2. Introduction
Stem Cells
Ability to self renew, to make
exact copies of itself through cell
division.
Ability to develop into different
types of mature cells.
Image adopted from:- http://clearlyexplained.com/nature/life/cells/stemcelltree.gif

3. Stem Cells
Have the ability to specialize in all postembryonic tissues and organs, and
also extra embryonic tissues. E.g.- zygote
Capable of giving rise to
most tissues of an
organism. E.g.- ES cells
Multipotent
Stem cells that can differentiate into many family of Can only differentiate into single type of cell
cells, E.g.– Hematopoietic stem cells but have the property of self-renewal, E.g.-
mucus membrane cells and skin stem cell
Adopted from:- http://www.wikipedia.org

5. Three methods of inducing pluripotency in
somatic cells.
©2008 by The Royal Society Adopted from:- Yamanaka S Phil. Trans. R. Soc. B 2008;363:2079-2087

6. Induced pluripotent stem cells (iPSCs) are
typically adult cells that have been
genetically reprogrammed to an embryonic
stem cell–like state by being forced to
express genes and factors important for
maintaining the defining properties of
embryonic stem cells.

7. iPSc-made from mouse-> lead
to the creation of beating cardiomyocyte, skin
and almost every kind of cell you can
imagine.
iPSc made from human-> also
lead to the creation of various cell types in
body, in addition even produced gamete!
iPSc created live organism->
“ Xiao Xiao / Tiny”

8. Adopted from: Cell 126, 663–676, August 25, 2006 ©2006 Elsevier Inc. 663

9. 24 key factors are responsible for
pluripotency
Tcl 1
ecat 1
Dnmt Ecat 15
3L
Ecat 8
Nanog
Gdf 3
Ecat β
15-2 Rex 1 catenin fth 17
Grb 2
lif
Sall 4
Sox 15
Fbx 15 Utf 1
esg 1
sox 2
Stat 3
c-myc
oct3/4 ERas
klf 4

11. Fbx 15 locus Fbx 15 locus
β-geo β-geo
mEF/mAF ES- like
cell
Retroviral
infection &
G418 selection
5’LTR cDNA 3’LTR

12. Dnmt ecat 1 Ecat 15 Nanog Tcl 1 Ecat 8 fth 17
3L
Sall 4
β
catenin
Fbx 15 locus Fbx 15 locus
Grb 2
β-geo β-geo
Rex 1
Gdf 3 mEF/mAF ES- like
cell
Retroviral
Ecat 15-2
infection &
esg 1 G418 selection
Utf 1
lif
5’LTR cDNA 3’LTR
Fbx 15
Sox 15 Klf 4 C-myc Oct3/4 Sox 2 ERas
Stat 3

14. Fbx 15 locus Fbx 15 locus
β-geo β-geo
mEF/mAF
iPSC
Retroviral
cocktail
infection &
G418 selection
5’LTR cDNA 3’LTR
Tcl 1 Fbx 15 Utf 1 Rex 1
Dnmt
Nanog 3L
β Ecat 8
esg 1 Ecat 15-2
Stat 3 Grb 2 catenin
fth 17
Ecat 15
lif
C-myc Oct3/4 ecat 1
Klf 4 Sox 2 ERas
Gdf 3 Sox 15 Sall 4

16. NOW THEY TRIED TO SEE THE MINIMUM
NUMBER OF FACTORS THAT COULD INDUCE
PLURIPOTENCY

17. Fbx 15 locus Fbx 15 locus
β-geo β-geo
mEF/mAF
iPSC
Retroviral
cocktail
infection &
G418 selection
5’LTR cDNA 3’LTR
Klf 4 C-myc Oct3/4 Sox 2

18. Results
Multiple number of
G418-resistant colonies
were observed after
transduction with a
combination of these four
factors
(oct3/4, sox2, klf4, c-myc)
Cell 126, 663-676, August 25, 2006 ©2006 Elsevier Inc.

19. VARIOUS TISSUES PRESENT IN
TERATOMAS DERIVED FROM iPSC
IMMUNOSTAINING CONFIRMING
DIFFERENTIATION INTO NEURAL
TISSUE (NEURON & GLIAL CELLS) AND
MUSCLES IN TERATOMAS DERIVED
FROM iPSC
IN VITRO EMBRYOID BODY FORMATION
Cell 126, 663-676, August 25, 2006 ©2006 Elsevier Inc.

20. RT- PCR ANALYSIS OF ESC
MARKER GENES IN IPSC
BISULFITE GENOME
SEQUENCING OF THE
PROMOTER REGIONS OF
GENES FROM IPSC
Cell 126, 663-676, August 25, 2006 ©2006 Elsevier Inc.

22.  In June 2007 Yamanaka et al, Kyoto University & two other
independent groups Harvard, MIT & UC, Los Angeles showed
successful reprogramming and production of viable chimeras
 Instead of Fbx15 they used Nanog as a marker of pluripotency
detection
 DNA methylation patterns were identical to ESCs

23. iPSc-made from mouse-> lead to
the creation of beating cardiomyocyte, skin
and almost every kind of cell you can
imagine.
iPSc made from human-> also
lead to the creation of various cell types
in body, in addition even produced
gamete!
iPSc created live organism->
“ Xiao Xiao / Tiny”!

24. For humans the same procedure was followed, excepting
the addition and substitution of a few other factors like:-
OCT 4, SOX 2, NANOG, LIN 28 genes & lentiviral
system- by Thompson J.
POU5F1(OCT4), C-MYC, KLF4, SOX2 genes &
retroviruses- by Yamanaka S.
PCM
Reprogramming
factors
FM
Fibroblasts iPSC (NANOG-GFP)
Adopted from:- http://www.systembio.com/downloads/StemCellposter.pdf

25. ADOPTED FROM:- http://www.nature.com/nm/index.html

26. iPSc-made from mouse-> lead to
the creation of beating cardiomyocyte, skin
and almost every kind of cell you can
imagine.
iPSc made from human-> also
lead to the creation of various cell types
in body, in addition even produced
gamete!
iPSc created live organism->
“ Xiao Xiao / Tiny” !

27. ADOPTED FROM: Stem cells: The magic brew, Janet Rossant. Nature 448, 260-262 (19 July 2007); doi:10.1038/448260a

28. TETRAPLOID COMPLEMENTATION
FORMS ONLY PLACENTA AND
2N EXTRA-EMBRYONIC MEMBRANE
FUSION 4N
2N
WHITE COAT
COLOUR
“XIAO XIAO “/
“ TINY “ /
AN ALL - iPSC
MOUSE
SKIN REPROGRAMMING iPSC
BLACK COAT
CAN FORM ALL POTENTIAL COLOUR
BLACK COAT
TYPES OF CELLS IN BODY
COLOUR

29. Defined factors
Motor neurons
Adopted from:- http://www.rndsystems.com/cb_detail_objectname_cb09i2_induced_pluripotent_stem_cells.aspx

31. Morphology-
cell have round shape, large nucleolus and scant cytoplasm; colony
morphology is also similar.
Growth properties-
iPSCs are mitotically active, actively self-renewing, proliferating, and dividing
at a rate equal to ESCs.
Pluripotent Stem cell markers:-
iPSCs express cell surface antigenic markers expressed on ESCs.
Pluripotent Stem Cell Genes-
Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and
hTERT.
Telomerase activity-
express high telomerase activity.

32. Neural differentiation-
βIII-tubulin, GFAP expressed (found specifically in neurons and glial cells).
Cardiac differentiation-
Differentiated into cardiomyocytes that spontaneously began beating.
Teratoma formation-
iPSCs injected into immunodeficient mice spontaneously formed teratomas
after nine weeks.
Embryoid body-
iPSCs also form embryoid bodies and have peripheral differentiated cells.
Chimeric mice-
Mice with iPSC derivatives incorporated all across their bodies with 10%-90%
chimerism.
Tetraploid complementation-
Whole, non-chimeric, fertile mice produced, although with low success rate.

33. Promoter demethylation-
Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and
Nanog, were demethylated in iPSCs, demonstrating their promoter
activity.
Histone demethylation-
H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated.

34. Adopted from:- cell 137, April 3, 2009, 13 ©2009 Elsevier Inc.

36. HbS /HbS
HbS /HbS
HbA /HbS
HbS /HbS
Scientists of MIT, have used iPS cells to cure
sickle cell anemia.
Adopted from:- 21 DECEMBER 2007 VOL 318 SCIENCE www.sciencemag.org

37. Whitehead Institute researchers rescued Parkinson's phenotype in rat, transplanting committed neurons free of
contaminating undifferentiated cell population & thus minimizing risk of tumor formation.
Reprogramming
Redifferentiation
Transplantation into adult brain of parkinson’s disease rat
model,after removal of contaminating pluripotent cells from
committed neurons using FACS

39. Whitehead Institute researchers produced Parkinson’s disease patient-specific stem cells free of
harmful reprogramming transgenes
Skin cells of parkinson’s patient
Retroviruses packaged with reprogramming genes
flanked by lox-p sequence
iPS cells with integrated transgenes
Retroviruses packaged with cre enzyme
iPS cells without transgenes
Differentiatng factors
Dopaminergic
neurons
Stain
Green-> class III beta-tubulin (neuron specific)
Red-> tyrosine hydroxylase (dopaminergic neuron specific)
Adopted from:- http://www.wi.mit.edu/news/archives/2009/rj_0305.html

40. Suneet Agarwal & George Daley from Children's Hospital Boston and the Harvard Stem Cell Institute were
studying Inherited premature aging disorder- Dyskeratosis congenita
 A progeroid disease
 Bone marrow failure
 Cells lose telomerase activity due to reduced
expression of TERC(Telomerase RNA Component)
BUT
 iPSC from skin of such patients shows TERC
expression three times more than diseased cell
 Thus simply turning the diseased skin cells into
iPS cells helped restore their damaged telomeres
at normal level
Published online in NATURE 17/2/2010

41. iPS Cells can be used for toxicological studies that
could save lives & money as these iPSC derived
tissues can be exposed to a vast library of chemical
compounds to test them as potential drugs.
Human disease can be studied now in human
tissues i.e. worth studying in mouse models increasing
the level of accuracy.
Making iPSC showed the link between stem cells
and cancer cells, as the reprogramming factors are in
general cancer critical genes (c-myc, klf4 and others).

42. No transplant rejection
No ethical issues involved as no
usage of embryos
As a method of cloning, the nonself
cytoplasmic inheritance is absent
here unlike SCNT

43. Oncogenic effect of retroviral integration
Due to c-myc 20% chimeric mice developed cancer
Forced reprogramming related safety issues
Teratoma related safety issues
Re-differentiation related safety issue

45. Trans-differentiation is the process when a differentiated
cell creates cells outside its established differentiation
path i.e. the cell fate switches, including the
interconversion of stem cells
Trans-differentiation was shown earlier by converting
pancreatic exocrine cell to beta cell
But now neurons are derived from fibroblasts !

46. Marius Wernig et al.demonstrated that
fibroblasts convert to functional neurons in vitro
by forced expression of three factors:-
Ascl1, Brn2, and Myt1l,
bypassing the pluripotent stage,
thus avoiding the risk of tumor formation.
?
•MAP2, NeuN, and
synapsin positive,
•Produces action
Brn2, Myt1l & Ascl1 potentials
12 days
mEF iNs
Nature. Published online January 27, 2010. doi:10.1038/nature08797

47. Instead of using retroviruses, researchers have tried using:
Plasmid expression vector- Yamanaka S. et al
ADVANTAGE- no evidence of transgene integration, no risk of
transformation due to integration.
DISADVANTAGE- low efficiency
Adenoviral vectors- Konrad H. et al
ADVANTAGE- no evidence of transgene integration, no risk of
transformation due to integration.
DISADVANTAGE- products get diluted out during successive cell divisions
in progeny cells; efficiency poor
Proteins channelized to cell via poly arginine anchors- Ding S. et al
ADVANTAGE- no evidence of transgene integration, no risk of
transformation due to integration.
DISADVANTAGE- products get diluted out during successive cell divisions
in progeny cells; efficiency poor
c-MYC was avoided due to its proto-oncogenic nature- Yamanaka S. et al
the cells took longer time to become iPSCs; low efficiency

48. NATURE METHOD OF THE YEAR : 2009->
iPS Cells

49. What are the exact sequence of molecular events that leads to this dramatic
reprogramming?
Whether additional changes, beyond the expression of the four transcription
factors, are involved or not?
The process of reprogramming is slow — colonies take up to 20 days to
develop into real ES-like cells, and their frequency is quite low.
Is this because only a few cells happen to express the right combination or
levels of the four factors because of the random integrations of the
retroviruses?
Or, are there additional events, perhaps associated with retroviral insertion,
that are required for full transformation?

51. “This will be the long-term solution”- Sir
Martin Evans
“This is the future of stem cell research. Its
hundered times more interesting than
SCNT”- Dr. Ian Wilmut
“A decade from now, this (hESC)
controversy will be just a funny historical
footnote.” – Dr. James Thompson.
“Embryonic stem cells are not safe, but at
the moment, iPS cells are more
dangerous.” – Shinya Yamanaka

52. Breakthrough of the year-2008; Science 322 ;2008;1766-1767
Catherine Verfaillie; The undoing of differentiation by four defined factors: A big step forward towards generating
patient specific pluripotent stem cells; Journal of Hepatology 49 (2008) 871–878
Daley G. Q. et al; Reprogramming of human somatic cells to pluripotency with defined factors; Nature, 451, 2008;
141-148
Dausman A.,Jaenisch R. et al; Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to
Pluripotency; , 2008; Cell 133, 250–264.
Dirk D. et al; Induced pluripotency with endogenous and inducible genes; Experimental Cell Research 314
(2008) 3255–3263
Douglas A. et al; Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2;
Nature Biotechnology 26, 2008; 1269-1278
Hongyan Z, Shili W et al; Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins; Cell Stem
Cell 4, 2009;381-385
Holm Z. and Hans R.; Induction of Pluripotency: From Mouse to Human; Cell 131, 2007; 834-836
James A. T. et al; Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells; Science 318,2007 ;
1916-1919
Keisuke O, Ichisaka T & Shinya Y; Generation of germline-competent induced pluripotent stem cells; Nature 448,
2007; 313-319
Konrad H. et al ; Defining Molecular Cornerstones during Fibroblast to iPS Cell Reprogramming in Mouse; Cell
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Marius W. et al; Nature. Published online 2010; doi:10.1038/nature08797

53. Matthias S.,Maherali N., David T.& Hochedlinger K; Defining Molecular Cornerstones during Fibroblast to iPS
Cell Reprogramming in Mouse; March 2008; Cell Stem Cell 2, 230–240, 2008
Method of the Year 2009; Nature Methods.7, 2010, DOI:10.1038/Nmeth.f.294
PAUL S. KNOEPFLER; Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine;
Stem Cells 2009;27:1050–1056
Rudolf J. et al; Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic
Cells; Cell Stem Cell 2, 151–159,2008; 151-160
Shinya Yamanaka; A Fresh Look at iPS Cells ; Cell 137, 2009 ;13-17
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322, 2008; 322-328
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Wernig M., Meissner A. , Cassady J. P. & Jaenisch R.; c-Myc Is Dispensable for Direct Reprogramming of Mouse
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Xiao-yang Z. et al; iPS cells produce viable mice through tetraploid Complementation; Nature,
doi:10.1038/nature08267
Yamanaka S. et al; Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors;
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