Subcloning, Spontaneous and controlled differentiation of hESCs.pdf

Subcloning, Spontaneous and controlled differentiation of HSC

1. DISCOVER . LEARN . EMPOWER

Lecture 2.1.2-2.1.4 Subcloning, Spontaneous and
controlled differentiation of hESCs
UNIVERSITY INSTITUTE OF BIOTECHNOLOGY

Master of Sciences (Biotechnology)
Course Name: Advances in Stem cell Technology
Course Code: 24BTT-725
Master Course Coordinator
Dr. Gajendra B. Singh
Professor

2. COURSE OBJECTIVE

•The course will provide students with knowledge of wide-ranging
topics related to stem cell and biotechnology, including: a brief
history of the field, cellular mechanisms and the political and ethical
issues surrounding the stem cell debate
2

3. COURSE OUTCOME
CO1 Recall key concepts in stem cell biology, including historical perspectives,
cellular features, and classification.
CO2 Understand the differences between embryonic and adult stem cells and
their relevance in research and therapy.
CO3 Apply knowledge of stem cell culture techniques to cultivate and differentiate
human embryonic stem cells in laboratory settings.
CO4 Analyze ethical considerations and regulatory issues surrounding stem cell
research and therapeutic applications.
3

4. SYLLABUS

5. Lecture No. Title
Unit- I
Chapter 1.1: Stem cells: Research history and sources
Lecture 1
Introduction to stem cell biology and stem cell
classification
Lecture 2, 3
Stem cell research and therapy- a historical
overview: 1, 2
Lecture 4, 5 Stem cell molecular markers: 1, 2
Lecture 6 Stem cell sorting
Lecture 7 Various sources of stem cells
Lecture 8 Stem cells and their developmental potential
Chapter 1.2: Stem cells: potency and types
Lecture 9 Stem cell division and differentiation
Lecture 10, 11 Pluripotent stem cells: 1, 2
Lecture 12, 13 Tissue specific stem cells: 1, 2
Lecture 14,15 Stem Cell Niches: 1, 2
Unit- II
Chapter 2.1: Human Embryonic Stem Cells and Culturing
Lecture 16 Characterization of human embryonic stem cells
Lecture 17 Subcloning of human embryonic stem cells
Lecture 18, 19
Spontaneous and controlled differentiation of
human embryonic stem cells: 1, 2
Lecture 20 In vitro differentiation of embryonic stem cells
Lecture 21 Feeder free culture of human embryonic stem cells
Lecture 22, 23
Reprogramming of somatic cells to a pluripotent
state: 1, 2
Chapter 2.2: Molecular bases of pluripotency
Lecture 24 Molecular bases of pluripotency
Lecture 25-28
Role of various signal transduction cascades in
maintaining pluripotency: 1, 2, 3, 4
Lecture 29 Mechanisms of stem cell selfrenewal
Lecture 30
Role of the embryonic stem cell environment in
maintaining pluripotency
Unit- III
Chapter 3.1: Ethical issues in stem cell research and manufacturing of stem
cells
Lecture 31, 32 Ethical considerations in stem cell research: 1, 2
Lecture 33 Pre-clinical regulatory considerations
Lecture 34, 35
Manufacturing and characterization issues pertaining to
stem cell products: 1, 2
Chapter 3.2: Therapeutic applications of stem cells
Lecture 36 Therapeutic applications of stem cells: General
Lecture 37 Therapeutic applications of stem cells: Leukaemia
Lecture 38, 39
Therapeutic applications of stem cells: Neurological
diseases part 1, 2
Lecture 40 Therapeutic applications of stem cells: Injuries/RA
Lecture 41, 42
Therapeutic applications of stem cells: Heart disease
part 1, 2
Lecture 43, 44 Therapeutic applications of stem cells: Diabetes part 1, 2
Lecture 45 Genetically engineered stem cells
LECTURE PLAN

6. Subcloning of hESCs
6
•hESC lines are derived from the ICM, a clump of pluripotent cells which may not
represent a homogenous cell population. Therefore, there is the possibility that the
pluripotency of the hESC lines reflects a collection of several distinct committed
multipotential cell types.
•In order to eliminate this possibility, parental lines must be single-cell cloned, and the
single-cell clone pluripotency has to be demonstrated.
•Several culture media have been tested to clone the first parental hESC lines: medium
supplemented with either FBS (Fetal bovine serum) or serum replacement (Serum
Replacement (KnockOut™ SR) is a more defined, FBS-free medium supplement that
supports the growth of pluripotent stem cells (PSCs) cultured on fibroblast feeder cells.),
either with or without human recombinant bFGF (basic fibroblast growth factor).

7. Subcloning of hESCs
7
•highest cloning efficiency has been obtained when a medium supplemented with 20%
serum replacement and bFGF is used.
•Resultant clones demonstrated hESC features, including pluripotency.
•Single-cell clones may have further advantages over parental lines.
oFirstly, they are easier to grow and manipulate in comparison to their parental lines.
oSecondly, research models based on gene knockout or targeted recombination
require homogeneous cell populations starting with a single cell harboring the
desired genotype.

8. Disadvantage of Subcloning of hESCs
8
•The main disadvantage of this strategy is the relatively low cloning efficiency (1%), this
is partly overcome by the prolonged culture abilities of the resultant clone, which
enable extended periods of research.

•Any future applications of hESCs for therapeutic purposes depend on their karyotypic
stability.

•Random karyotypic instability incidents were reported for hESCs. In the case 17q
trisomy (telomeric duplication 17q) and 12 chromosome trisomy (12p duplication), If
the problem is identified, single-cell cloning of clones harboring normal karyotypes may
prevent the loss of cell lines.

9. Spontaneous Differentiation of hESCs
9
Teratomas
•Differentiation of hESCs occurs once they are removed from culture conditions which
support their pluripotency and self-renewal. In vivo spontaneous differentiation can be
achieved by the injection of undifferentiated hESCs into immuno-compromised mice.
•This results in the formation of a teratoma, a benign tumor containing complex
structures and composed of differentiated cell types representing derivatives of the
three major embryonic lineages.
•A more recent study suggested that a teratoma generated from hESCs may be utilized as
an in vivo human microenviroment model for the study of tumor invasion, angiogenesis
and metastasis.
•It should be noted that the capability of undifferentiated hESCs to form teratomas is a
risk factor to consider when applying hESC-derivatives to cellular therapy.

10. Spontaneous Differentiation of hESCs
10
Embryoid bodies (EBs) (3D aggregates of pluripotent stem cells.)
(Another similar term organoid is a miniaturized and simplified version of an organ produced in vitro in three
dimensions that shows realistic micro-anatomy.)
Formation and growth
•After removal from their undifferentiated supporting culture conditions, the differentiation of
hESCs in vitro can be activated in two main ways:
•two-dimensional (2-D) culturing on a differentiation-inducing layer (matrix or culture), or
•formation of embryo-like aggregates in suspension, termed embryoid bodies (EBs).
Human EB (hEB) formation can be achieved by culturing hESC aggregates in suspension (i.e. using
a non-adherent dish) or within hanging-drops, followed by their cultivation in Petri dishes.
Forty eight hours after initiation of cellular aggregation, hEBs complete the agglomeration
process, which results in the formation of simple EBs by day 3, followed by cavitated EBs after 7–
10 days, and cystic EBs after two weeks of differentiation.

11. Spontaneous Differentiation of hESCs
11
Ref: Amit, Michal & Gerecht-Nir, Sharon & Itskovitz-Eldor, Joseph. (2005). Culture, Subcloning, Spontaneous and Controlled Differentiation of Human Embryonic Stem Cells.
10.1142/9789812569370_0006.

12. 12
Just For Information

13. 13
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418120/
Methods to form embryoid bodies

•The suspension culture in the petri dish is the simplest
method to make a large amount of EBs at the same time.
Generally, ESCs are harvested from a feeder layer and
counted and suspended inside a non-coated petri dish to
allow them to aggregate and form EBs.
•Hanging drop is a method to generate homogenous-size
EBs by suspending single cells in drops, which hang on the
cover of a petri dish, and each hanging drop contains a
certain number of ESCs.
•The ESCs aggregate at the bottom of droplets upon being
affected by gravity.
•The hanging drop method successfully prevents EBs from
attaching to the surface of the container and controls the
size and the shape of EBs.
•However, this method has disadvantage that it cannot
produce a high number of EBs at the same time because
each drop only contains a few EBs.

14. 14
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418120/
Methods to form embryoid bodies
•Microwell/capsule methods were adopted to
control the size, shape, and homogeneity of
EBs. Round-bottom 96-well plates have been
used to form EBs because these plates were
found to be better than the flat-bottom 96-
well plates for the formation of EBs.
•Polyacrylamide hydrogel made microwells
good for culturing EBs in 3D.
•Polyacrylamide hydrogel microwells are stable
in an EB medium, and the surface of
polyacrylamide hydrogel can prevent ESCs
from attaching to the surface of the well.
•In addition, they can create a hydrated niche
condition for ESC differentiation.

15. Spontaneous Differentiation of hESCs
15
Embryoid bodies (EBs)
Lineage differentiation
•With the use of EB formation, hESC differentiation into several lineages has been extensively
studied.
•Cardiomyocytes: Seeding hEBs on an adherent substrate may result in the appearance of
beating areas. These beating areas display structural and functional properties of early-stage
cardiomyocytes and exhibit properties of early-stage cardiac phenotype.
•Hematopoietic: 15-day-old spontaneously differentiated hEBs were shown to express low levels
of CD45 (1.4% ± 0.7%). Most of these cells coexpress CD34 (1.2% ± 0.5%), a phenotype similar
to the first definitive hematopoietic cells detected within the wall of the dorsal aorta of human
embryos.
•Vasculature: During hEB differentiation, an increase in expression of several endothelial cell-
specific genes and the development of extensive vasculature-resembling structures within them
have been reported. Sorted CD31+ cells (2%) were shown to possess embryonic endothelial cell
features as well as a potential for microvessel formation both in vitro and in vivo.

16. Controlled Directed Differentiation of hESCs
16
•The spontaneously differentiated EBs are not sufficient to serve as a source of differentiated
human cells for clinical therapy. This is because of the vague nature of tissue differentiation, the
non-homogenous cell population and the rather small number of specific cells that can be
generated by this method.
•To overcome this problem, methods for enrichment, selection, or direct differentiation to
specific lineages were developed.
•Utilizing specific growth factor combinations and cell–cell induction systems, can enhance
differentiation of hEBs into a desired lineage.
•The ability of one group of cells to affect the fate of another is called “induction.” The cells that
produce the signals are referred to as “inducing cells,” whereas the receiving cells are termed
“responders”.
•The ability of cells to respond to the inducers, referred to as “competence”, usually reflects the
presence of a receptor at the top of a pathway that regulates the expression of specific
transcription factors in the responding cells.
•Eg. Insulin-producing cells can be generated from hESCs through Controlled Differentiation,
HESCs could be directed to a CD34+ progenitor population etc.

17. 17
Directed differentiation of human embryonic stem cells (hESCs) derived from blastocyst and
human pluripotent stem cells (hPSC) to β cell like cells
Neurogenin-3 pro-endocrine TF
Maf family of TF: regulates
pancreatic beta cell-specific
expression of the insulin gene
PDX1 pancreatic and duodenal homeobox 1,
also known as insulin promoter factor 1
NK6 Homeobox 1 TF
PTF1A pancreas associated transcription factor 1a
SOX9: SRY-Box Transcription Factor 9
FOXA2: Forkhead box protein A2 plays an important
role during development, in mature tissues and,
when dysregulated or mutated.
https://doi.org/10.1007/s10529-022-03247-w
SOX17: SRY-Box Transcription Factor 17 regulator of
human primordial germ cells-like cells specification

18. ASSESSMENT MODEL
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19. REFERENCES
•Amit, M., Gerecht-Nir, S., & Itskovitz-Eldor, J. (2005). Culture, subcloning,
spontaneous and controlled differentiation of human embryonic stem cells. Stem
Cells: From Bench to Bedside, 98.
•Amit, M., Segev, H., Manor, D., & Itskovitz-Eldor, J. (2003). Subcloning and
alternative methods for the derivation and culture of human embryonic stem
cells. In Human embryonic stem cells (pp. 127-141). Humana Press, Totowa, NJ.
•https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040128/
•https://www.reprocell.com/what-is-alvetex-i70


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20. TEXT/REFERENCE BOOKS
Text Books
•Slack, Jonathan MW. The science of stem cells. John Wiley & Sons, 2018.
•Lanza, Robert, et al., eds. Essentials of stem cell biology. Elsevier, 2013.
Reference Books
•Sell, Stewart, ed. Stem cells handbook. Springer Science & Business Media, 2013.
•Lanza, Robert, et al., eds. Handbook of Stem Cells, Two-Volume Set: Volume 1-
Embryonic Stem Cells; Volume 2-Adult & Fetal Stem Cells. Elsevier, 2004.

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21. THANK YOU
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