me
It must suffice only to say, ’tis she.
Beaumont & Fletcheê.
“Heêe I am again, dear Miss Enna,” said my darling, pretty friend,
Kate Wilson, to me the other morning.
I have already introduced Kate to you, dear reader; and if you
had looked into the deep wells of her beaming, bright eye as often
as I have, and heard the rich, gushing music of her laugh, you would
hail her approach, even though she did plunge unceremoniously into
your sanctum, and interrupt you in your studies, or your deep
divings into your imagination for something particularly clever, out of
which to form a “readable story” for “Mr. Graham’s next Monthly.”
I felt a little annoyed, I must admit, on the morning in question,
for I was very earnestly engaged—not in writing, dear reader; oh,
no! I spared you that one morning—but in looking over an old
writing-desk, that I had not opened for years. It was one that
belonged to my mother; and one part I had devoted to her
treasured gifts, in the other, for it is a large, capacious, old-fashioned
A PACKAGE FROM MY OLD WRITING-DESK.
———
BY ENNA DUVAL.
———
She can show art rules to astonish her.
How like the nimble winds, which play upon
The tender grass, yet press it not, or fly
Over the crystal face of smoothest streams,
Leaving no curl behind them.
She makes
Motion the god of every excellence,
And what the muses would with study find,
She teaches in her dancing——
To me
It must suffice only to say, ’tis she.
Beaumont & Fletcheê.
“Heêe I am again, dear Miss Enna,” said my darling, pretty friend,
Kate Wilson, to me the other morning.
I have already introduced Kate to you, dear reader; and if you
had looked into the deep wells of her beaming, bright eye as often
as I have, and heard the rich, gushing music of her laugh, you would
hail her approach, even though she did plunge unceremoniously into
your sanctum, and interrupt you in your studies, or your deep
divings into your imagination for something particularly clever, out of
which to form a “readable story” for “Mr. Graham’s next Monthly.”
I felt a little annoyed, I must admit, on the morning in question,
for I was very earnestly engaged—not in writing, dear reader; oh,
no! I spared you that one morning—but in looking over an old
writing-desk, that I had not opened for years. It was one that
belonged to my mother; and one part I had devoted to her
treasured gifts, in the other, for it is a large, capacious, old-fashioned
affair, not at all like the little rosewood, mother-of-pearl inlaid thing
which has usurped its place on my writing-table; in the other part I
have stored gifts, letters, and remembrances of my school-girl days;
and this part I was exploring as Kate entered. I had just been
sighing over a package, containing letters, a bracelet of hair, and a
faded bunch of flowers—mementoes of a dear friend, long since laid
in the cold grave, and was almost weeping over remembrances of
the past. To me that is the only sad thing in growing old. If those we
love could only live to cheer and comfort us, old age would have no
terrors. A single woman feels this particularly; for if a woman
marries, she forms new connections, and looks forward to a new life,
and new interests, in the future of her children; but “we poor old
maids” are oftentimes very lonely.
Brothers and sisters, and dear friends, will marry; and however
pretty, fascinating, and agreeable a woman may have been, there
comes a time when the little decided opinions and caprices that
were deemed so pretty and cunning at eighteen, are pronounced by
the saucy new-comers on life’s stage, “prim, old-maidish
whimsicalities;” and even the fathers and mothers, who had formerly
considered this same dear, single friend, the realization of womanly
perfection when she was the belle of their young days, they also are
often found, coinciding with their children in these saucy opinions.
Now, members of my dear sisterhood, let me give you a little advice.
True, I am but a new comer amongst you. I know I have not yet
seen fifty summers—I only own to thirty-five, and scarcely to that,
excepting when in company with those well “booked-up” on the
subject of my age—I have no gray hairs or wrinkles, and yet I have
experience; and my single-blessedness bids fair to be a happy state.
Seek companions amongst the young. I do not mean for you to
affect juvenile manners. Oh, heaven forbid! a youthful old-maid is, in
truth, ridiculous. But mingle with the young; sympathize with them;
cultivate their friendship and love; make your presence a sunshine to
them; be to them a friend, a confidant, and an adviser. Keep your
feelings, your heart, your spirit young—your mind, by pleasant, but
regular study, in a healthful state; in this way you will secure
which has usurped its place on my writing-table; in the other part I
have stored gifts, letters, and remembrances of my school-girl days;
and this part I was exploring as Kate entered. I had just been
sighing over a package, containing letters, a bracelet of hair, and a
faded bunch of flowers—mementoes of a dear friend, long since laid
in the cold grave, and was almost weeping over remembrances of
the past. To me that is the only sad thing in growing old. If those we
love could only live to cheer and comfort us, old age would have no
terrors. A single woman feels this particularly; for if a woman
marries, she forms new connections, and looks forward to a new life,
and new interests, in the future of her children; but “we poor old
maids” are oftentimes very lonely.
Brothers and sisters, and dear friends, will marry; and however
pretty, fascinating, and agreeable a woman may have been, there
comes a time when the little decided opinions and caprices that
were deemed so pretty and cunning at eighteen, are pronounced by
the saucy new-comers on life’s stage, “prim, old-maidish
whimsicalities;” and even the fathers and mothers, who had formerly
considered this same dear, single friend, the realization of womanly
perfection when she was the belle of their young days, they also are
often found, coinciding with their children in these saucy opinions.
Now, members of my dear sisterhood, let me give you a little advice.
True, I am but a new comer amongst you. I know I have not yet
seen fifty summers—I only own to thirty-five, and scarcely to that,
excepting when in company with those well “booked-up” on the
subject of my age—I have no gray hairs or wrinkles, and yet I have
experience; and my single-blessedness bids fair to be a happy state.
Seek companions amongst the young. I do not mean for you to
affect juvenile manners. Oh, heaven forbid! a youthful old-maid is, in
truth, ridiculous. But mingle with the young; sympathize with them;
cultivate their friendship and love; make your presence a sunshine to
them; be to them a friend, a confidant, and an adviser. Keep your
feelings, your heart, your spirit young—your mind, by pleasant, but
regular study, in a healthful state; in this way you will secure
happiness. Then, to escape ridicule—ah! that is the hardest task of
all—admit your age; it is the only safe way, believe me. Walk up to
the cannon’s mouth boldly. Show them you do not care any thing
about it, and the saucy opinions and laughs of these young ones will
be averted; and depend upon it they will flutter around you, love
you, and almost imagine you still retain the youthful charms and
agreeability with which your cotemporaries so kindly invested you. I
have found this plan successful, and have surrounded myself with a
troop of young things. With one who is a fanatic, a pretty devotee to
the divine study of sweet sounds, I practice music; and instead of
falling back upon the “music of my day,” I find beauties in the music
of her day. Mozart, Hayden, and Beethoven, Steibell, Clementi, and
Dessek, are now banished from my music-stand, and only cheer my
solitary hours, to make way for Bellini, Donizetti, and Auber,
Thalberg, Herz, and Litz. With another, a gentle, little, imaginative
creature, whose transparent cheek, and brilliant eye, warns us she is
not long to dwell with us, I read old poets. But of all my youthful
friends, there is not one among them who loves me better, or who is
more companionable to me, than dear Kate Wilson. I have known
her from her babyhood. I knew her mother before Kate was born;
true, I was a tiny girl when Kate’s mother was married. She is a
beautiful, rich belle, “petted, fêted, courted, and caressed;” and yet
she daily comes to her “dear Miss Enna,” as she calls me, as she did
in her little girl days, and cheers many an hour that would otherwise
be lonely. I find myself forgetting, when with her, as she so
flatteringly does, that I am no longer young; and I very much fear
Kate makes me a little too youthful in dress; but the darling,
bewitching creature, has such a saucy, decided way with her, that I
always yield to her wishes.
“What are you at?” she asked, as she closed the door; “looking
over an old writing-desk, as I live. What piles of letters and old
things—that is, indeed, delightful. Stores of love-letters, I’ll wager a
bright, golden guinea. Come, let me help you toss it over, and tell
me the love-history of each discarded one.”
all—admit your age; it is the only safe way, believe me. Walk up to
the cannon’s mouth boldly. Show them you do not care any thing
about it, and the saucy opinions and laughs of these young ones will
be averted; and depend upon it they will flutter around you, love
you, and almost imagine you still retain the youthful charms and
agreeability with which your cotemporaries so kindly invested you. I
have found this plan successful, and have surrounded myself with a
troop of young things. With one who is a fanatic, a pretty devotee to
the divine study of sweet sounds, I practice music; and instead of
falling back upon the “music of my day,” I find beauties in the music
of her day. Mozart, Hayden, and Beethoven, Steibell, Clementi, and
Dessek, are now banished from my music-stand, and only cheer my
solitary hours, to make way for Bellini, Donizetti, and Auber,
Thalberg, Herz, and Litz. With another, a gentle, little, imaginative
creature, whose transparent cheek, and brilliant eye, warns us she is
not long to dwell with us, I read old poets. But of all my youthful
friends, there is not one among them who loves me better, or who is
more companionable to me, than dear Kate Wilson. I have known
her from her babyhood. I knew her mother before Kate was born;
true, I was a tiny girl when Kate’s mother was married. She is a
beautiful, rich belle, “petted, fêted, courted, and caressed;” and yet
she daily comes to her “dear Miss Enna,” as she calls me, as she did
in her little girl days, and cheers many an hour that would otherwise
be lonely. I find myself forgetting, when with her, as she so
flatteringly does, that I am no longer young; and I very much fear
Kate makes me a little too youthful in dress; but the darling,
bewitching creature, has such a saucy, decided way with her, that I
always yield to her wishes.
“What are you at?” she asked, as she closed the door; “looking
over an old writing-desk, as I live. What piles of letters and old
things—that is, indeed, delightful. Stores of love-letters, I’ll wager a
bright, golden guinea. Come, let me help you toss it over, and tell
me the love-history of each discarded one.”
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knowledge seekers. We believe that every book holds a new world,
offering opportunities for learning, discovery, and personal growth.
That’s why we are dedicated to bringing you a diverse collection of
books, ranging from classic literature and specialized publications to
self-development guides and children's books.
More than just a book-buying platform, we strive to be a bridge
connecting you with timeless cultural and intellectual values. With an
elegant, user-friendly interface and a smart search system, you can
quickly find the books that best suit your interests. Additionally,
our special promotions and home delivery services help you save time
and fully enjoy the joy of reading.
Join us on a journey of knowledge exploration, passion nurturing, and
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