1. Programming and reprogramming
Lecture 2
Embryonic stem cells
Programming and reprogramming
You should understand
What is an ES cell
ES cells from reprogramming of somatic cells
Appplications of ES cell technology

2. Embryonic Stem (ES) Cells
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
Terminology for differentiative capacity of stem cells/progenitors;
Totipotent; differentiates into all cell types,including extraembryonic lineages
e.g. morula cells
Pluripotent; differentiates into all cell types of the three germ layers, ectoderm, mesoderm
and endoderm eg primitive ectoderm cells of the blastocyst.
Multipotent; differentiates into limited number of cell types, e.g. adult stem cells

3. Embryonal carcinoma (EC) cells
Teratocarcinoma
Teratocarcinomas; malignant tumours from germ cells comprising multiple cell types from all three germ layers, indicating the presence of a pluripotent stem cell population.
Occur at high frequency in 129 strain of mouse or produced by injecting early
embryo cells into testis or kidney capsule of syngeneic host.
Cell lines derived from teratocarcinomas, termed embryonal carcinoma (EC) cells,
derived by growth on inactivated feeder cells and serum. EC cells express high levels
of alkaline phosphatase, similar to germ cells.
EC cells can self-renew indefinitely and can undergo lineage differentiation in vitro and
to a limited extent, in vivo, following transfer into recipient blastocysts. Karyotypically
abnormal so cannot contribute to germline
Martin and Evans (1974), Cell 2, p

4. ES cells Derived from blastocyst stage embryos
Contribute to the germ-line of chimeric animals (blastocyst injection) and can therefore be
transmitted to subsequent generations.
Derived from blastocyst stage embryos
Grow as ‘clumps’ or ‘colonies’ by culturing with fetal calf-serum (FCS) on layer of inactivated
primary embryonic fibroblast cells (PEFs).
Have stable normal karyotype
Alkaline phosphatase positive
Contribute to all three germ layers (but not trophectoderm) when differentiated in vitro or
when transferred to recipient blastocyst – pluripotent.
Efficient at homologous recombination allowing development of gene knockout technology.
Evans and Kaufman (1984) Nature 292, p154-6

5. Transcription factor circuitry in ES cells
Unlimited availability of ES cells has facillitated genome wide analysis;
Oct4, Nanog and Sox2 co-occupy a large proportion of target genes.
Oct4, Nanog and Sox2 participate in positive feedback loops with themselves and one another.
Oct4, Nanog and Sox2 participate in negative regulatory loops to block expression of core transcription factors of trophectoderm and primitive endoderm lineages.
Other targets can be either activated or repressed.
Repressed targets associated with differentiation into different lineages. Held in a ‘poised’ configuration by epigenetic mechanisms (e.g. Polycomb).
Boyer et al (2005) Cell 122, p947-56

6. What is an ES cell?
No self-renewing pool of embryonic stem cells in ICM or epiblast – ES cells are ‘synthetic’.
Single cell transcriptomics suggest closest to ICM (primitive ectoderm) cells of the blastocyst.
Pluripotent stem cell lines can also be derived from later stage epiblast, E.5.5 – termed EpiSC
EpiSC form chimeras but have reduced developmental potential (no germline contribution).
EpiSCs have hallmarks of later stage cells (transcriptome and inactive X chromosome). ES
EpiSC

7. Homogeneous expression of Oct4 but not Nanog
Immunostaining of ES cell colonies
Immunostaining of blastocyst
Eed

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

9. Signalling pathways regulating self-renewal
and differentiation of mouse ES cells
Evidence suggests LIF +BMP blocks autostimulation of differentiation by FGF4
LIF/STAT3 (JAK/STAT)
and BMP/Smad/Id
FGFs
Via ERK1/2 pathway
2i - Small molecule
inhibitors of ERK
GSK inhibition
(wnt?)
Ying et al (2008) Nature 453, p519-23
LIF/STAT3 and
BMP/Smad/Id

10. Ground state pluripotency
2i ES cells more closely resemble embryo precursors of the blastocyst
Without 2i
With 2i
LIF+
serum 2i
Somatic cells
ES cells
2i ES cells
% meCpG
70-80
25-30%
Reduced DNA methyltransferases (Dnmt3a, Dnmt3b), and enhanced levels of
enzymes that actively remove methyl groups from CpG.
Leitch et al (2013) Nat Struct Mol Biol 20, p311-6

11. Applications of the 2i method
2i method has opened up the possibility of obtaining ESCs from any mouse strain and from other species, notable success being rat (Buehr et al, 2008, Cell 135, p ).

12. ES cells from differentiated somatic cells - reprogramming
Cell fate becomes restricted through development (modified chromatin/epigenetic states).
Adult stem cells retain some degree of plasticity.
Cells of the early embryo differentiate into many cell types – plasticity.
Reversal of differentiation back to embryonic state = reprogramming (blue line).
Interconversion of differentiated cells = transdifferentiation (red line)
Embryonic progenitor/ES cell
Differentiated cells
Adult stem cell

13. Reprogramming in preimplantation development
Demethylation of the genome occurs between 1-cell
and blastocyst stage.
Methylation is re-established from blastocyst stage onwards.
X inactivation initiated at 2-4 cell stage. Reactivation of inactive
X chromosome occurs in ICM cells.

14. Primordial germ cell (PGC) development
PGCs arise from mesoderm tissue at around E6.5-E7.5;
Repression of somatic program and reactivation of pluripotency program
Changes in global histone modification status
Loss of DNA methylation (active/passive?) including erasure of parental imprints
and reactivation of inactive X chromosome

15. Experimental Reprogramming
Nuclear transfer experiment suggested by Spemann in 1938, was performed for blastocyst
cells by Briggs and King, 1952, and for tadpole and then adult cells by Gurdon, 1957.
Experimental reprogramming in mammalian cells achieved in one of three ways; cloning, cell
fusion, and using induced pluripotency (iPS) technology.
Briggs and King (1952) Proc Natl Acad Sci U S A. 38, p55-63; Gurdon et al (1958) Nature 182, p64-5

16. Cloning
Many failed attempts to clone mammals led to the belief this wouldn’t be possible - until Dolly
Methodology now extended to mouse, cat, cow and many other mammalian species.
Cells are reprogrammed back to a totipotent state.
Cloned mouse blastocysts can be used to derive ES cell lines.
Cloning of a mouse from a lymphocyte affirms cloning of terminally differentiated cell
Frequency of success (liveborn) remains poor, less than 1/100.
Cloned animals often have serious health problems
Campbell, Wilmut and colleagues, 1996
Campbell et al (1996) Nature 380, p64-6; Wakayama et al (1998), Nature 394, p ;
Hochedlinger and Jaenisch (2002) Nature 415, p1035-8

17. Cell fusion of somatic and pluripotent cells
Cell type A
Cell type B
Sendai virus
PEG
Electroshock
Heterokaryon
4N hybrid
Same or different species
2N hybrid
Experiments by Henry Harris in demonstrated dominance - suppression of transformed
phenotype following fusion of transformed cells and certain normal cells.
Blau and colleagues demonstrated fusion can convert fibroblasts to myoblasts
Ruddle, Takagi, Martin and others show EC cell hybrids with somatic cells have pluripotent
differentiative capacity and reactivate inactive X chromosome.
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, p329-33

18. X Induced pluripotent stem (iPS) cells
Fibroblast cells
iPS cells
Introduce genes for ES cell factors
X24 then narrowed down to;
Oct4, Sox2, Klf4, c-myc
+ LIF + feeders + neomycin
Approx 2 weeks…..
Neomycin resistance ORF
Fbx15
Nanog
etc X
Neomycin resistance ORF
Fbx15
Nanog
etc
iPS cells induce endogenous pluripotency genes and switch off fibroblast program.
Mouse iPS cells contribute to chimeras and can be passed through the germline
Reactivation of somatic cell inactive X chromosome.
Takahashi and Yamanaka (2006) Cell 126, p663-76

19. Molecular foundations of pluripotency
Switching on master transcription factors for the pluripotency network
i.e. Oct4, Nanog, Sox2.
Erasure of epigenetic information eg DNA methylation,
X chromosome inactivation etc.

20. Human ES cells
Pluripotent human ESCs derived from blastocysts explanted onto mouse feeder cells.
Human ESCs resemble mouse EpiSCs eg post X inactivation.
Human ESCs can be generated using iPS technology.
Significant potential for regenerative medicine.
(Thomson et al, 1998, Science 282, p )

21. 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

22. Directed differentiation of ES cells in vitro

23. Repair of coronary infarct with ES cell derived cardiomyocytes

24. ES cell derived cardiomyocytes live cell imaging

25. Regenerative cell therapy - challenges
Cell type heterogeneity
Graft rejection
Teratoma forming potential

26. Hatching
Four days after fertilization the blastocyst hatches from the zona pellucida
ready to implant in the uterine wall.

27. Development of the egg cylinder)

28. Overview of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?