Reprogramming refers to erasure and remodelling of epigenetic marks, such as DNA methylation, during mammalian development. Exposure of a differentiated cell nucleus to the cytoplasm of less differentiated cell leads to erasure of the stable epigenetic code that maintains the differentiated cell’s phenotype. Gradually, the nucleus acquires a new epigenetic code that is characteristic of the dedifferentiated cell donating the cytoplasm, a process termed cellular reprogramming.

1. CELLULAR REPROGRAMMING
Current Technology, Perspectives and Generation of
iPS cells
M U N N A L A L YA D AV
Protein Science Lab
ABTC
NDRI KARNAL

2. Introduction
 Reprogramming refers to erasure and remodelling of epigenetic marks, such as DNA
methylation, during mammalian development.
 Exposure of a differentiated cell nucleus to the cytoplasm of less differentiated cell
leads to erasure of the stable epigenetic code that maintains the differentiated cell’s
phenotype.
 Gradually, the nucleus acquires a new epigenetic code that is characteristic of the
dedifferentiated cell donating the cytoplasm, a process termed cellular
reprogramming.

3. Strategies for cellular Reprogramming
Konrad Hochedlinger et al., Nature, 200

4. Direct Reprogamming
Fig. 3. Steps involved in direct reprogramming to pluripotency. The starting, intermediate and end stages of reprogramming
to pluripotency that can be identified during the generation of iPS cells are shown. „Intermediate cells‟ appear only
transiently before converting into iPS cells, whereas „partially reprogrammed cells‟ can be stably propagated and converted
into iPS cells upon treatment with DNA demethylating agents and knockdown of lineagespecific genes. Although not
proven, it is assumed that partially reprogrammed cells originate from transient intermediate cells. The defining molecular
Konrad Hochedlinger et al, Devlopment, 2009
and cellular characteristics are shown above and below each cell population.

5. Chromatin Connections to Pluripotency and Cellular
Reprogramming
Figure. Networks and Their Interconnections in ESCs Protein-protein interactions derived from microsequencing of protein complexes
purified from ESCs are shown on the upper left. The network is a consensus view of proteins from Mallanna et al., 2010; Pardo et
al., 2010; van den Berg et al., 2010; and Wang et al., 2006. The triad of core pluripotency factors, Oct4, Nanog, and Sox2, are circled in
red. Components of chromatin-remodeling or modifying complexes are highlighted in green circles. In the upper right, the transcriptional
regulatory network as established through ChIp-ChiP and ChIP-seq studies is summarized (Boyer et al., 2005; Kim et al., 2008; Loh et
al., 2006). The Myc network to the lower right refers to the protein-protein and protein-DNA networks reported in Kim et al., 2010. The
factors in the core and c-myc regulatory networks crossregulate each other and regulate, and are regulated by, chromatin factor al.,2011
Stuart H. Orkin et
components illustrated in the lower left. The output of these complex regulatory interactions is maintenance of self-renewal and blocking

6. Open and Closed Chromatin and Cellular
Reprogramming
Figure. Properties of Open and Closed Chromatin At the top, simplified views of open and closed
chromatin are depicted. As differentiation proceeds, chromatin becomes closed. Cellular reprogramming
reverses the chromatin state. The table summarizes the protein characteristics of open and closed
chromatin and the factors that promote each state.
Stuart H. Orkin et al.,2011

7. Cellular Reprogramming and Pluripotency
Figure: Features that influence pluripotency and reprogramming. Depicted is a murine
preimplantation blastocyst -the origin of pluripotent cells and features that influence
pluripotency and reprogramming.

8. The core embryonic stem cell transcriptional
circuit
Stemness is maintained by a network centered on NANOG, which
is controlled by the four factors.
Left: protein-protein interaction network of genes upregulated when cell is in
a “stem” state.
Right: genes downregulated when middle box (3 of 4MacArthur + NANOG)
factors et al., Nature, 2009

9. Transcription factor induced mechanism of
reprogramming

10. Cont......
Figure. ES cell transcription factor network and implications for reprogramming. (A) The reprogramming factors
Oct4, Sox2 and Klf4 (light blue) often co-bind promoter regions with other transcription factors, including Nanog, Nr0b1
(nuclear receptor subfamily 0, group B, member 1), Esrrb (estrogen-related receptor, beta), Zfp281 (zinc finger protein
281) and Nac1 (nucleus accumbens associated 1; all of which have been purified in large protein complexes with Oct4
or Nanog), as well as with Stat3 and Smad1 (transcription factors downstream of the Bmp4 and Lif signaling pathways
that maintain ES cell self-renewal and pluripotency). The recruitment of co-activators, such as the histone
acetyltransferase (HAT) p300 is often observed (yellow). This binding pattern is found in transcriptionally active genes in
ES cells. ES cell target groups and implications for reprogramming are also indicated. (B) In ES cells, genes bound by
either Oct4, Sox2 or Klf4 are often repressed, potentially through the recruitment of Polycomb group (PcG) proteins or
histone deacetylases (HDACs), but become activated upon differentiation. (C) cMyc is proposed to bind and activate
Konrad transcription factors.
largely different sets of genes to Oct4, Klf4 and Sox2, but in collaboration with other Hochedlinger et al, Devlopment, 2009

11. Mechanisms of DNA demethylation &
pluripotency
Figure. Pathways to DNA demethylation of key pluripotency genes. (A) The establishment of symmetric DNA methylation
patterns could be prevented passively during replication by the steric hindrance of Dnmt1 due to the stochastic binding of the
reprogramming factors to target sites or by inhibiting Dnmt1 function indirectly. Hemimethylation of the DNA would result in a
progressive loss of methylation upon further rounds of cell division. (B) Alternatively, DNA methylation could be actively
removed by the recruitment of a demethylating enzyme.
Konrad Hochedlinger et al, Devlopment, 200

12. Connection between miRNAs and the core transcription
factors
Figure. Noncoding RNAs Modulate ESC Self-Renewal, Differentiation, and Cellular Reprogramming Shown are examples of
microRNAs (in red) and lncRNAs (in black) that are occupied and either activated by Oct4, Sox2, and Nanog or silenced by
the same factors in combination with PRC2 in pluripotent cells, as well as their roles in self-renewal and differentiation.
Manipulation of several noncoding RNAs in the context of iPSC formation has been shown to enhance cellular
reprogramming. Note that some miRNAs, such as members of the miR-200 family, may directly target PRC1 and PRC2
components, such as Bmi-1 and Suz12, respectively. Expression of the miR-302/367 cluster has been suggested to be
Stuart H. Orkin et al, Cell. 2011

13. Cont......
Figure. Connection between miRNAs and the core transcription factors regulating pluripotency. Diagrammatic representation
of the interconnectivity between miRNAs and genes known to affect pluripotency, including some of the direct targets of
miRNAs. The core transcription factors promote the expression of ESC-specific genes and miRNA expression and, at the
same time, repress developmental genes. The combined effect of OCT4, SOX2, NANOG and ESC-specific miRNA
repression upregulates expression of ESC-specific genes. OCT4, SOX2 and NANOG form an autoregulatory loop that leads
to a sustained positive feed-forward pathway. The miRNA let-7 is downregulated by high levels of LIN28 in pluripotent cells. 2012
Frederick Anokye-Danso et al., Cell Science,

14. p53-microRNA (miRNA) network in regulating cell
behaviours.
Fig. Schematic representation of the role of the p53-microRNA (miRNA) network in regulating cell behaviors. The
p53 tumor-suppressor gene is a well-known master regulator that helps downregulate genes required for proliferation and
survival. Meanwhile, along with other p53 targets, such as p21 and BAX, the miR-34 family of miRNAs promote growth
arrest and cell death in response to various stress signals. Particularly, the p53-miR145 network (red) could disrupt the core
reprogramming network in ESCs and thereby inhibit self-renewal and reprogramming. Exploiting details of the p53-miR145
network and it interesting mechanisms involved in pluripotency may help to understand Sun, Ageing Research
Xiaoyan the molecular underpinnings in

15. miRNA-induced pluripotency and cellular
reprogramming
Fig. Strategies for miRNA-induced pluripotency in cellular reprogramming. Research into miRNAs represents a potential tool
for reprogramming in both cancer and normal cells. Clusters of miRNAs involved in p53 signaling could inhibit the p53 tumorsuppressor network, indirectly drive the endogenous expression of the key pluripotency factors, and increase the cell division
rate to accelerate the efficiency and kinetics of the reprogramming process, but without genetic modification of the donor cells.
This suggests that screening the miRNA regulatory network involved in p53 signaling would help to identify possible regulators
Xiaoyan Sun, Ageing Research
that could facilitate or improve the efficiency of reprogramming.

16. Cellular Reprogramming and Generation of iPS
cells
 The initial derivation of iPSC by Shinya Yamanaka‟s group was
achieved by overexpressing four transcription factors first in
mouse and then human fibroblasts:
(Takahashi and
Yamanaka, 2006).
 Octamer binding transcription factor 4 (Oct4)
 Sex-determining region Y HMG box 2 (Sox2)
 Kruppel like factor 4 (Klf4)
 v-myc myelocytomatosis viral oncogene homolog (c-Myc),
 Often referred as the „Yamanaka factors‟

17. Cont........
 Subsequent reports from many labs have contributed to a growing list of
reprogramming factors used for iPSC generation, including
 Estrogen-related receptor beta (Esrrb)
 Sal-like 4 (Sall4)
 microRNAs (miRNA)
 simian virus 40 large-T antigen (SV40LT)
 human telomerase reverse transcriptase (hTERT)

18. Current methods for cellular reprogramming
Fig. The current reprogramming strategies used to induce pluripotent stem cells from adult somatic
cells. The first method introduced was the viral delivery system involving the use of
adenovirus, retrovirus and lentivirus. But since then, non-viral methods have been explored. For
example, episomal plasmids for gene delivery and creloxP and piggyBac transposon are used as the
excision strategy. Protein-tagging, cell culture manipulationsLai et al., J Assist are non-DNA modification 2011
Mei I. and miRNAs Reprod Genet, Stem Cell Biology,

19. Efficiency of iPS induction methods in human
fibroblasts
Retroviruse has high efficiency of transduction and iPS induction with
low safety.
Sendai virus has high efficiency of transduction and iPS induction with
high safety.
Shinya Yamanaka et al., 2011

20. Mechanism for induction of pluripotency in somatic
cells
Figure: Putative mechanism for the induction of pluripotency in somatic cells. Downregulation of p21 by c-Myc promotes the
oncogenic activity of Klf4, which serves to inhibit p53-dependent apoptosis induced by c-Myc overexpression. The coordinated
actions of c-Myc and Klf4 suppress apoptosis and senescence and promote cell cycle progression. Through interactions with
epigenetic modifiers, c-Myc and Klf4 contribute to chromatin decondensation and promoter demethylation. These epigenetic
changes may represent the rate-limiting step in the induction of pluripotency. This permissive chromatin conformation allows
Oct4 and Sox2 to bind to the promoters of target genes. Genes associated with pluripotency are expressed, including the
endogenous “core” pluripotency factors: Oct4, Sox2, and Nanog. These 3 factors form an autoregulatory feed-forward loop that
regulates the pluripotent transcriptional network and suppresses genes associated with differentiation, leading to the induction
Judi L. Azevedo et al., Genes & Cancer, 2
of pluripotency and the formation of iPSCs.

21. Generation of iPS Cells from Adult Human Fibroblasts
by Defined Factors
Figure. Induction of iPS Cells from Adult HDF
(A) Time schedule of iPS cell generation. (B)
Morphology of HDF. (C) Typical image of nonES cell-like colony. (D) Typical image of hES
cell-like colony. (E) Morphology of established
iPS cell line at passage number 6 (clone
201B7). (F) Image of iPS cells with high
magnification. (G) Spontaneously differentiated
cells in the center part of human iPS cell
colonies. (H–N) Immunocytochemistry for
SSEA-1 (H), SSEA-3 (I), SSEA-4 (J), TRA-1-60
(K), TRA-1-81 (L), TRA-2-49/6E (M), and
Nanog (N). Nuclei were stained with Hoechst
33342 (blue). Bars = 200 mm (B–E, G), 20 mm
(F), and 100 mm (H–N).
Kazutoshi Takahashi et al., Cell, 2007

22. Promoters of ES Cell-Specific Genes Are Active in Human
iPS Cells
Figure 3. Analyses Promoter Regions of
Development-Associated Genes in Human
iPS Cells(A) Bisulfite genomic sequencing
of
the
promoter
regions
of
OCT3/4, REX1, and NANOG. Open and
closed circles indicate unmethylated and
methylated CpGs . (B) Luciferase assays.
The luciferase reporter construct driven by
indicated promoters were introduced into
human iPS cells or HDF by lipofection. The
graphs show the average of the results
from four assays. Bars indicate standard
deviation.
(C)
Chromatin
Immunoprecipitation of histone H3 lysine 4
and lysine 27 methylation.
Kazutoshi Takahashi et al., Cell, 2007

23. High Telomerase Activity and Exponential Growth of
iPS Cells
Figure . High Levels of Telomerase Activity and Exponential Proliferation of Human iPS Cells
(A) Detection of telomerase activities by the TRAP method. Heat-inactivated (+) samples were
used as negative controls. IC, internal control. (B) Growth curve of iPS cells. Shown are
averages and standard deviations in quadruplicate.
Kazutoshi Takahashi et al., Cell, 2007

24. Teratoma Formation from Human iPS Cells
Figure. Teratoma derived from human iPS cells. Hematoxylin and eosin staining of
teratoma derived from iPS cells (clone 201B7). Cells were transplanted subcutaneously
into four parts of a SCID mouse. A tumor developed from oneKazutoshi Takahashi et al., Cell, 2007
injection site.

25. miRNA-Mediated Somatic Cell Reprogramming
miR-302/367 gene cluster alone is sufficient to induce pluripotency in fibroblasts
by targeting the epigenetic regulators AOF1, AOF2 (LSD1, KDM1A), MECP1p66, and MECP2).
The miR302/367 cluster is
located in intron 8 of the Larp7
gene on chromosome 3 and is
transcribed as a single
polycistronic primary transcript.
The sequences of the
miR302/367 miRNAs are
highly conserved across
species.
Figure: Potential miR-302-367 targets that affect cellular reprogramming. miR302–367, and other related miRNAs, target multiple
cellular processes as shown. The combined repression of these targets affects a global change in cell proliferation, epigenetic
state, MET and suppression of developmental factors, which leads to reprogramming of the cell phenotype. It is likely that the
combined action of most, if not all, of these processes is required for efficient cellular reprogramming. This diagram shows how the
miR-302-367 cluster coordinates multiple cellular processes that are important for reprogramming of somatic cells into pluripotent
Frederick Anokye-Danso et al., Cell Science,
stem cells as well as maintaining the pluripotent stem cell phenotype. Some of the targets known to be affected in each process 2

26. miRNA-Mediated Somatic Cell Reprogramming in
mouse
Figure . miR302/367 Can Reprogram Mouse Fibroblasts to a Pluripotent Stem Cell Phenotype (A) The sequences of the
miR302/367 cluster showing the similarity between members of the miR302a/b/c/d subfamily. miR367 has a different seed
sequence than miR302a/b/c/d. (B) Schematic of viral expression protocol for miR302/367 iPSC reprogramming with VPA.
Day 0 is the start of viral transduction. (C) Oct4-GFP-positive miR302/367 clones at 7 days after starting viral transduction.
(D) AP staining of a primary induction plate of miR302/367 iPSC clones at 8 days after starting viral transduction. (E)
Immunostaining for Nanog, Oct4, Sox2, and SSEA1 in both mouseFrederick Anokye-Danso et al., Cell Stem Cell, 2011
ES and primary induction samples of miR302/367
iPSCs at day 10, showing expression of pluripotent genes.

27. miR302/367 Plus VPA is more efficient than OSKM factors in iPSC
Reprogramming
Figure. miR302/367 Plus VPA is two orders of magnitude more efficient than OSKM factors in iPSC Reprogramming of Mouse
Fibroblasts (A) miR302/367 iPSC clones are readily observed 6 to 7 days after starting viral transduction and express high
levels of Oct4-GFP while OSKM-induced clones are not observed until 8–10 days, are very rare, and do not express significant
levels of GFP from the Oct4 locus. (B) Counts of clones with ES-like morphology from transduction of 1.75 ×104 Oct4-GFP
MEFs with equivalent amounts of either OSKM or miR302/367 virus at 8 and 10 days after viral transduction. (C) Percentage
of Oct4-GFP-positive clones 10 days after viral transduction with OSKM or miR302/367. (D) Q-PCR of the indicated pluripotent
Frederick Anokye-Danso et al., Cell Stem Cell,
factors comparing OSKM versus miR302/367 during the first 8 days after viral transduction.

28. miR367 Expression is Required for miR302/367 iPSC
Reprogramming
Figure. miR367 Expression is Required for miR302/367 iPSC Reprogramming (A) The miR302a/b/c/d pre-miRNA is
expressed at high levels in transduced MEFs. (B) Number of colonies generated after 10 days of miR302a/b/c/d or
miR302/367 expression. Data are the average of four assays ± SEM. (C) Pluripotent gene expression from primary
induction plates 8 days after viral induction of miR302a/b/c/d or miR302/367 viruses. Note lack of Oct4 gene expression in
miR302a/b/c/d-expressing cells (red arrow). Data are the average of three assays ± SEM. (D) FACS analysis of Oct4-GFP
Frederick Anokye-Danso et al., Cell Stem
MEFs 8 days after transduction with either miR302a/b/c/d or miR302/367 viruses.

29. Low Levels of Hdac2 Permit miR302/367
Reprogramming
Mouse embryonic
fibroblast have high Hdac2
protein level
Valproic acid (VPA) causes
Hdac2 degradation and promote
reprogamming
MiR302/367 alone can not
generate iPS cells in mouse
Figure . VPA Specifically Degrades Hdac2 Protein, and Suppression of Hdac2 is Required for iPSC Reprogramming by
miR302/367. Q-PCR for pluripotent stem cell marker genes shows enhanced expression of pluripotency markers at day
8 of reprogramming by miR302/367 in wild-type (Hdac2+/+) and Hdac2-/- MEFs versus WT MEFs without VPA
treatment. Data are the average of three assays ± SEM.
Frederick Anokye-Danso et al., Cell Stem Cell,

30. miRNA-Mediated Somatic Cell Reprogramming in
Human
Figure . miR302/367 Reprograms Human Fibroblasts to a Pluripotent State More Efficiently Than OSKM Factors (A–E) Colony
morphology and OCT4, SSEA4, TRA-1-60, and TRA-1-81 immunostaining of miR302/367-reprogrammed human fibroblasts.
(F) Q-PCR of pluripotent stem cell marker genes in three different miR302/367-reprogrammed human fibroblast lines as
compared to the human ES line HUES13. (G–I) Hematoxylin and eosin staining of teratomas derived from miR302/367 human
iPSC clones showing endoderm (gut)-, mesoderm (muscle)-, and ectoderm (neural epithelium)-like structures. These data
represent the results from seven human miR302/367 iPSC clones. (J–L) Immunostaining of miR302/367 human iPSC-derived
Frederick Anokye-Danso muscle, and β-tubulinteratoma tissues showing expression of E-cadherin-positive endodermal cells, MF20-positive striatedet al., Cell Stem Cell, 20

31. Human foreskin fibroblasts express lower levels of Hdac2 than
MEFs.
VPA is not necessary for reprogramming of human foreskin or dermal
fibroblast.
Protein levels of Hdac2 were not affected by VPA in these cells
These data suggest that low levels of
Hdac2 may significantly enhance or
even be required for miR302/367
reprogramming
Figure. VPA Specifically Degrades Hdac2 Protein, and Suppression of Hdac2 is Required for iPSC
Reprogramming by miR302/367. Human foreskin fibroblasts express much lower levels of Hdac2 than
MEFs.

32. Cellular Reprogramming and Future Perspectives
 Cell therapy
 Disease modelling
 Drug Screening & Discovery
 Toxicological testing
 Regeneration & repair of lost organs/tissues

33. Therapeutic potential of iPSCs for Spinal muscular
atrophy (SMA)
Figure: Potential applications of iPSCs. Shown are the potential applications of iPSC
technology for cell therapy and disease modeling using Matthias Stadtfeld et al., Genes & Development, 2
SMA as an example.

34. Human iPS cells in modelling cardiac and neural
diseases
Figure. Human iPS cells in modelling cardiac and neural diseases. Schematic diagram of disease modelling with human induced pluripotent
stem (iPS) cells, showing amelioration of the disease phenotype. a | Skin fibroblasts from a patient affected by type 1 long QT syndrome
(LQT1), carrying a mutation in the KCNQ1 potassium channel gene, were reprogrammed into iPS cells by retroviral transduction of the genes
encoding the four reprogramming factors OCT4, SOX2, Krüppel-like factor 4 (KLF4) and MYC3. iPS cells were then differentiated as
embryoid bodies. Spontaneous contraction indicated the presence of cardiomyocytes that were micro-dissected and plated separately.
β-adrenergic stress was mimicked by isoprenaline application, which induced arrhythmic events in these cells, which is the phenotype seen in
the heart of patients with LQT1. Treatment with the β-blocker propranolol suppressed arrhythmia. b | Skin fibroblasts from a patient affected
by Rett syndrome (RTT) that carry a mutation in the epigenetic regulator methyl CpG binding-protein 2 (MECP2) gene were reprogrammed
into human iPS cells by retroviral transduction of OCT4, SOX2, KLF4 and MYC. iPS cells were then differentiated as embryoid bodies.
Appearance of rosettes structures (not shown) indicated the presence of neural precursors that were further differentiated into glutamatergic
neurons. These cells showed reduced glutamatergic synapse number (red dots) and Milena Bellin et al.,the somaReviews Molecular of the
reduced the size of Nature (that is, the cell body

35. Conclusion
 Although significant differences between mouse and human
pluripotent stem cell biology
“what works in mouse may not be successful in
human.......”
Unique & distinct signal transduction pathways
II.
Gene expression properties
III. In vitro cell culture demands and
IV. Self renewal dependency on cytokines
I.
To establish and maintenance of pluripotency have to be
considered. When a new protocol developed.
 Reprogramming towards functional pluripotency will have
long term implications in regenerative medicine, gene
therapy.

36. References
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Reprogramming of Mouse and Human Somatic Cells to Pluripotency. Cell Stem Cell 8, 376–388.
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Raymond C.B. Wong, Ellen L. Smith and Peter J. Donovan (2011). New Techniques in the
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38. Thank You