Induced pluripotent stem cells

1. INDUCED PLURIPOTENT
STEM CELLS
Presented by:
VIDUR BHATIA
M. Sc. (F), KUK.

2. ContentsContents
IntroductionIntroduction
HistoryHistory
Reprogramming of Reprogramming of
somatic cellssomatic cells
What are iPSCsWhat are iPSCs
Genes responsibleGenes responsible
ProductionProduction
first generationfirst generation
Second generationSecond generation
Human iPSCsHuman iPSCs
ComplicationsComplications
Identity to natural Identity to natural
pluripotent stem cellspluripotent stem cells
Applications Applications
ConclusionsConclusions
ReferencesReferences

3. Introduction
 Most of the cells of a multicellular organism become
more and more restricted to specific cell lineages.
For the treatment of many genetic diseases Human
embryonic stem cells can be used , But due to some
ethics we can’t use embryo for this purpose.
 To avoid this problem artificially induced pluripotent
stem cells came in picture, which can be created from
normal somatic cells by the ectopic expression of some
genes which are responsible for the pluripotency.

4. History History
First generated by Shinya Yamanaka et al. First generated by Shinya Yamanaka et al.
At Kyoto in Japan in 2006.; At Kyoto in Japan in 2006.;
Second generated in mice in 2006 by same group.Second generated in mice in 2006 by same group.
Alexender Meissner showed that induction of Alexender Meissner showed that induction of
pluripotency is a slow and gradual process, 2008.pluripotency is a slow and gradual process, 2008.
Yang Chao showed that p53 siRNA and UTF1 Yang Chao showed that p53 siRNA and UTF1
enhances the efficiency of pluripotency, in enhances the efficiency of pluripotency, in
Nov.2008.Nov.2008.

5. Conti…..Conti…..
Cesor A. Sommer used a single lentivirus for all the Cesor A. Sommer used a single lentivirus for all the
genes required pluripotency in 2008genes required pluripotency in 2008
Yomiuri Shimbun has created the mouse kidney by Yomiuri Shimbun has created the mouse kidney by
the use of iPS cells 10 march,2009.the use of iPS cells 10 march,2009.
James A Thomson made the use of plasmid for James A Thomson made the use of plasmid for
pluripotency induction 26 march 2009.pluripotency induction 26 march 2009.

6. Embryonic Stem cells
Totipotent
Pluripotent
Multipotent
Unipotent

7. Reprogramming of somatic cells to ES Reprogramming of somatic cells to ES
cellscells
Somatic cell nuclear transfer
Cell fusion
Treatment with the extract
of the pluripotent stem cells
Stable expression of
defined factors
(Cowan et al.,2005)
(Wilmut et al.,1997)
(Takahashi and Yamanaka, 2006.)

9. Cell Fusion TechnologyCell Fusion Technology

10. Treatment with the extract of the Treatment with the extract of the
pluripotent cellspluripotent cells
Permeabilised cells are exposed to cell-free extract of Permeabilised cells are exposed to cell-free extract of
pluripotent cells.pluripotent cells.
LimitationsLimitations :- :-
a. Limited experience with primary cells. a. Limited experience with primary cells.
b. Reprogrammed cells regain only some ofb. Reprogrammed cells regain only some of
the properties of pluripotent cellthe properties of pluripotent cell

11. Ecat1
Dppa5(Esg1)
Fbox15
Nanog
Eras
Dnmt31
Ecat8
Gdf3
Sox15
Dppa4
Dppa2
Fthl17
Sall4
Oct3/4
Sox2
Rex1
Utf1
Tcl1
Dppa3
Klf4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
SymbolNo.
Twenty four candidate
genes play pivotal roles in
the maintenance of ES cell
identity base on their
hypothesis.
(Takahashi and Yamanaka,2006.)
Myb,
Kit,
Gdf3,
Esrrb
21
22
23
24
Stable expression of Stable expression of
defined factorsdefined factors

12. Exogenous expression of Oct3/4, Sox2, Exogenous expression of Oct3/4, Sox2,
Klf4 and c-Myc and factors are essentiallyKlf4 and c-Myc and factors are essentially
required.required.
But large number of factors are also required But large number of factors are also required
for pluripotency …..for pluripotency …..
(Takahashi and Yamanaka, August 25, 2006.)

13. iPSCsiPSCs, are a type of , are a type of pluripotentpluripotent stem cellstem cell
artificially derived from a non-artificially derived from a non-pluripotentpluripotent cell, cell,
typically an adult typically an adult somatic cellsomatic cell, by inducing a , by inducing a
"forced" expression of certain "forced" expression of certain genesgenes..
first produced in 2006 from mouse cells and in first produced in 2006 from mouse cells and in
2007 from human cells2007 from human cells
WHAT ARE iPSCs ?

14. Genes responsible for pluripotencyGenes responsible for pluripotency
Group 1Group 1
ES cell-Specific transcription factorsES cell-Specific transcription factors
Essential for pluripotency in ES cell & early embryosEssential for pluripotency in ES cell & early embryos
Oct¾, Sox2, Nanog…Oct¾, Sox2, Nanog…
Group 2Group 2
(Proto-oncogene's)(Proto-oncogene's)
Important for proliferation of ES cells, but not in early embryosImportant for proliferation of ES cells, but not in early embryos
TCL1, Stat3, c-Myc, ERas, Klf4…TCL1, Stat3, c-Myc, ERas, Klf4…
Group 3Group 3
Less famousLess famous
Specifically expressed in ES cellSpecifically expressed in ES cell
But less defined functionBut less defined function
ECAT1, Esg1,Fbx15, …ECAT1, Esg1,Fbx15, …
(Takahashi and Yamanaka, 2006.)

15. Oct3/4:Oct3/4:
Involve in the maintenance of self renewal of pluripotent Involve in the maintenance of self renewal of pluripotent
cells.cells.
Repression in ES cells leads to the formation of Repression in ES cells leads to the formation of
trophoectoderm.trophoectoderm.
Overexpression leads to the formation of various lineages Overexpression leads to the formation of various lineages
includingincluding
primitive endodermprimitive endoderm..
Sox2:Sox2:
Essential for embryonic developmentEssential for embryonic development
Downregulation by siRNA silencing leads to the Downregulation by siRNA silencing leads to the
differentiation of cell in murine ES cellsdifferentiation of cell in murine ES cells..

16. Klf4Klf4
Klf4 repress p53 directly
p53 protein suppress Nanog during ES cell differentiation
Klf4 contributes to activation of Nanog and other ES cell-specific
genes
(Rowland et al., 2005; Lin et al., 2004)
Klf4 acts as an inhibitor of c-Myc-induced apoptosis through the
repression of p53
(Zindy et al., 1998)
Klf4 activates p21CIP1, thereby suppressing cell proliferation .This
antiproliferation function of Klf4 inhibited by c-Myc, which
suppresses the expression of p21CIP1
(Zhang et al., 2000; Seoane et al., 2002)

17. NanogNanog:: In embryonic stem cells, Nanog, along In embryonic stem cells, Nanog, along
with Oct-3/4 and Sox2, is necessary in with Oct-3/4 and Sox2, is necessary in
promoting pluripotency. promoting pluripotency.
LIN28LIN28:: LIN28 is an LIN28 is an mRNA binding proteinmRNA binding protein
expressed in expressed in embryonic stem cellsembryonic stem cells and and
embryonic carcinoma cellsembryonic carcinoma cells associated with associated with
differentiation and proliferation. (Thomson et differentiation and proliferation. (Thomson et
al.) al.)

18. Production of iPSCsProduction of iPSCs
Typically derived by Typically derived by transfectiontransfection of certain stem of certain stem
cell-associated genes into non-pluripotent cells, cell-associated genes into non-pluripotent cells,
such as adult such as adult fibroblastsfibroblasts. .
Transfection is typically achieved through viral Transfection is typically achieved through viral
vectors, such as vectors, such as retrovirusesretroviruses..
After 3–4 weeks, small numbers of transfected cells After 3–4 weeks, small numbers of transfected cells
begin to become morphologically and begin to become morphologically and
biochemically similar to pluripotent stem cells, and biochemically similar to pluripotent stem cells, and
are typically isolated through morphological are typically isolated through morphological
selection, doubling time, or through a selection, doubling time, or through a
reporter genereporter gene and antibiotic selection. and antibiotic selection.

19. (1)Isolate and culture donor
cells. (2)Transfect stem cell-
associated genes into the
cells by viral vectors.
(3)Harvest and culture the
cells according to ES cell
culture, (4)A small subset of
the transfected cells become
iPS cells and generate ES-
like colonies.

20. First generationFirst generation
First generated by First generated by Shinya YamanakaShinya Yamanaka's team at 's team at
Kyoto UniversityKyoto University, Japan in 2006., Japan in 2006.
four key pluripotency genes essential for the four key pluripotency genes essential for the
production of pluripotent stem cells were used; production of pluripotent stem cells were used;
Oct-3/4, Sox2, c-Oct-3/4, Sox2, c-MycMyc, and , and Klf4Klf4. .
RetrovirusesRetroviruses was used to transfect mouse was used to transfect mouse
fibroblasts. fibroblasts.
Cells were isolated by antibiotic selection of Cells were isolated by antibiotic selection of
Fbx15Fbx15+ cells. + cells.

21. Limitations:-Limitations:-
This iPS line showed DNA methylation errors This iPS line showed DNA methylation errors
compared to original patterns in ESC lines and compared to original patterns in ESC lines and
failed to produce viable failed to produce viable chimeraschimeras if injected into if injected into
developing embryosdeveloping embryos

22. Second generation in miceSecond generation in mice
In June 2007, by the same group.In June 2007, by the same group.
These cell lines were also derived from mouse These cell lines were also derived from mouse
fibroblast by retroviral mediated reactivation of the fibroblast by retroviral mediated reactivation of the
same four endogenous pluripotent factors, but same four endogenous pluripotent factors, but
Instead of Fbx15, they used Instead of Fbx15, they used NanogNanog which is an which is an
important gene in ESCs. important gene in ESCs.
DNA methylation patterns and producing viable DNA methylation patterns and producing viable
chimeras (and thereby contributing to subsequent chimeras (and thereby contributing to subsequent
germ-line production) indicated that Nanog is a germ-line production) indicated that Nanog is a
major determinant of cellular pluripotency.major determinant of cellular pluripotency.

23. Limitations:-Limitations:-
One of the four genes used (namely, c-Myc) is One of the four genes used (namely, c-Myc) is
oncogeniconcogenic, and 20% of the chimeric mice , and 20% of the chimeric mice
developed cancer. developed cancer.
(In a later study, Yamanaka reported that one (In a later study, Yamanaka reported that one
can create iPSCs even without c-Myc, although can create iPSCs even without c-Myc, although
process takes longer and is not as efficient, but process takes longer and is not as efficient, but
the resulting chimeras didn't develop cancer).the resulting chimeras didn't develop cancer).

24. Human induced pluripotent stem Human induced pluripotent stem
cellscells
Produced in November 2007. Produced in November 2007.
With the same principle used earlier in mouse With the same principle used earlier in mouse
models, Yamanaka had successfully transformed models, Yamanaka had successfully transformed
human fibroblasts into pluripotent stem cells using human fibroblasts into pluripotent stem cells using
the same four pivotal genes: Oct3/4, Sox2, Klf4, the same four pivotal genes: Oct3/4, Sox2, Klf4,
and c-Myc with a and c-Myc with a retroviralretroviral system. system.

25. Pluripotency induction is a slow and Pluripotency induction is a slow and
gradual processgradual process
After transfecting the cells with all of the four factors After transfecting the cells with all of the four factors
(Sox2,OCct3/4,Klf4 and c-Myc) initially only(Sox2,OCct3/4,Klf4 and c-Myc) initially only
eight colonies were picked at 11th day and ten eight colonies were picked at 11th day and ten
colonies on 16th day.colonies on 16th day.
Only one out of eight colonies from 11 day and four Only one out of eight colonies from 11 day and four
colonies from ten colonies from 16 day old colonies colonies from ten colonies from 16 day old colonies
gave rise the ES like cells.gave rise the ES like cells.
(Alexender Meissner et al.;2007)

26. ComplicationsComplications
Because of viral transfection systems,Because of viral transfection systems, the created the created
cells might be cells might be prone to form tumors.prone to form tumors.
However However Konrad HochedlingerKonrad Hochedlinger and his Harvard and his Harvard
University research team successfully used an University research team successfully used an
adenovirusadenovirus to transport the requisite four genes to transport the requisite four genes
into the DNA of skin and liver cells of mice.into the DNA of skin and liver cells of mice.
Since the adenovirus does not combine any of Since the adenovirus does not combine any of
its own genes with the targeted host, the danger its own genes with the targeted host, the danger
of creating tumors is eliminated.of creating tumors is eliminated.

27. IdentityIdentity
The generated iPSCs were remarkably similar to The generated iPSCs were remarkably similar to
naturally-isolated pluripotent stem cellsnaturally-isolated pluripotent stem cells

Cellular biological properties: Cellular biological properties:
MorphologyMorphology, , Growth propertiesGrowth properties, , Stem Cell MarkersStem Cell Markers, ,
Stem Cell GenesStem Cell Genes, , Telomerase Activity:Telomerase Activity:
Pluripotency of iPSCs :Pluripotency of iPSCs :
Neural Differentiation, Cardiac DifferentiationNeural Differentiation, Cardiac Differentiation, ,
Teratoma FormationTeratoma Formation, , Embryoid Body, Embryoid Body,

28. Conti…..Conti…..
Epigenetic reprogramming :Epigenetic reprogramming :
Promoter DemethylationPromoter Demethylation,,Histone Histone
demethylationdemethylation etc. etc.

29. APPLICATIONS APPLICATIONS

30. iPS Cells - the Wave of Future
iPSC regarded as holy grail stem cell research
Studying disease models in vitro
Drug screening
Toxicological testing of new drugs
Generating patient specific & disease specific pleuripotent
stem cells
Allow unprecedented access to all stages of human biology
Studying development & function of human tissue
Regenerative medicine

31. iPS CELLS TO CURE SC ANEAMIAiPS CELLS TO CURE SC ANEAMIA

32. Cell replacement therapy seems particularly suitable for Cell replacement therapy seems particularly suitable for
Parkinson’s disease,Parkinson’s disease,
A common neurodegenerative disease caused by loss of A common neurodegenerative disease caused by loss of
midbrain dopamine neurons . Transplantation of fetal midbrain midbrain dopamine neurons . Transplantation of fetal midbrain
cells has been shown to restore dopamine function in animal cells has been shown to restore dopamine function in animal
models and in human patients . models and in human patients .
((Parish CL, Parish CL, et alet al. (2008)). (2008))
iPS can be used for treatment of
Parkinson’s disease

33. Mouse kidneys created using iPS cells
A team of scientists has successfully used
induced pluripotent stem (iPS) cells to create
kidneys inside a mouse whose parents were
genetically engineered so their offspring
would not be born with the organ.
(Hiromitsu Nakuchi; Mar. 10, 2009))

34. Stem cells scientists at UCLA showed for the first Stem cells scientists at UCLA showed for the first
time that human induced pluripotent stem (iPS) cells time that human induced pluripotent stem (iPS) cells
can be differentiated into electrically active motor can be differentiated into electrically active motor
neurons, a discovery that may aid in studying and neurons, a discovery that may aid in studying and
treating neurological disorders.treating neurological disorders.
The motor neurons derived from the iPS cells The motor neurons derived from the iPS cells
appeared to be similar in function and efficiency to appeared to be similar in function and efficiency to
those derived from human embryonic stem cells, those derived from human embryonic stem cells,
although further testing needs to be done to confirm although further testing needs to be done to confirm
that. that.
iPSc can be used to create
Electrically Active Neuron
(Michael Scott on February - 25 – 2009)

35. Somatic cells can be used to generate Somatic cells can be used to generate
the the β-cells-cells
Pancreatic Exocrine cells can be converted into
β-cells closely related to the islet β-cells.
Can be used to cure diabetes.
(Qiao Zhou et al.,2008)

36. ConclusionConclusion
SCNT and cell fusion may use to produce the SCNT and cell fusion may use to produce the
pluripotent cells but can be used only for pluripotent cells but can be used only for
animals, these processes can’t be shifted to animals, these processes can’t be shifted to
human beings.human beings.
iPS may be the answer of the all question of iPS may be the answer of the all question of
ethics and may avoid the problem of transplant ethics and may avoid the problem of transplant
rejection. In future these technique may help us rejection. In future these technique may help us
in the field genetic study, research and to fight in the field genetic study, research and to fight
against disease. against disease.

37.  Takahashi & K. Yamanaka; Induction of
pluripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors, Cell
2006;126:663–676.
 Yamanaka S. & et al. ; Generation of germline-
competent induced pluripotent stem cells, Nature
2007;448:313-317.
 Maherali N & et. al.; Directly reprogrammed
fibroblasts show global epigenetic remodeling and
widespread tissue contribution, Cell Stem Cell
2007;1:55–70 .
References

38.  Stadtfeld M., Nagaya M., Utikal J., Weir G.,
Hochedlinger K.Induced Pluripotent Stem
Cells Generated without Viral Integration.
Science 2008 Sep. 25. pp.212-220.
 Okita K., Nakagawa M., Hyenjong H.,
Ichisaka T,Yamanaka S. Generation of
Mouse Induced Pluripotent Stem Cells
Without Viral Vectors, Science, 2008 Oct 9. pp.167-178.

39. Thank youThank you