1. Programming and Reprogramming
Lecture 2
Embryonic stem cells,
Programming and Reprogramming
The discovery of ES cells
Developmental origin of ES cells
Signalling pathways regulating ES cells in vitro
2i and the concept of ground state pluripotency
What is reprogramming
Reprogramming in normal development
Experimental reprogramming and cloning
Molecular basis of pluripotency
Human ES cells and their application in medicine
You should understand
What are ES cells and where do they come from
Methods for reprogramming somatic cells
Appplications of ES cell technology

2. Definitions – stem-cells and progenitors
Stem cell; unlimited capacity to self-renew
and produce differentiated derivatives
Progenitor cell; limited capacity to self-renew
and produce differentiated derivatives
Terminally differentiated cell

3. Potency of stem cells and progenitors
Totipotent; can differentiate into all cell types, including extraembryonic
lineages e.g. morula cells
Pluripotent; can differentiate into all cell types of the three germ layers,
ectoderm, mesoderm and endoderm e.g. ES cells
Multipotent; can differentiate into limited number of cell types, e.g.
haematopoietic stem cells

4. Embryonal carcinoma (EC) cells
Teratocarcinoma
Teratocarcinomas are malignant tumours that derive from pluripotent germ cells
Tumour cells differentiate to ectoderm, mesoderm and endoderm cell types
129 strain of mouse is useful model used to derive cell lines –
EC cells.
EC cells are pluripotent stem cells but are karyotypically abnormal
Martin and Evans (1974), Cell 2, p

5. ES cells from mouse embryos
Alkaline phosphatase
positive
Grown from (129/Sv) mouse blastocysts using fetal calf-serum (FCS) on
layer of inactivated primary embryonic fibroblast cells (PEFs).
Self-renew and pluripotent.
Stable normal karyotype.
Germline competent.
Efficient homologous recombination.
Evans and Kaufman (1984) Nature 292, p154-6

6. Pathways regulating self-renewal and differentiation
of mouse ES cells
LIF/STAT3 (JAK/STAT)
+ BMP/Smad/Id
FGFs
Via ERK1/2 pathway
LIF/STAT3 +
BMP/Smad/Id
LIF + BMP blocks autostimulation of differentiation by FGF4

7. What do ES cells correspond to?
ES cells are ‘synthetic’.
Closest to ICM cells (single-cell RNA-seq).
Distinct from EpiSC - pluripotent stem cell lines from later stage (E.5.5).
EpiSC; no germline contribution and XX female cells have inactive X. ES
EpiSC

8. Transcription factor circuitry in ES cells
Genome wide analysis; RNA-seq, ChIP-seq etc
Nanog, Oct4, and Sox2 (NOS) co-occupy many target genes.
Targets can be either activated or repressed.
NOS positively regulate one another
NOS negatively regulate master TFs for other lineages eg trophectoderm and primitive endoderm lineages.
Repressed targets ‘poised’ by epigenetic mechanisms (e.g. Polycomb).
Boyer et al (2005) Cell 122, p947-56

9. Serum/LIF ES cells are not identical to ICM cells
Oct4
Nanog
Immunostaining of ES cell colonies blastocyst
Eed

10. DNA methylation in serum/LIF ES cells
Santos et al (2002) Dev Biol 241,
Somatic cells
ES cells
% meCpG
70-80

11. 2i ES cells and ground state pluripotency
LIF/STAT3 (JAK/STAT)
+ BMP/Smad/Id
FGFs
Via ERK1/2 pathway
2i (Small molecule
inhibitors of ERK
+Wnt signalling)
LIF/STAT3 +
BMP/Smad/Id
2i (small molecule
inhibitors of ERK
+Wnt signalling)
LIF +BMP blocks autostimulation of differentiation by FGF4
Ying et al (2008) Nature 453, p519-23

12. Ground state pluripotency
Without 2i
With 2i
LIF+
serum 2i
Somatic cells
ES cells
2i ES cells
% meCpG
70-80
25-30%
Down-regulation of DNA methyltransferases etc.
Leitch et al (2013) Nat Struct Mol Biol 20, p311-6

13. Applications of the 2i method
ESCs from any mouse strain and from other species, eg rat
(Buehr et al, 2008, Cell 135, p )

14. Differentiation and Reprogramming
Progressive restriction of cell fate through development.
Adult stem cells retain a degree of plasticity.
ES/early embryo cells are plastic.
Differentiated cells can transdifferentiate (red arrows) or reprogram (blue arrows)
Embryonic progenitor/ES cell
Differentiated cells
Adult stem cell

15. Reprogramming in normal development
1. Epigenetic erasure in pre-implantation embryos
Genome demethylation between 1-cell and blastocyst stage.
Reactivation of inactive X chromosome occurs in ICM cells.

16. Reprogramming in normal development
2. Primordial Germ Cell (PGC) development
PGCs reactivate pluripotency program and repress somatic program
Erasure of DNA methylation, parental imprints and reactivation of
inactive X chromosome

17. Experimental Reprogramming
Cloning of Frog (Xenopus); Briggs and King, 1952 (blastocyst cells).
Gurdon, 1957 (tadpole and adult cells).
Briggs and King (1952) Proc Natl Acad Sci U S A. 38, p55-63; Gurdon et al (1958) Nature 182, p64-5

18. Cloning in Mammals 1996; a sheep (Dolly) was the first cloned mammal
Extended to mouse, cat, cow and many other mammalian species.
Cloning of a mouse from a lymphocyte proves cloning of a terminally
differentiated cell.
Low success rate (less than 1/100) and cloned animals have health issues.
Campbell et al (1996) Nature 380, p64-6; Wakayama et al (1998), Nature 394, p ;
Hochedlinger and Jaenisch (2002) Nature 415, p1035-8

19. Reprogramming by cell fusion
Cell type A
Cell type B
Sendai virus/PEG/Electroshock
Heterokaryon
4N hybrid
2N hybrid
1969, Henry Harris shows normal cells can supress transformed
phenotype- dominance.
Blau demonstrates fusion can convert fibroblasts to myoblasts
Ruddle, Takagi, and Martin make hybrids with EC and somatic cells.
Pluripotent and reactivate inactive X chromosome.
Requirement for induction of DNA synthesis by dominant cell type.
Harris et al (1969) J. Cell Sci. 4, p ; Blau et al (1985) Science 230, p ; Miller and Ruddle (1976) Cell 9, p45-55;
Takagi et al (1983) Cell 34, p ; Martin et al (1978) Nature 271, p Tsubouchi T, et al (2013) Cell 152,p

20. X Induced pluripotent stem (iPS) cells iPS cells Fibroblast cells
24 x ES cell specific factors
(Oct4, Sox2, Klf4, c-myc)
+ LIF + feeders + neomycin
Approx 2 weeks…..
Neomycin resistance ORF
Fbx15
Nanog
etc X
Neomycin resistance ORF
Fbx15
Nanog
etc
Endogenous pluripotency genes on/fibroblast genes off.
Mouse iPS cells can passthrough germline
Reactivation of somatic cell inactive X chromosome.
Takahashi and Yamanaka (2006) Cell 126, p663-76

21. Molecular basis of reprogramming to pluripotency
Induce DNA synthesis/replication in somatic cell.
Express key master TFs i.e. Oct4, Nanog, Sox2.
Epigenetic plasticity (erase/reverse DNA methylation/X inactivation etc).

22. Human ES cells
Human ESCs derived from blastocysts grown on mouse feeder cells.
Resemble mouse EpiSCs eg post X inactivation.
Can also be generated using iPS technology.
Significant potential for regenerative medicine.
(Thomson et al, 1998, Science 282, p )

23. Directed differentiation of ES cells in vitro
GFP
Cell type specific promoter driving GFP
eg Flk1, Nkx2.5, Isl1 for cardiomyocytes
GFP
ES Cell
+Ligand/inhibitor

24. Directed differentiation of ES cells in vitro

25. Repair of coronary infarct with ES cell derived
cardiomyocytes

26. ES cell derived cardiomyocytes live cell imaging

27. Regenerative cell therapy - challenges
Cell type heterogeneity
Graft rejection
Teratocarcinoma forming potential