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

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

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, p163-172

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

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

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

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

DNA methylation in ES cells Santos et al (2002) Dev Biol 241, 172-182. Somatic cells ES cells % meCpG 70-80

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

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

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, p1287-98).

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

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.

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

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

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, p369-74 ; Hochedlinger and Jaenisch (2002) Nature 415, p1035-8

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 1969 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, p449-525; Blau et al (1985) Science 230, p758-766; Miller and Ruddle (1976) Cell 9, p45-55; Takagi et al (1983) Cell 34, p1053-62; Martin et al (1978) Nature 271, p329-33

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

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.

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, p1145-47)

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

Directed differentiation of ES cells in vitro

Repair of coronary infarct with ES cell derived cardiomyocytes

ES cell derived cardiomyocytes live cell imaging

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

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

Development of the egg cylinder )

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