WO2014174047A1

WO2014174047A1 - Cell-cycle directed differentiation of pluripotent cells

Info

Publication number: WO2014174047A1
Application number: PCT/EP2014/058400
Authority: WO
Inventors: Siim PAUKLIN; Ludovic Vallier
Original assignee: Cambridge Enterprise Limited
Priority date: 2013-04-25
Filing date: 2014-04-24
Publication date: 2014-10-30
Also published as: GB201307486D0

Abstract

This invention relates to the differentiation human pluripotent cells (hPSCs) into progenitor cells of the endoderm or neuroectoderm germ layers by respectively either inhibiting or increasing CyclinD- CDK4/6 activity in the cells. This may be useful in the in vitro production of definitive endoderm and neuroectoderm cells for research and therapeutic applications.

Description

Cell-Cycle Directed Differentiation of Pluripotent Cells

This invention relates to the in vitro differentiation of

pluripotent human cells into lineages of specific germ layers.

Tissue differentiation and maintenance is ultimately regulated by coordination between differentiation and proliferation of specific stem cells or progenitor populations. The importance of cell cycle regulation in these major cell fate choices has been well documented in early development and in a diversity of organs such as the skin, brain, gut and hematopoietic system (Fuchs, E. Cell 137, 811-819, doi : 10.1016/j . cell .2009.05.002 (2009); Li, L. & Clevers, H. Science 327, 542-545, doi : 10.1126/science .1180794 (2010) and Lange, C. et al Cell Cycle 9, 1893-1900 (2010)) . Since the molecular analysis of these mechanisms remains technically challenging in vivo, especially in humans, (PSCs) could represent an advantageous system to model these regulations in vitro. Indeed, embryonic stem cells (ESCs) derived from embryo at the blastocyst stage or Induced Pluripotent Stem cells (IPSCs) generated from reprogrammed somatic cells have the property to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a broad number of cell types. Studies in mouse and human ESCs have shown that a truncated Gl phase is a hallmark of their pluripotent state while their differentiation is associated with an increase of the same phase of the cell cycle (Neganova, I. et al Oncogene 28, 20-30, doi : onc2008358 [pii]

10.1038/onc.2008.358 (2009); Savatier, P.L et al Oncogene 12, 309- 322 (1996) . Thus, cell cycle regulation appears to be intrinsically linked with cell fate decisions in PSCs. This invention relates to the finding that human pluripotent cells (hPSCs) differentiate into progenitor cells of the endoderm germ layer when the Gl phase of the cell cycle is extended through inhibition of CyclinD-CDK4 /6 activity, and into progenitor cells of the neuroectoderm germ layer when the Gl phase of the cell cycle is reduced through increasing CyclinD-CDK4/6 activity. This may be useful in the in vitro production of definitive endoderm and neuroectoderm cells for research and therapeutic applications.- An aspect of the invention provides a method for producing human progenitor cells of a single germ layer comprising;

providing a population of human pluripotent cells,

modulating CyclinD-CDK4 /6 activity in said cells, and;

allowing said cells to differentiate into progenitor cells.

Another aspect of the invention provides a method for producing human definitive endoderm (DE) progenitor cells comprising;

providing a population of human pluripotent cells,

inhibiting CyclinD-CDK4 /6 activity in said cells, and;

allowing said cells to differentiate into definitive endoderm progenitor cells.

Inhibition of CyclinD-CDK4/6 activity in the pluripotent cells extends the Gl phase of the cell cycle in the cells and induces endodermal differentiation.

Following endodermal differentiation, the definitive endoderm progenitor cells may be further differentiated into more specific endodermal lineages and/or differentiated endoderm cells. For example, the definitive endoderm progenitor cells may be further differentiated into hepatic lineages, for example resulting in albumin-expressing hepatocytes; pancreatic lineages, for example resulting in insulin-producing beta cells; and lineages resulting in thyroid, oesophagus, liver, biliary, stomach, small-intestine or colon cells.

Endoderm cells produced by the further differentiation of definitive endoderm progenitor cells may be useful, for example, in drug screening, toxicological tests or for therapeutic purposes, such as cell therapy.

Another aspect of the invention provides a method for producing human neuroectoderm progenitor cells comprising;

providing a population of human pluripotent cells,

increasing CyclinD-CDK4 /6 activity in said cells, and; allowing said cells to differentiate into neuroectoderm progenitor cells.

Increasing CyclinD-CDK4/6 activity in the pluripotent cells reduces the Gl phase of the cell cycle in the cells and induces

neuroectodermal differentiation.

Following neuroectodermal differentiation, the neuroectoderm progenitor cells may be further differentiated into more specific neuroectodermal lineages and/or differentiated neuroectoderm cells. For example, the neuroectoderm progenitor cells may be further differentiated into neuronal or glial lineages.

Neuroectoderm cells produced by the further differentiation of neuroectoderm progenitor cells may be useful, for example, in drug screening, toxicological tests or for therapeutic purposes, such as cell therapy.

Human pluripotent cells are cells which exhibit an undifferentiated phenotype and are potentially capable of differentiating into any foetal or adult cell type of any of the three germ layers (endoderm, mesoderm and endoderm) . A pluripotent cell is distinct from a totipotent cell and cannot give rise to extraembryonic cell

lineages. The population of pluripotent cells may be clonal i.e. genetically identical cells descended from a single common ancestor cell .

Pluripotent cells may express one or more of the following

pluripotency associated markers: Oct4, Sox2, Alkaline Phosphatase, POU5fl, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc, preferably one or more of POU5fl, NANOG and SOX2. A human pluripotent cell may lack markers associated with specific differentiative fates, such as Bra, Soxl7, FoxA2, o;FP, Soxl, NCAM, GATA6, GATA4 , Handl and CDX2. In particular, a human pluripotent cell may lack markers associated with endodermal fates. Human pluripotent cells may include embryonic stem cells (ESCs) and non-embryonic stem cells, for example foetal and adult stem cells and induced pluripotent stem cells (IPSCs) . Embryonic stem cells may be obtained using conventional techniques. For example, ESCs cells may be obtained from a cultured ESC cell line, for example a hESC line. Numerous cultured hESC lines are publically available from repositories (e.g. NIH Human Embryonic Stem Cell Registry), such as CHB-1 to CHB-12, RUES1 to RUES 3 , HUES1 to HUES28, HUES45, HUES48, HUES49, HUES53, HUES62 to HUES66, WA01 (HI) , WA07 (H7) , WA09 (H9) , WA13 (H13) , A14 (H14) , NYUES1 to

NYUES7, MFS5, and UCLAl to UCLA3. Further examples of suitable human embryonic stem cell lines are described in Thomson JA et al Science 282: 1145-1147 (1998); Reubinoff et al. Nat Biotechnol 18:399-404 (2000); Cowan, C.A. et al. N. Engl. J. Med. 350, 1353-1356(2004), Gage, F.H., et al . Ann. Rev. Neurosci. 18 159-192 (1995); and

Gotlieb (2002) Annu. Rev. Neurosci 25 381-407); Carpenter et al. Stem Cells. 5(1) : 79-88 (2003) . Potentially clinical grade hESCs are described in Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005) and Ludwig, T.E. et al . Nat. Biotechnol. 24, 185-187 (2006) .

Suitable hESCs may be obtained for use in the invention without either destroying a human embryo or using a human embryo for an industrial or commercial purpose. For example, hESCs may be obtained by blastomere biopsy techniques (Klimanskaya (2013) Semin Reprod Med. 31(1) : 49-55; Klimanskaya et al Nature (2006)

444 (7118) 81-5) . iPSCs are pluripotent cells which are derived from non-pluripotent, fully differentiated ancestor cells. Suitable ancestor cells include somatic cells, such as adult fibroblasts and peripheral blood cells.

Ancestor cells are typically reprogrammed by the introduction of pluripotency genes or proteins, such as Oct4, Sox2 and Soxl into the cell. The genes or proteins may be introduced into the

differentiated cells by any suitable technique, including plasmid or more preferably, viral transfection or direct protein delivery.

Other genes, for example Kif genes, such as Kif-1, -2, -4 and -5; Myc genes such as C-myc, L-myc and N-myc; nanog; and Lin28 may also be introduced into the cell to increase induction efficiency.

Following introduction of the pluripotency genes or proteins, the ancestor cells may be cultured. Cells expressing pluripotency markers may be isolated and/or purified to produce a population of iPSCs. Techniques for the production of iPSCs are well-known in the art (Yamanaka et al Nature 2007; 448:313-7; Yamanaka 6 2007 Jun 7; l(l) :39-49; Kim et al Nature. 2008 Jul 31; 454 ( 7204 ) : 646-50 ;

Takahashi Cell. 2007 Nov 30; 131 (5) : 861-72. Park et al Nature. 2008 Jan 10; 451 (7175) : 141-6; Kimet et al Cell Stem Cell. 2009 Jun

5; 4 (6) : 472-6; Vallier, L., et al. Stem Cells, 2009. 9999(999A) : p. N/A) . iPSCs may be derived from somatic cells, such as fibroblasts, which have a normal (i.e. non-disease associated) genotype, for example cells obtained from an individual without a genetic disorder. The iPSCs may be used as described herein to produce definitive endoderm progenitors with a normal (i.e. non-disease associated) genotype. These definitive endoderm progenitors may be further differentiated into pancreatic, hepatic or other endodermal lineages, which may be useful in therapy, modelling or other applications.

In some embodiments, the iPSCs may be derived from somatic cells obtained from an individual with a distinct genetic background. For example, iPSCs may be produced from cells from an individual having a disease condition, an individual having a high risk of a disease condition and/or an individual with a low risk of a disease

condition. Definitive endoderm progenitor cells produced as

described herein from individuals with distinct genetic backgrounds, or cells differentiated therefrom in vitro, may be useful in studying the mechanisms of disease conditions, such as diabetes and liver disease, and in identifying therapeutic targets. iPSCs may be derived from somatic cells, such as fibroblasts, which have a disease-associated genotype, for example cells obtained from an individual with a genetic disorder. Genetic disorders may include disorders of endodermal tissue, such as pancreatic and hepatic disorders, and may be monogenetic disorders. Any cell with the disease genotype, for example a genetic mutation or defect, may be used to produce iPSCs, although samples of fibroblasts, e.g.

dermal fibroblasts, may be conveniently obtained. iPSCs which are produced from cells obtained from an individual with a genetic disorder may be used as described herein to produce definitive endoderm progenitor cells which have the genotype of the genetic disorder. These definitive endoderm progenitor cells may further differentiated into pancreatic, hepatic or other endodermal lineages which possess the disease genotype. These endoderm cells may be useful, for example, in modelling the genetic disorder.

In some embodiments, a population of pluripotent cells may be obtained from a pluripotent cell line. Conventional techniques may be employed for the culture and maintenance of human pluripotent cells (Vallier, L. et al Dev. Biol. 275, 403-421 (2004), Cowan, C.A. et al. N. Engl. J. Med. 350, 1353-1356 (2004), Joannides, A. et al . Stem Cells 24, 230-235 (2006) Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005), Ludwig, T.E. et al. Nat. Biotechnol. 24, 185-187 (2006) ) . Pluripotent cells for use in the present methods may be grown in defined conditions or on feeder cells. For example, pluripotent cells may be conventionally cultured in a culture dish on a layer of feeder cells, such as irradiated mouse embryonic fibroblasts (MEF) , at an appropriate density (e.g. 10⁵ to 10⁶ cells/60mm dish) , or on an appropriate substrate with feeder conditioned or defined medium. Pluripotent cells for use in the present methods may be passaged by enzymatic or mechanical means. Suitable culture media for pluripotent cells are well-known in the art and include; Knockout Dulbecco's Modified Eagle's Medium (KO- DMEM) supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, ImM L-Glutamine, 0. ImM β-mercaptoethanol and 4ng/ml to lOng/ml FGF2; or Knockout (KS) medium supplemented with 4 ng/ml FGF2; or KO-DMEM supplemented with 20% Serum Replacement, 1% Non- Essential Amino Acids, ImM L-Glutamine, 0. ImM β-mercaptoethanol and 4ng/ml to lOng/ml human FGF2; or DMEM/F12 supplemented with 20% knockout serum replacement (KSR) , 6 ng/ml FGF2 (PeproTech) , lmM L- Gln, 100 m non-essential amino acids, 100 μ 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin. In preferred embodiments, a population of pluripotent cells for use in the present methods may be cultured in chemically defined medium (CDM) .

A chemically defined medium (CDM) is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A CDM is devoid of undefined components or constituents which include undefined components, such as feeder cells, stromal cells, serum, matrigel, serum albumin and complex extracellular matrices. In some embodiments, the chemically defined medium is humanised. A humanised chemically defined medium is devoid of components or supplements derived or isolated from non- human animals, such as Foetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA) , and mouse feeder cells. Conditioned medium includes undefined components from cultured cells and is not chemically defined.

Suitable chemically defined basal medium, such as Advanced

Dulbecco's modified eagle medium (DMEM) (Price et al Focus (2003) 25 3-6), Iscove's Modified Dulbecco's medium (IMDM) and RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508) are known in the art and available from commercial sources (e.g. Sigma-Aldrich MI USA; Life Technologies USA) .

In some preferred embodiments, a population of pluripotent cells for use in the present methods may be cultured in a CDM which comprise a chemically defined basal medium supplemented with one or more additional components, for example transferrin, 1-thioglycerol and defined lipids and optionally polyvinyl alcohol; polyvinyl alcohol and insulin; serum albumin; or serum albumin and insulin. A suitable CDM is described in Johansson and Wiles (1995) Mol Cell Biol 15, 141-151) . Johansson and Wiles CDM consists of: 50% IMDM (Gibco) plus 50% F12 NUT-MIX (Gibco) ; 7μg/ml insulin; 15μς/πι1 transferrin; 1 mg/ml polyvinyl alcohol (PVA) ; 1% chemically defined lipid

concentrate ( Invitrogen) ; and 450μΜ 1-thiolglycerol . Other suitable medium are described in the experimental section below. In order to maintain pluripotency, a CDM may further comprise FGF2 (for example, 10 to 20 ng/ml, e.g. 12ng/ml) and activin A (for example, 10 ng/ml)

(Vallier et al. 2005 J Cell Sci 118:4495-4509; Brons et al Nature.

(2007) Jul 12; 448 (7150) : 191-5) . Other suitable media include CDM- MA, as described below. A population of pluripotent cells suitable for use in the present methods may be substantially free from one or more other cell types. Pluripotent cells may, for example, be separated from other cell types, using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and magnetic beads or fluorescence activated cell sorting (MACS or FACS) including the use of antibodies against extracellular regions of molecules found on stem cells, such as SSEA4. In some preferred aspects of the invention, CyclinD-Cdk4/6 activity is inhibited in the population of human pluripotent cells.

Inhibition of CyclinD-Cdk4 /6 activity may induce, drive or

facilitate endodermal differentiation of the human pluripotent cells to produce definitive endoderm progenitor cells.

A definitive endoderm progenitor cell has reduced differentiation potential compared to a pluripotent cell and exhibits a partially differentiated endodermal phenotype. Definitive endoderm progenitor cells are committed to lineages in the endoderm primary germ layer and are potentially capable of further differentiation into any foetal or adult cell type of the endodermal germ layer. For example, a definitive endoderm progenitor may differentiate under appropriate conditions into all cell types in the liver, pancreas, lungs, gut, and thyroid. In some embodiments, definitive endoderm progenitors may be termed "multipotent" . Definitive endoderm progenitor cells cannot give rise to extraembryonic, mesoderm or neuroectoderm cell lineages . Definitive endoderm progenitors may express one or more of the following endoderm associated markers: Soxl7, foxA2, GSC, Mixll, Lhxl, CXCR4 , GATA4, eomesodermin (EOMES ) , Mixll, FoxA2, goosecoid and Hex. A definitive endoderm progenitor may lack markers

associated with pluripotency, such as Oct4, Sox2, Alkaline

Phosphatase, POU5fl, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c- myc. A definitive endoderm progenitor may lack markers associated with extraembryonic, mesoderm or neuroectoderm cell lineages.

The inhibition of CyclinD-CDK4/6 activity as described herein increases the proportion of human pluripotent cells in the

population which differentiate into endoderm progenitors. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the population of human pluripotent cells may differentiate into definitive endoderm progenitor cells following inhibition of CyclinD-Cdk4 /6 activity. The remainder of the cells in the population following differentiation may display markers indicative of mesodermal (for example, 10-15%) and/or extra- embryonic (for example, 5-10%) lineages. Increasing differentiation efficiency through CyclinD-CDK4/6 inhibition as described herein may be particularly useful for the differentiation of hIPSCs, which are known to exhibit poor differentiation capacity. In other aspects of the invention, CyclinD-Cdk4/6 activity is increased in the population of human pluripotent cells. Increased CyclinD-Cdk /6 activity may induce, drive or facilitate

neuroectodermal differentiation of the human pluripotent cells into neuroectoderm progenitor cells.

A neuroectoderm progenitor cell has reduced differentiation

potential compared to a pluripotent cell and exhibits a partially differentiated neuroectodermal phenotype. Neuroectoderm progenitor cells are committed to lineages in the neuroectoderm primary germ layer and are potentially capable of further differentiation into any foetal or adult cell type of the neuroectodermal germ layer. For example, a neuroectodermal progenitor may differentiate under appropriate conditions into any cell type in the central and peripheral nervous systems. In some embodiments, neuroectoderm progenitors may be termed "multipotent" . Neuroectodermal progenitor cells cannot give rise to extraembryonic, mesoderm or endoderm cell lineages .

Neuroectoderm progenitors may express one or more of the following neuroectoderm associated markers: Sox2, Soxl, Pax6 and Nestin. A neuroectoderm progenitor may lack markers associated with

pluripotency, such as Oct4, Sox2, Alkaline Phosphatase, POU5fl,

SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc. A neuroectoderm progenitor may lack markers associated with extraembryonic, mesoderm or endoderm cell lineages.

An increase in CyclinD-CDK4 /6 activity as described herein increases the proportion of human pluripotent cells in the population which differentiate into neuroectoderm progenitors. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the population of human pluripotent cells may

differentiate into neuroectoderm progenitor cells following

increases in CyclinD-Cdk4/6 activity. Increasing differentiation efficiency through increased CyclinD-CDK4/6 activity as described herein may be particularly useful for the differentiation of hIPSCs, which are known to exhibit poor differentiation capacity.

CyclinD-CDK4/6 activity is mediated in the pluripotent cells by complexes which comprise Cyclin D and CDK4 or Cyclin D and CDK6. The activity of CyclinD-CDK4 and CyclinD-CDK6 complexes (i.e. cyclinD- CDK4/6 activity) modulates the progression of the cell through the Gl phase of the cell cycle.

Inhibition of CyclinD-CDK4 and/or CyclinD-CDK6 activity (i.e.

CyclinD-CDK4/6 activity) increases the duration of the Gl phase in the human pluripotent cells and is shown herein to direct endodermal differentiation. In some preferred embodiments, both CyclinD-CDK4 and/or CyclinD-CDK6 may be inhibited, for example by inhibition of CDK4 and CDK6. Inhibition of CyclinD-CDK4 and/or CyclinD-CDK6 activity in a cell results in localization of Smad2/3 onto chromatin; transcriptional activation of endodermal genes; and phosphorylation of the

retinoblastoma protein. The extent of CyclinD-CDK4/6 inhibition in a cell may be determined by measuring one or more of these parameters. Suitable techniques are available in the art. For example, CyclinD- CDK4/6 inhibition in a cell may be determined be measuring the amount or extent of retinoblastoma protein phosphorylation in the cell, for example by western blotting using antibodies specific for phosphorylated retinoblastoma protein.

CyclinD-CDK4/6 activity may be inhibited in a cell by suppressing the expression or directly inhibiting the activity of one or more of i) Cyclins Dl to D3, ii) CDK4 and iii) CDK6.

In some preferred embodiments, the expression of cyclins Dl and D2; cyclins Dl and D3; cyclins D2 and D3; or cyclins Dl, D2 and D3 may be suppressed or their activities inhibited in a cell.

In some preferred embodiments, the expression of CDK4 and CDK6 may be suppressed or the activity of CDK4 and CDK6 may be inhibited in a cell . Human Cyclin Dl to D3, CDK4 and CDK6 are well-characterised in the art and reference sequences are available from public databases.

Cyclin Dl (CCND1; Gene ID: 595) has the reference amino acid sequence of NCBI database entry NP_444284.1 GI: 16950655 and is encoded by the reference nucleotide sequence of NCBI database entry NM_053056.2 GI:77628152.

Cyclin D2 (CCND2; Gene ID: 894) has the reference amino acid sequence of NCBI database entry NP_001750.1 GI: 4502617 and is encoded by the reference nucleotide sequence of NCBI database entry NM 001759.3 GI : 209969683. CyclinD3 (CCND3; Gene ID: 896) has the reference amino acid sequence of NCBI database entry NP_001129489.1 GI:209862835 and is encoded by the reference nucleotide sequence of NCBI database entry

N _001136017.2 GI : 209915554.

Cyclin-dependent kinase 4 (Cdk4 Gene ID: 1019) has the reference amino acid sequence of NCBI database entry NP_000066.1 GI: 4502735 and is encoded by the reference nucleotide sequence of NCBI database entry N _000075.3 GI : 345525417.

Cyclin dependent kinase 6 (Cdk6 Gene ID: 1021) has the reference amino acid sequence of NCBI database entry NP_001250.1 GI: 4502741 and is encoded by the reference nucleotide sequence of NCBI database entry NM_001259.6 GI : 223718130.

Preferably, CyclinD-CDK4 / 6 activity is inhibited in the human pluripotent cells in the absence of growth differentiation factors (GDFs) , such as GDF-8. Preferably, CyclinD-CDK4/6 activity is inhibited in the human pluripotent cells without activation of the Wnt signalling pathway. The Wnt signalling pathway is described in Logan and Nusse (2004) , Annu. Rev. Cell Dev. Biol. 20, 781-810 and Wodarz and Nusse (1998) , Annu. Rev. Cell Dev. Biol. 14, 59-88. For example, CyclinD-CDK4 /6 activity may be inhibited in the absence of Wnt ligands which activate the Frizzled (Fz) receptor; GSK-3 inhibitors; β-catenin or activators of β-catenin or other molecules which activate of the Wnt signalling pathway. CyclinD-Cdk4/6 activity may be inhibited in the population of human pluripotent cells by any suitable method. In some preferred

embodiments, cyclinD-Cdk4 / 6 activity may be inhibited by contacting, treating or exposing the human pluripotent cells to a cyclinD-Cdk4 / 6 inhibitor . The cyclinD-Cdk4/6 inhibitor may be a small chemical molecule, for example a non-polymeric organic compound having a molecular weight of less than 800 Daltons . Suitable small molecule cyclinD-Cdk4/6 inhibitors include PD0332991 ( 6-Acetyl-8 -cyclopentyl-5-methyl-2- ( 5-piperazin-l-yl-pyridin-2- ylamino) -8H-pyrido [2, 3-d] pyrimidin-7-one hydrochloride; RL

Sutherland et al Breast Cancer Res. 2009, 11(6), 112-113), Ro09- 3003, roscovitine (Seliciclib: 6-Benzylamino-2 [ (R) - ( 1 ' -ethyl-2 ' - hydroxyethylamino) ] -9-isopropylpurine) , olomoucine ( 6-Benylamino-2- (2-hydroxyethylamino) -9-methyl- purine); butyrolactone

(dihydrofuran-2 (3H) -one) , flavopiridol (2- (2-Chlorophenyl) -5, 7- dihydroxy-8- [ (3S, 4R) -3-hydroxy-l-methyl-4-piperidyl] chromen-4-one ) and purvalanol ( ( 2R) -2- [ [ 6- [ ( 3-Chlorophenyl ) amino] -9- ( 1- methylethyl) -9H-purin-2-yl] amino] -3-methyl-l-butanol) . AT-7519 (4-

(2, 6-Dichloro-benzoylamino) -lH-pyrazole-3-carboxylic acid piperidin- 4-ylamide methanesul fonic acid) , P276-00 ( 2-Aromatic-Substituted- 5, 7-dihydroxy-8- (2- (hydroxymethyl ) -l-methylpyrrolidin-3-yl ) -4H- chromen-4-one) , SNS-032 (BMS 387032; N-(5-(((5-(l, 1-dimethylethyl ) - 2-oxazolyl) methyl) thio) -2-thiazolyl ) -4-piperidinecarboxamide) , ZK

304709, R-547 (Ro-4584820; [ 4-Amino-2- [ ( 1-methylsulfonylpiperidin-4- yl ) amino] yrimidin-5-yl ] (2 , 3-difluoro-6-methoxyphenyl ) methanone) and AG-24322. P1446A-05 SCH 727965 (dinaciclib; ( S ) -3- ( ( ( 3-ethyl-5- ( 2- ( 2-hydroxyethyl ) piperidin-l-yl ) pyrazolo [1, 5-a] pyrimidin-7 - yl) amino)methyl)pyridine 1-oxide) , BAY1000394, CCI-779 ( ( 1R, 2R, 4S ) - 4-{ (2R) -2- [ (3S, 6R, 7E, 9R, 10R, 12R, 14S, 15E, 17E, 19E,21S, 23S,26R,27R, 34aS) -9, 27-dihydroxy-10,21-dimethoxy-6, 8, 12 , 14 , 20 , 26-hexamethyl- 1, 5, 11,28, 29-pentaoxo-l, 4, 5, 6, 9, 10, 11, 12, 13, 14, 21, 22, 23, 24, 25, 26, 27, 28,29,31,32,33,34, 34a-tetracosahydro-3H-23 , 27-epoxypyrido [2,1- c] [l,4] oxazacyclohentriacontin-3-yl] propyl } -2-methoxycyclohexyl 3- hydroxy-2- (hydroxymethyl) -2-methylpropanoate) , LY2835219,

Terameprocol (EM-1421; meso-1, 4-Bis (3, 4- dimethoxyphenyl ) dimethylbutane ) ; flavopiridol; 2-Bromo-12 , 13- dihydro-5H-indolo [2 , 3-a] pyrrolo [ 3 , 4-c] carbazole-5 , 7 ( 6H) -dione ; 5- (N- ( 4-Methylphenyl ) amino) -2-methyl-4 , 7-dioxobenzothiazole; 1,4-

Dimethoxyacridine-9 (10H) -thione; 4- ( ( ( 4-Hydroxy-5-propoxy-pyridin-2- ylmethyl) -amino) -methylene) -6-iodo-4H-isoquinoline-l , 3-dione; trans- 4- ( ( 6- ( ethylamino) -2- ( (1- (phenylmethyl ) -lH-indol-5-yl ) amino) -4- pyrimidinyl ) amino) -cyclohexanol , CINK4; and LY2835219.

Other suitable Cdk4/6 inhibitors are well-known in the art.

Suitable Cdk4/6 inhibitors may be obtained from commercial suppliers or synthesised using known synthetic pathways.

In some embodiments, PD0332991 ( 6-Acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-l-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d] pyrimidin-7- one hydrochloride may be preferred. For example, the pluripotent cells may be exposed to 0.1 to ΙΟμΜ, preferably 0.2 to 2 μ , most preferably 0.5 to ΙμΜ, for example about 0.75 μΜ, PD0332991. In other preferred embodiments, cyclinD-Cdk4 / 6 activity may be inhibited in the population of human pluripotent cells by nucleic acid suppression. For example, the cells may be transfected with a nucleic acid molecule, such as an siRNA or shRNA, which reduces or suppresses CyclinD-Cdk4 /6 activity, for example by suppressing the expression of one or more of Cyclin Dl, Cyclin D2, Cyclin D3, CDK4 or CD 6. In some preferred embodiments, the expression of Cyclin Dl, Cyclin D2 and Cyclin D3 may be suppressed or the expression of CDK4 and CDK6 may be suppressed. The use of nucleic acid suppression techniques such as anti-sense and RNAi suppression, to down-regulate gene expression is well- established in the art.

Transfection may be stable or transient. Suitable techniques for transfecting human pluripotent cells with suppressor nucleic acid molecules are well known in the art and include microinjection, electroporation and chemical-mediated techniques, such as cationic- lipid mediated transfection. Following transfection with the suppressor nucleic acid molecule, the pluripotent cells may be cultured in a definitive endoderm (DE) medium and allowed to differentiate into definitive endoderm cells. Definitive endoderm (DE) media are described in more detail elsewhere herein. When cyclinD-Cdk4/6 activity is inhibited by nucleic acid suppression, there is no need for the definitive endoderm (DE) differentiation medium to be additionally supplemented with a cyclinD-Cdk4/6 inhibitor .

In some embodiments, the human pluripotent cells are transfected with a heterologous nucleic acid which encodes a suppressor nucleic acid, such as an siRNA or shRNA. Expression of the heterologous nucleic acid in the cells leads to production of the suppressor nucleic and suppression of cyclinD-Cdk4 /6 activity. The heterologous nucleic acid may be contained in a vector, for example an adenoviral vector or lentiviral vector.

A nucleic acid molecule which suppresses CyclinD-Cdk4/6 activity (i.e. a suppressor nucleic acid molecule) may comprise or consist of all or part of the nucleotide coding sequence for the target polypeptide (i.e. Cyclin Dl, Cyclin D2, Cyclin D3, CDK4 or CDK6) , or its complement. For example, a nucleic acid molecule may comprise or consist of all or part (for example, 18 to 25 nucleotides) of the reference nucleotide coding sequence for Cyclin Dl, Cyclin D2,

Cyclin D3, CDK4 or CDK6 which is set out above, or its complement. CyclinD-Cdk4 /6 activity is suppressed in the pluripotent cells by down-regulation of the production of active target polypeptide. RNAi involves the expression or introduction into a cell of an RNA molecule which comprises a sequence which is identical or highly similar to a target gene. The RNA molecule interacts with mRNA which is transcribed from the target gene, resulting in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of the mRNA. This reduces or suppresses expression of the target gene (Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675- 3684; Voinnet & Baulcombe (1997) Nature 389: pg 553) .

The RNA molecule is preferably double stranded RNA (dsRNA) (Fire A. et al Nature 391, (1998)) . Synthetic siR A duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001) } .

Suitable RNA molecules for use in RNAi suppression include short interfering RNA (siRNA) . siRNA are double stranded RNA molecules of 15 to 40 nucleotides in length, preferably 15 to 28 nucleotides or 19 to 25 nucleotides in length, for example 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, two unmodified 21 mer oligonucleotides may be annealed together to form a siRNA. A siRNA molecule may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The overhang lengths of the strands are independent, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.

Other suitable RNA molecules for use in RNAi include small hairpin RNAs (shRNAs) . shRNA are single-chain RNA molecules which comprise or consist of a short (e.g. 19 to 25 nucleotides) antisense

nucleotide sequence, followed by a nucleotide loop of 5 to 9 nucleotides, and the complementary sense nucleotide sequence (e.g.

19 to 25 nucleotides) . Alternatively, the sense sequence may precede the nucleotide loop structure and the antisense sequence may follow. The nucleotide loop forms a hairpin turn which allows the base pairing of the complementary sense and antisense sequences to form the shRNA.

The siRNA, shRNA or other nucleic acid molecule may suppress the expression of one or more of Cyclin Dl, Cyclin D2, Cyclin D3, CDK4 or CDK6. The nucleic acid molecule may comprise or consist of a sequence which is identical or substantially identical (i.e. at least 90%, at least 95% or at least 98% identical) to a sequence of 15 to 28 contiguous nucleotides in the target gene. Suitable reference sequences of Cyclin Dl, Cyclin D2, Cyclin D3, CDK4 and CDK6 for the design of suppressor nucleic acids are publically available and described above. Nucleic acid molecules, such as siRNAs and shRNAs, useful for targeting Cyclin Dl to D3, CDK4 or CDK6 expression may be readily designed using reference sequences and software tools which are widely available in the art and may be produced using routine techniques. For example, a nucleic acid molecule may be chemically synthesized; produced recombinantly in vitro or cells (Elbashir, S. M. et al., Nature 411:494-498 (2001); Elbashir, S. M., et al., Genes & Development 15:188-200 (2001)) or obtained from commercial sources (e.g. Cruachem (Glasgow, UK), Dharmacon Research (Lafayette, Colo., USA)) .

Nucleic acid suppression may also be carried out using anti-sense techniques. Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences. The construction of anti-sense sequences and their use is well known in the art (Peyman and Ulman, Chemical Reviews,

90:543-584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol. 32:329- 376, (1992) ) .

Anti-sense oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse

orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene. The complementary anti- sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein.

The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example, fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.

In other preferred embodiments, cyclinD-Cdk4 /6 activity is inhibited by expressing a heterologous nucleic acid in the population of human pluripotent cells which encodes a protein factor which inhibits CyclinD-Cdk4/6 activity. For example, the population of pluripotent cells may be transfected with the heterologous nucleic acid and the transfected cells cultured in a definitive endoderm (DE) medium under suitable conditions for expression of the protein factor. The protein factor may inhibit one or more of Cyclin Dl, Cyclin D2, Cyclin D3, CDK4 or CDK6.

Suitable protein factors include pl5, pl6, pl8, pl9 and p27. P15 (CDKN2B; gene id 1030) has the reference amino acid sequence

NP_004927.2 GI : 17981694 and is encoded by the reference nucleotide sequence NM_004936.3 GI:47132608. P16 (CDKN2A; gene id 1029) has the reference amino acid sequence NP_000068.1 GI: 4502749 and is encoded by the reference nucleotide sequence NM_000077.4 GI : 300863097.

P18 (CDKN2C; gene id 1031) has the reference amino acid sequence NP_001253.1 GI: 4502751 and is encoded by the reference nucleotide sequence NM_001262.2 GI:17981697. P19 (CDKN2D; gene id 1032) has the reference amino acid sequence NP_001791.1 GI : 4502753 and is encoded by the reference nucleotide sequence NM_001800.3 GI: 39995074. P21 (CDKN1A; gene ID 1026) has the reference amino acid sequence

NP_000380.1 GI: 11386203 and is encoded by the reference nucleotide sequence NM_000389.4 GI : 310832422. P27 (CDK1B; gene ID 1027) has the reference amino acid sequence NP_004055.1 GI : 4757962 and is encoded by the reference nucleotide sequence NM_004064.3 GI : 207113192. Increased CyclinD-CDK4 and/or CyclinD-CDK6 activity (i.e. CyclinD- CDK4/6 activity) is shown herein to direct neuroectodermal

differentiation. In some preferred embodiments, both CyclinD-CDK4 and/or CyclinD-CDK6 activity may be increased, for example by overexpression of recombinant CDK4 and CDK6.

CyclinD-CDK4/6 activity may be increased in a cell by increasing the expression or activity of one or more of i) Cyclins Dl to D3, ii) CDK4 and iii) CDK6.

In some preferred embodiments, the expression or activity of cyclins Dl and D2; cyclins Dl and D3; cyclins D2 and D3; or cyclins Dl, D2 and D3 may be increased in a cell.

In some preferred embodiments, the expression or activity of CDK4 and CDK6 may be increased in a cell.

Preferably, CyclinD-CDK4 /6 activity is increased in the human pluripotent cells in the absence of growth differentiation factors (GDFs) , such as GDF-8.

Preferably, CyclinD-CDK / 6 activity is increased in the human pluripotent cells without activation of the Wnt signalling pathway. The Wnt signalling pathway is described in Logan and Nusse (2004), Annu. Rev. Cell Dev. Biol. 20, 781-810 and Wodarz and Nusse (1998), Annu. Rev. Cell Dev. Biol. 14, 59-88. For example, CyclinD-CDK4/6 activity may be increased in the absence of Wnt ligands which activate the Frizzled (Fz) receptor; GSK-3 inhibitors; β-catenin or activators of β-catenin or other molecules which activate of the Wnt signalling pathway.

CyclinD-Cdk4 / 6 activity may be increased in the population of human pluripotent cells by recombinant means. For example, cyclinD-Cdk4 / 6 activity may be increased by expressing a heterologous nucleic acid in the population of human pluripotent cells which encodes one or more of Cyclin Dl, Cyclin D2, Cyclin D3, CDK4 or CDK6 or a co-factor or activator thereof. For example, the population of pluripotent cells may be transfected with the heterologous nucleic acid and the transfected cells cultured in a neuroectoderm medium under suitable conditions for expression of the heterologous nucleic acid. The heterologous nucleic acid may be contained in a vector, for example an adenoviral vector or lentiviral vector.

A method may comprise transfecting the pluripotent cells with a nucleic acid molecule, such as an RNA or DNA, which encodes one or more of Cyclin Dl, Cyclin D2 , Cyclin D3, CDK4 or CDK6 or a co-factor or activator thereof, under conditions in which the cells express the nucleic acid molecule.

Transfection may be stable or transient. Suitable techniques for transfecting human pluripotent eelIs with suppressor nucleic acid molecules are well known in the art and include microin ection, electroporation and chemical-mediated techniques, such as cationic- lipid mediated transfection. Foilowing transfection with the nucleic acid molecule, the pluripotent cells may be cultured in a neuroectoderm medium and allowed to differentiate into neuroectoderm cells. Suitable neuroectoderm media are described in more detail elsewhere herein.

For the production of recombinant protein factor or nucleic acid molecule, nucleic acid sequences encoding the protein factor or nucleic acid molecule may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing

appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the encoding nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in a range of expression systems are well- known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts, such as E. coli and/or in eukaryotic cells, such as yeast, insect or mammalian cells. Vectors suitable for use in expressing a protein factor or nucleic acid molecule in pluripotent cells include plasmids and viral vectors e.g. adenoviral, or lentiviral vectors. Suitable techniques for expressing a protein factor or nucleic acid molecule in pluripotent cells are well known in the art (see for example; Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press or Protocols in Molecular Biology, Second Edition, Ausubel et al. eds . John Wiley & Sons, 1992; Recombinant Gene Expression

Protocols Ed RS Tuan (Mar 1997) Humana Press Inc) .

In some embodiments, the pluripotent cells may be differentiated into definitive endoderm (DE) progenitor cells in a DE

differentiation medium.

The DE differentiation medium is preferably a chemically defined medium (CDM) . A suitable DE differentiation medium may comprise a chemically defined nutrient medium which comprises a basal medium and optionally one or more additional defined components.

In other embodiments, the pluripotent cells may be differentiated into neuroectoderm progenitor cells in a neuroectoderm

differentiation medium.

The neuroectoderm differentiation medium is preferably a chemically defined medium (CDM) . A suitable neuroectoderm differentiation medium may comprise a chemically defined nutrient medium which comprises a basal medium and optionally one or more additional defined components.

Suitable chemically defined basal media include Iscove's Modified Dulbecco' s Medium (IMDM), Ham's F12, Advanced Dulbecco' s modified eagle medium (DMEM) (Price et al Focus (2003), 25 3-6), and RPMI- 1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508) . The basal medium may be supplemented by serum-free culture medium supplements and/or additional components in the DE differentiation medium. Suitable additional components may include L-glutamine or substitutes, such as GlutaMAX-1™, chemically defined lipids, albumin, 1-thiolglycerol , polyvinyl alcohol, insulin and

transferrin .

Suitable chemically defined nutrient media include CDM-PVA

(Johansson and Wiles (1995) Mol Cell Biol 15, 141-151), which comprises a basal medium supplemented with polyvinyl alcohol, insulin, transferrin and defined lipids. For example, a CDM-PVA medium may consist of: 50% Iscove's Modified Dulbecco's Medium (IMDM) plus 50% Ham's F12 with GlutaMAX-1™ supplemented with 1% chemically defined lipid concentrate, 450μΜ 1-thiolglycerol, 15 g/ml transferrin, 1 mg/ml polyvinyl alcohol, 7μg/ml Insulin.

Suitable CDM-PVA media are described in Vallier et al 2009 PLoS ONE 4: e6082. doi : 10.1371; Vallier et al 2009 Stem Cells 27: 2655-2666, Touboul 2010 51: 1754-1765. Teo et al 2011 Genes & Dev. (2011) 25: 238-250 and Peterson & Loring Human Stem Cell Manual: A Laboratory Guide (2012) Academic Press.

Other suitable chemically defined nutrient media include hESC maintenance medium (CDMA) which is identical to the CDM-PVA

described above with the replacement of PVA with 5 mg/ml BSA; and

RPMI basal medium supplemented with B27 and Activin (for example at least 50ng/ml) .

In some embodiments, CDM-PVA may be preferred for the

differentiation of endoderm cells and CDMA may be preferred for the differentiation of neuroectoderm cells.

In some embodiments, the DE differentiation medium or neuroectoderm medium may be devoid of differentiation factors. For example, a DE differentiation medium may consist of a chemically defined nutrient medium, such as CDM-PVA as described above and, optionally, a cyclin D-Cdk4/6 inhibitor (depending on the approach used to inhibit cyclin D-Cdk4/6 activity) . A neuroectoderm differentiation medium may consist of a chemically defined nutrient medium, such as CDMA- as described above. Differentiation factors are factors which modulate, for example promote or inhibit, a signalling pathway which mediates

differentiation in a mammalian cell. Differentiation factors may include growth factors, cytokines and inhibitors which modulate one or more of the Activin/Nodal , FGF, Wnt or BMP signalling pathways. Examples of differentiation factors include FGFs, BMPs, retinoic acid, GF ligands, such as Activin, TGF or Nodal, GDFs, LIF, IL, activin and phosphatidylinositol 3-kinase (PI3K) inhibitors.

A definitive endoderm (DE) or neuroectoderm differentiation medium may, for example, be devoid of GF , Bone Morphogenic Proteins

(BMPs) , PI3K inhibitors, activin/TGFp antagonists; retinoic acid; BMP antagonists; hedgehog signalling inhibitors; notch signalling inhibitors, glycogen synthase kinase 3β, and growth differentiation factors (GDFs), such as GDF-8.

In some embodiments, the DE differentiation medium may be devoid of Activin and Fibroblast Growth Factors. For example, the human pluripotent cells may be differentiated in the absence of Activin and FGF.

In other embodiments, the DE differentiation medium may comprise one or preferably both of Activin and FGF, for example one or both of Activin A and FGF2. The medium may be devoid of other

differentiation factors. For example, the DE differentiation medium may consist of a chemically defined nutrient medium, such as CDM-PVA as described herein, supplemented with i) Activin ii) FGF or iii) Activin and FGF; and optionally a cyclin D-Cdk4/6 inhibitor, depending on the approach used to inhibit cyclin D-Cdk4/6 activity. Activin (Activin A: NCBI GenelD: 3624 nucleic acid reference sequence NM_002192.2 GI : 62953137, amino acid reference sequence NP_002183.1 GI : 4504699) is a dimeric polypeptide which exerts a range of cellular effects via stimulation of the Activin/Nodal pathway (Vallier et al . Cell Science 118:4495-4509 (2005) ) . Activin is readily available from commercial sources (e.g. Stemgent Inc. MA USA) .

When present in the DE differentiation medium, low concentrations of Activin may be employed. For example, concentrations which are reduced relative to media which induce endodermal differentiation in the absence of CyclinD-Cdk4/6 inhibition. For example, the medium may be supplemented with a maximal amount of 1 to 20 ng/ml Activin A, preferably lOng/ml Activin A.

Fibroblast growth factor (FGF) is a protein factor which stimulates cellular growth, proliferation and cellular differentiation by binding to a fibroblast growth factor receptor (FGFR) . Suitable fibroblast growth factors include any member of the FGF family, for example any one of FGFl to FGF14 and FGF15 to FGF23.

Preferably, the FGF is FGF2 (NCBI GenelD: 2247, nucleic acid sequence NM_002006.3 GI : 41352694, amino acid sequence NP_001997.4 GI : 41352695); FGF7 (also known as keratinocyte growth factor (or KGF) , NCBI GenelD: 2247, nucleic acid sequence NM_002006.3 GI :

41352694, amino acid sequence NP_001997.4 GI : 41352695); or FGFl 0 (NCBI GenelD: 2247, nucleic acid sequence NM_002006.3 GI : 41352694, amino acid sequence NP_001997.4 GI : 41352695) . Most preferably, the fibroblast growth factor is FGF2.

Fibroblast growth factors, such as FGF2, FGF7 and FGF10, may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g. R&D Systems, Minneapolis, MN; Stemgent Inc, USA) .

When present in the medium, low concentrations of fibroblast growth factor may be employed. For example, concentrations which are reduced relative to media which induce endodermal differentiation in the absence of CyclinD-Cdk4 / 6 inhibition. For example, the medium may be supplemented with less than 40ng/ml FGF2, less than 30 ng/ml or preferably less than 20ng/ml FGF2, with a minimal amount of 12ng/ml FGF2.

The absence or low concentrations of Activin and FGF and the absence of cytokines and other differentiation factors may be useful in reducing the cost and technical difficulty of the methods described herein compared to known endoderm differentiation methods.

In some preferred embodiments, the definitive endoderm (DE)

differentiation medium may further comprise the cyclin D-Cdk4/6 inhibitor. For example, if the cyclin D-Cdk4/6 inhibitor is a small chemical molecule, the cyclin D-Cdk4/6 activity in the human pluripotent cells may be inhibited by culturing the cells in a DE differentiation medium which comprises the cyclin D-Cdk4/6

inhibitor.

The cyclin D-Cdk4/6 inhibitor may be present in the DE

differentiation medium in an amount or concentration which is sufficient to increase the duration of the Gl phase of the cell cycle in the pluripotent cells and/or induce endodermal

differentiation of the pluripotent cells. Cyclin D-Cdk4/6 inhibition in a cell may be determined, for example, by measuring

retinoblastoma protein phosphorylation, as described above. In other embodiments, following transfection of the human

pluripotent cells with the cyclin D-Cdk4/6 inhibitor or nucleic acid encoding the cyclin D-Cdk4/6 inhibitor, the cells may be allowed to differentiate in a DE differentiation medium which does not comprise the cyclin D-Cdk4/6 inhibitor. For example, following transfection with an siRNA or shRNA which inhibits cyclin D-Cdk4/6 activity, the pluripotent cells may be cultured in a DE differentiation medium which does not comprise a cyclin D-Cdk4/6 inhibitor.

The human pluripotent cells may be cultured in the DE

differentiation medium for 3 to 8 days following inhibition of

CyclinD-Cdk4/6 activity, preferably about 6 days, to produce the population of DE progenitor cells. In some embodiments, the neuroectoderm differentiation medium may be devoid of activin/TGF antagonists and Fibroblast Growth Factors. For example, the human pluripotent cells may be differentiated in the absence of SB-431542 and FGF.

In other embodiments, the DE differentiation medium may comprise one or preferably both of activin/TGFp antagonist and FGF, for example one or both of SB-431542 and FGF2. The medium may be devoid of other differentiation factors. For example, the neuroectodermal

differentiation medium may consist of a chemically defined nutrient medium, such as CDM-BSA or CDMA as described herein, supplemented with i) activin/TGF antagonist, such as SB-431542 ii) FGF or iii) 3θίί ίη/Τ6Ρβ antagonist and FGF.

When present in the medium, low concentrations of fibroblast growth factor may be employed. For example, concentrations which are reduced relative to media which induce neuroectodermal

differentiation in the absence of increased CyclinD-Cdk4 / 6 activity. For example, the medium may be supplemented with less than 40ng/ml FGF2, less than 30 ng/ml or preferably less than 20ng/ml FGF2, with a minimal amount of 12ng/ml FGF2.

The absence or low concentrations of 3θί ί ίη/Τ6Ρ antagonist and FGF and the absence of cytokines and other differentiation factors may be useful in reducing the cost and technical difficulty of the methods described herein compared to known endoderm differentiation methods . The human pluripotent cells may be cultured in the neuroectodermal differentiation medium for 3 to 8 days following the increase in CyclinD-Cdk4 / 6 activity, preferably about 6 days, to produce the population of neuroectodermal progenitor cells. The extent of differentiation of the pluripotent cell population may be determined during cell culture by monitoring and/or detecting the expression of one or more cell markers in the population of differentiating cells. For example, an increase in the expression of markers characteristic of definitive endoderm or neuroectoderm or a decrease in the expression of markers characteristic of pluripotent cells may be determined. Endoderm markers characteristic of DE progenitor cells are described above and may include S0X17, CXCR4 and GSC. DE progenitor cells may lack expression of pluripotency markers or markers associated with ectodermal or mesodermal

lineages. Neuroectoderm markers characteristic of neuroectoderm progenitor cells are described above and may include Sox2, Soxl, Pax6 and Nestin. Neuroectodermal progenitor cells may lack

expression of pluripotency markers or markers associated with endodermal or mesodermal lineages. For example the definitive endoderm or neuroectoderm cells may lack expression of one or more, preferably all, of the following; Oct4, Sox2, alkaline phosphatase, SSEA-3, Nanog, SSEA-4, Tra-1-60 and KLF-4.

The expression of cell markers may be determined by any suitable technigue, including immunocytochemistry, immunofluorescence, RT- PCR, immunoblotting, fluorescence-activated cell sorting (FACS) , and enzymatic analysis.

The methods described above may further comprise monitoring and/or detecting the presence of one or more definitive endoderm or neuroectoderm markers and/or the absence of one or more pluripotency markers in the population of cells.

The methods described above may further comprise identifying one or more cells in the population as definitive endoderm progenitor cells, for example from the presence of expression of one or more endoderm markers; or identifying one or more cells in the population as neuroectoderm progenitor cells, for example from the presence of expression of one or more neuroectoderm markers

Differentiation of human pluripotent cells as described herein may produce a population of human progenitor cells which is

substantially free from other cell types. For example, the

population may contain 85% or more, 90% or more, 95% or more, or 98% or more definitive endoderm or neuroectodermal progenitor cells, following culture in the medium. Preferably, the population of definitive endoderm or neuroectodermal progenitors is sufficiently free of other cell types that no purification is required. If required, the definitive endoderm or neuroectodermal progenitor cells may be separated from other cell types in the population using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and magnetic beads or fluorescence activated cell sorting (MACS or FACS) including the use of antibodies against extracellular regions of characteristic markers as described above.

The methods described above may further comprise culturing,

expanding or maintaining the population of definitive endoderm or neuroectodermal progenitor cells. Suitable techniques for the culture of definitive endoderm progenitor cells are well-known in the art (see, for example, Basic Cell Culture Protocols, C.

Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of

Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol

Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) Basic Cell Culture Protocols' by J. Pollard and J. M. Walker (1997), 'Mammalian Cell Culture: Essential Techniques' by A. Doyle and J. B. Griffiths (1997), 'Human Embryonic Stem Cells' by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside' by A. Bongso (2005), Peterson & Loring (2012)Human Stem Cell Manual: A Laboratory Guide Academic Press and 'Human Embryonic Stem Cell Protocols' by K. Turksen (2006) . Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems) . Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37°C, 21% Oxygen, 5% Carbon Dioxide. Media is preferably changed every two days and cells allowed to settle by gravity. In some embodiments, the definitive endoderm or neuroectodermal progenitor cells may be further differentiated.

For example, the definitive endoderm progenitor cells may be cultured in a differentiation medium and allowed to differentiate into an endoderm lineage. For example, the DE progenitor cells may be differentiated into cells of a pancreatic lineage, such as dorsal foregut cells, pancreatic endoderm cells or pancreatic endocrine progenitors or cells of a hepatic lineage, such as ventral foregut cells, hepatic endoderm cells or hepatocytes. Populations of endoderm cells produced as described herein may display increased homogeneity relative to populations produced by other techniques.

In some embodiments, the population of definitive endoderm cells may be differentiated into cells of a hepatic lineage, for example hepatocytes or hepatic progenitor cells, such as ventral foregut cells or hepatic endoderm cells. Suitable methods for hepatic differentiation are available in the art (see for example

O2012/025725; Yusa et al Nature. 2011 Oct 12; 478 (7369) : 391-4; Cho et al Diabetologia . 2012 Dec; 55 ( 12 ) : 328 -95 ; Hannan et al Nat Protoc. 2013 Jan 31 ; 8 ( 2 ) : 430-7 ; Touboul et al Hepatology. 2010 May; 51 (5) : 1754-65; Si-Tayeb et al Hepatology. 2010 Jan; 51 ( 1 ) : 297- 305; Song et al Cell Res. 2009 Nov; 19 ( 11 ) : 1233-42 ; Zhao et al PLoS One. 2009 Jul 31 ; 4 ( 7 ) : e6468 ; Hay et al Proc Natl Acad Sci U S A. 2008 Aug 26; 105 ( 34 ) : 12301-6. Baharvand et al Differentiation. 2008 May; 76 (5) : 465-77. Agarwal et al Stem Cells. 2008 May; 26 ( 5) : 1117-27. Cai et al Hepatology. 2007 May; 45 (5) : 1229-39; Cai, J., et al J Mol Cell Biol 2(1) : 50-60; D'Amour, K. A. et al (2006) , Nat Biotechnol 24(11) : 1392-401; Jiang, W. et al . (2007) Cell Res 17(4) : 333-44.

In brief, any one of methods described above may further comprise; culturing the population of DE progenitor cells produced as described above in a hepatic induction medium to produce a

population of hepatic progenitor cells,

wherein the hepatic induction medium is a chemically defined medium which stimulates SMAD2 and SMAD3 mediated signalling

pathways . A suitable hepatic induction medium may comprise a chemically defined basal medium supplemented with one or more additional factors, preferably recombinant human factors, which induce the DE progenitor cells to differentiate into hepatic progenitor cells.

Suitable chemically defined basal media include RPMI-1640, which is described above, preferably supplemented with B27 supplement. The CDM may be supplemented with a TGFp ligand which stimulates SMAD2 and SMAD3 mediated signalling pathways, such as TGF or activin, as described above. Preferably, the medium is supplemented with 5 to

500 ng/ml of TGFfi ligand, such as activin, preferably about 50ng/ml.

The population of DE progenitor cells may be cultured for 4 to 6 days, preferably about 5 days, to produce the population of hepatic progenitor cells.

In some preferred embodiments, the hepatic induction medium may be supplemented with Activin alone for 2-3 days, preferably about 3 days, and then Activin, BMP4 and FGF10 for 2-3 days, preferably about 3 days (see Cho et al Diabetologia . 2012 Dec; 55 (12) : 3284-95) .

The hepatic progenitor cells may be further differentiated into hepatocytes. For example, a method may further comprise;

culturing the population of hepatic progenitor cells in a hepatic maturation medium to produce a population of hepatocytes.

A suitable hepatic maturation medium may consist of a chemically defined basal medium supplemented with additional factors,

preferably recombinant human factors, to induce the hepatic

progenitor cells to mature into hepatic progenitor cells. Suitable chemically defined basal media include CMRL and hepatozyme SFM.

(GIBCO^M; Invitrogen Inc) . CMRL basal medium is a serum-free basal medium which is well known in the art and readily available from commercial sources (e.g. Cat No: 11530037 Invitrogen; Product #C0422 Sigma) . Hepatozyme SFM is a serum-free basal medium which is available from commercial sources (e.g. Cat No 17705; Invitrogen) . The chemically defined basal medium may be supplemented with one or more factors which induce differentiation and maturation of hepatic progenitors into hepatocytes. For example, the basal medium may be supplemented with hepatocyte growth factor (HGF) or epidermal growth factor (EGF) . The chemically defined basal medium may also be supplemented with one or more factors which induce differentiation and maturation of hepatocyte, such as oncostatin-M .

The population of hepatic progenitor cells may be cultured for 10 to 40 days, preferably about 25 days, to produce the population of hepatocytes .

Suitable techniques, media and reagents or differentiation of DE progenitor cells into hepatic progenitors and hepatocytes are described in O2012/025725; Yusa et al Nature. 2011 Oct 12;

478 (7369) : 391-4 and Cho et al Diabetologia . 2012 Dec; 55(12) :3284- 95.

In some embodiments, the population of definitive endoderm cells may be differentiated into pancreatic progenitor cells. Suitable methods for pancreatic differentiation are available in the art (see for example Cho et al Diabetologia. 2012 Dec; 55 ( 12 ) : 3284-95; D'Amour et al., 2006), Jiang et al., 2007, Cai et al . , 2010) . In brief, methods described above may further comprise;

culturing the population of definitive endoderm cells in a first pancreatic induction medium comprising an activin antagonist;

FGF; retinoic acid; and a BMP inhibitor to produce a population of dorsal foregut cells;

culturing the dorsal foregut cells in a second pancreatic induction medium comprising FGF, retinoic acid, a BMP inhibitor, and a hedgehog signalling inhibitor and then;

culturing the cells in a third pancreatic induction medium differentiation factors comprising FGF;

thereby producing a population of pancreatic progenitor cells. A suitable first pancreatic induction medium may be a chemically defined medium (CDM) which comprises an activin/TGF antagonist; FGF; retinoic acid; and a BMP antagonist. In some embodiments, these may be the only differentiation factors in the medium.

For example, the first pancreatic induction medium may consist of a chemically defined basal medium, such as advanced DMEM, supplemented with an 3θί ίνίη/Τ0Γβ antagonist, preferably SB-431542 (for example, 5 to 25 μΜ, preferably about 10 μΜ) , FGF, preferably FGF10 ( for example 5 to lOOng/ml, preferably about 50ng/ml) , retinoic acid (for example at 0.5 to 20 μΜ, preferably about 2 μΜ) and a BMP

antagonist, preferably noggin (for example 100 to 500ng/ml) .

Preferably, the population of definitive endoderm cells may be cultured for 2 to 4 days, most preferably 3 days to produce the population of dorsal foregut cells.

A suitable second pancreatic induction medium may be a chemically defined medium (CDM) which comprises FGF, a BMP inhibitor, retinoic acid, and a hedgehog signalling inhibitor. In some embodiments, these may be the only differentiation factors in the medium. For example, the second pancreatic induction medium may consist of a chemically defined basal medium, such as advanced DMEM, supplemented with an FGF, preferably FGF10 (for example at 5 to lOOng/ml, preferably about 50ng/ml); retinoic acid, (for example at 0.5 to 20 μΜ, preferably about 2 μΜ) ; hedgehog signalling inhibitor,

preferably KAAD-cyclopamine (for example 0.1 to 1 μΜ, preferably 0.25 μΜ) ; and a BMP antagonist, preferably noggin (for example 5 to 500ng/ml or 100 to 500 ng/ml, preferably about 50ng/ml) .

The dorsal foregut cells may be cultured in the second pancreatic induction medium for 2 to 4 days, most preferably 3 days.

A suitable third pancreatic induction medium may be a chemically defined medium (CDM) which comprises FGF. In some embodiments, FGF may be the only differentiation factor in the medium. For example, the third pancreatic induction medium may consist of a chemically defined basal medium, such as advanced DMEM, supplemented with an FGF, preferably FGF10 (for example at 5 to lOOng/ml, preferably about 50ng/ml) . The cells may be cultured in the third pancreatic induction medium for 2 to 4 days, most preferably 3 days to produce a population of pancreatic progenitor cells.

Optionally, the pancreatic progenitor cells may be further

differentiated and/or matured into pancreatic endocrine cells. For example, a method may further comprise;

culturing the population of pancreatic progenitor cells in a first endocrine induction medium and a second endocrine induction medium to produce a population of pancreatic endocrine cells.

Suitable protocols, reagents and media for the maturation of pancreatic endocrine cells are available the art (see Kroon E et al . (2008) Nat Biotechnol 26: 443-452 and Cho et al Diabetologia . 2012 Dec; 55 (12) : 3284-95) .

A suitable first endocrine induction medium may be a chemically defined medium (CDM) supplemented with a serum-free media

supplement, such as B27; which further comprises a Notch signalling inhibitor. In some embodiments, the Notch signalling inhibitor may be the only differentiation factor in the medium.

Suitable serum-free media supplements include B27 (Brewer et al Brain Res (1989) 494 65-74; Brewer et al J. Neurosci Res 35 567-576 (1993); Brewer et al Focus 16 1 6-9; Brewer et al (1995) J.

Neurosci. Res. 42:674-683; Roth et al J Trace Elem Med Biol (2010) 24 130-137) and NS21 (Chen et al J. Neurosci Meths (2008) 171 239- 247) . Serum-free media supplements, such as B27 and N21, are well known in the art and widely available commercially (e.g. Invitrogen; Sigma Aldrich Inc) .

For example, the first endocrine induction medium may consist of a chemically defined basal medium, such as advanced DMEM, supplemented with B27 and Notch signalling inhibitor, preferably DAPT (for example at 0.1 to 10 mM, preferably about 1 mM) .

The pancreatic progenitor cells may be cultured in the first endocrine induction medium for 2 to 4 days, most preferably 3 days.

A suitable second endocrine induction medium may be a chemically defined medium (CDM) , such as advanced DMEM, supplemented with B27, without additional differentiation factors.

The pancreatic progenitor cells may be cultured in the second endocrine induction medium for 2 to 4 days, most preferably 3 days.

Suitable techniques, media and ingredients thereof for

differentiation of DE progenitor cells into pancreatic progenitors and pancreatic exocrine cells are described in Cho et al 2012 supra.

Neuroectoderm progenitor cells produced as described above may be cultured in a differentiation medium and allowed to differentiate into a neuroectoderm lineage. For example, the neuroectoderm progenitor cells may be differentiated into cells of the central or peripheral nervous system, including neurons of all types and glial cells. Populations of neuroectodermal cells produced as described herein may display increased homogeneity relative to populations produced by other techniques.

Suitable methods for the differentiation of neuroectoderm progenitor cells are well known in the art. Another aspect of the invention provides a definitive endoderm differentiation medium, for example for use in a method described above, comprising;

a chemically defined nutrient medium and;

an inhibitor of cyclin D-CDK4/6 activity.

Suitable DE differentiation media are described in more detail above . The definitive endoderm differentiation medium supports the

differentiation of human pluripotent cells into DE progenitor cells, as described above.

The DE differentiation medium may be devoid of i) activin A, ii) FGF or iii) both activin A and FGF. More preferably, the DE

differentiation medium may be further supplemented with activin A (for example, lOng/ml) and FGF2 (for example, 10 to 20ng/ml, e.g. 12ng/ml) . Other than the optional inclusion of activin and/or FGF, the DE differentiation medium is preferably devoid of

differentiation factors. For example, the definitive endoderm (DE) differentiation medium may be devoid of cytokines, Bone Morphogenic Proteins (BMPs) , PI3K inhibitors, glycogen synthase kinase 3β inhibitors, and growth differentiation factors (GDFs), such as GDF- 8.

The DE differentiation medium comprises a cyclin D-CDK4/6 inhibitor. Suitable inhibitors of cyclin D-CDK4/6 activity are described above and include small chemical molecule inhibitors of Cdk4 and/or Cdk6. In some preferred embodiments, the CDK4/6 inhibitor is PD0332991. For example, the cell culture medium may comprise 0.1 to 10μΜ

PD0332991, preferably about 0.75μΜ. As described above, a chemically defined nutrient medium may comprise a chemically defined basal medium and one or more defined supplements or other components

Chemically defined basal media are well known in the art and include 50% IMDM 50% F12 NUT-MIX. The basal medium may be supplemented, for example, with transferrin, 1-thioglycerol , defined lipids, polyvinyl alcohol and optionally insulin.

For example, the chemically defined nutrient medium may comprise a basal medium consisting of 50% IMDM (Gibco) and 50% F12 NUT-MIX (Gibco) which is supplemented with 7μg/ml insulin; 15μg/ml

transferrin; 1 mg/ml polyvinyl alcohol (PVA; 1% chemically defined lipid concentrate (Invitrogen) ; and 450μΜ 1-thiolglycerol (Johansson and Wiles (1995) Mol Cell Biol 15, 141-151) . Other suitable medium are described above and in the experimental section below. In some preferred embodiments, the DE differentiation medium may comprise or consist of 50% IMDM plus 50% F12 NUT-MIX; Ίμς/ιαΐ insulin; 15μς/πι1 transferrin; 1 mg/ml polyvinyl alcohol (PVA) ; 1% chemically defined lipid concentrate; 450μΜ 1-thiolglycerol and 0.1 to 10μΜ PD0332991. Other chemically defined nutrient media may comprise or consist of the constituents set out above with the PVA replaced by BSA (hESC maintenance medium; CDM-BSA) .

The DE differentiation medium may be formulated in deionized, distilled water. The medium will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. The culture medium may be frozen (e.g. at -20°C or -80°C) for storage or transport. The medium may contain one or more antibiotics to prevent contamination. The DE differentiation medium may be a lx formulation or a more concentrated formulation, e.g. a 2x to 250x concentrated medium formulation. In a lx formulation each ingredient in the medium is at the concentration intended for cell culture, for example a

concentration set out above. In a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture. Concentrated culture media is well known in the art. Culture media can be concentrated using known methods e.g. salt precipitation or selective filtration. A concentrated medium may be diluted for use with water (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.

The culture medium may be contained in a hermetically-sealed vessel. Hermetically-sealed vessels may be preferred for transport or storage of the culture media, to prevent contamination. The vessel may be any suitable vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag. Another aspect of the invention provides a culture medium supplement comprising a cyclin D-CDK4/6 inhibitor for use in producing a DE differentiation medium as described above.

A culture medium supplement is a mixture of ingredients that cannot itself support human pluripotent cell growth or differentiation, but which enables or improves human pluripotent cell culture and differentiation when combined with other cell culture ingredients. The supplement may therefore be combined with other cell culture ingredients to produce a functional DE differentiation medium as described herein. The use of culture medium supplements is well known in the art. The culture medium supplement may comprise an inhibitor of cyclin D- CDK4/6 activity. The supplement may comprise any of the cyclin D- CDK4/6 inhibitors described above, including PD0332991. The

supplement may also comprise one or more additional cell culture ingredients as disclosed herein. For example, the supplement may comprise one or more cell culture ingredients selected from the group consisting of amino acids, vitamins, inorganic salts, carbon energy sources, buffers, transferrin, 1-thioglycerol , defined lipids, polyvinyl alcohol and insulin. The supplement may further comprise activin A (for example, lOng/ml) and/or FGF2 (for example, 10 to 20ng/ml, e.g. 12ng/ml) .

A culture medium supplement may be a concentrated liquid supplement (e.g. a 2x to 250x concentrated liquid supplement) or may be a dry supplement. Both liquid and dry supplements are well known in the art .

A supplement may be lyophilised. A supplement as described herein will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. A culture medium supplement may be frozen (e.g. at -20°C or -80°C) for storage or transport.

Another aspect of the invention provides a kit comprising a cyclin D-CDK4/6 inhibitor for use in formulating a DE differentiation medium as described above.

Cyclin D-CDK4/6 inhibitors are described above. The kit may further comprise a chemically defined basal medium, a supplement as described above and/or one or more other cell culture ingredients selected from the group consisting of amino acids, vitamins, inorganic salts, carbon energy sources, buffers, activin, FGF, transferrin, 1-thioglycerol , defined lipids, polyvinyl alcohol and optionally insulin.

The components of the kit may be contained in separate hermetically- sealed vessels. Another aspect of the invention provides a kit comprising a DE differentiation medium as described above and one or more antibodies which bind to definitive endoderm markers.

Suitable antibodies are well known in the art.

Kits as described above for endodermal differentiation of

pluripotent cells may further comprise a cell culture vessel.

Suitable cell culture vessels, such as flasks, single or multiwell plates, single or multiwell dishes, bottles, jars, vials, bags and bioreactors, are well-known in the art.

Another aspect of the invention provides the use of a cyclin D-CDK 4/6 inhibitor as described above in a method for the in vitro differentiation of pluripotent cells into definitive endoderm progenitors. Another aspect of the invention provides the use of a DE differentiation medium as described above in a method for the in vitro differentiation of pluripotent cells into definitive endoderm progenitors .

Suitable methods and cyclin D-CDK 4/6 inhibitors are described above .

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments

described above with the term "comprising" replaced by the term "consisting essentially of". It is to be understood that the application discloses all

combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise.

Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific

disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below. Figure 1 shows that the differentiation capacity of hESCs varies during cell cycle progression. The figure shows cell cycle dependent binding of Smad2/3 to endoderm genes. ChIP analyses in sorted Fucci- hESCs showing Smad2/3 binding on endoderm genes in early Gl . Figure 2 shows cell-cycle dependent differentiation of hESCs. Q-PCR analysis for the expression of germ layer markers in FACS sorted Fucci-hESCs incubated for 12 hours in culture condition inductive for neuroectoderm, endoderm or, mesoderm differentiation.

Figure 3 shows early Gl phase directs endoderm whereas late Gl promotes neuroectoderm differentiation. Flow cytometry analysis for the expression of germ layer markers in FACS sorted Fucci-hESCs incubated for up to three days in culture condition inductive for endoderm or neuroectoderm differentiation. Scale bar, 100mm. All data are shown as mean+s.d. (n=3) . Student's t-test was performed. P<0.05 marked with (*) .

Figures 4-6 show that cyclin Ds are necessary for pluripotency .

Figure 4 shows cyclin D expression during early differentiation of hESCs. Cyclin Dl-3 protein expression during day 1 to 3 on

neuroectoderm, endoderm and mesoderm differentiation shown by western blot analysis.

Figure 5 shows triple knockdown of Cyclin D induces endoderm differentiation. Cyclin Dl/3 double knockdown cells were transfected with a Cyclin D2 shRNA construct expressing GFP and then FACS sorted for Q-PCR analyses. Figure 6 LH panel shows double knockdown of Cyclin D causes endoderm differentiation and blocks neuroectoderm differentiation. Cyclin D double knockdown cells were analysed for germ layer marker expression by western blot. Figure 6 middle panel shows triple knockdown of Cyclin D causes endoderm differentiation. Cyclin Dl/3 double knockdown cells were transfected with a Cyclin D2 shRNA construct expressing GFP and then FACS sorted for western blot analyses. Figure 6 RH panel shows cyclin D knockdown causes the accumulation of Smad2/3 on chromatin. Relative amount of Smad2/3 protein in cytoplasm and on chromatin in Cyclin Dl-3 knockdown cells compared to Scramble shRNA overexpressing cells. Scale bar, 100mm. Data shown as meanls.d. (n=3) . Student's t-test was performed.

P<0.05 marked with (*) .

Figures 7 to 9 show cyclin Ds promote neuroectoderm and block endoderm. Figure 7 shows cyclin Dl T286A mutant hESCs exhibit neuroectodermal morphology. Representative colonies of GFP

expressing control hESCs and Cyclin Dl mutant expressing cells.

Figure 8 (top R panel) shows Cyclin Dl T286A mutant accumulates in the nucleus of hESCs through Western blot analysis for Cyclin Dl expression in nuclear extracts of hESCs stably overexpressing Cyclin Dl T286A compared to control over-expressing GFP cells. Figure 8 (bottom R panel and L panel) show cyclin D overexpression causes neuroectoderm differentiation. Expression of neuroectoderm markers in Cyclin D overexpressing cells shown by Q-PCR analyses (bottom R) and western blot (L) . Figure 9 shows Cyclin D overexpression results in Smad2/3 accumulation in the cytoplasm. Smad2/3 localisation in cytoplasm and on chromatin was analysed in Cyclin Dl, D2 and D3 overexpressing cells by western blot. UD - undifferentiated. Scale bar, 100mm. Data shown as meanls.d. (n=3) . Student's t-test was performed. P<0.05 marked with (*) . UD - undifferentiated.

Figures 10-13 show cyclin Ds regulate differentiation genes. Figure 10 shows cell cycle dependent binding of Cyclin Ds to neuroectoderm genes and Figure 11 shows endoderm genes. Cyclin D1-D3 ChIP was performed on FACS sorted Fucci-hESCs cells and then Q-PCR was performed to detect the genes denoted. Figure 12 shows that cyclins D1-D3 interact with p300 and HDAC1 on chromatin of hESCs. Cyclin D proteins were immunoprecipitated from chromatin fraction and analysed for the presence of p300 and HDAC1 by western blot. Figure 13 shows cyclin D proteins recruit transcriptional co-regulators to genes controlling germ layer specification. ChIP of histone H3K4me3, H3K27me3, p300 and HDAC1 was performed in Cyclin D double knockdown hESCs and then Q-PCR performed to detect genomic regions corresponding to key developmental genes. Scale bar, 100mm. Data shown as meanls.d. (n=3) . Student's t-test was performed. P<0.05 marked with (*) . UD - undifferentiated.

Figures 14-16 shows endoderm production by CDK4/6 inhibition. Figure

14 (top R panel) shows that Smad2/3 interacts with Cyclin D

proteins. Smad2/3 was immunoprecipitated and analysed for the presence of Cyclin Dl-3 by western blot. Figure 14 (LH panel) shows that Cyclin D proteins interact with Smad2/3. Cyclins Dl-3 were immunoprecipitated and analysed for the presence of Smad2/3 by western blot. Figure 14 (bottom R panel) shows that CDK4/6

inhibition by small molecule results in Smad2/3 accumulation on chromatin. hESCs cells were treated with CDK4/6 inhibitor (CDKi, or 0.75mM PD0332991) for 2 h or 8 h and then Smad2/3 localisation in cytoplasm and on chromatin was analysed using western blot. Figure

15 shows the morphology of hESCs grown for 6 days in the presence of CDKi (0.75mM PD0332991) . Representative colonies of untreated hESCs and CDKi treated cells. Figure 16 shows that CDK4/6 inhibition results in endoderm differentiation. hESCs grown for 6 days in the presence of CDKi (0.75mM PD0332991) were analysed for the expression of germ layer markers using Q-PCR.

Figure 17 to 21 show that CDK4/6 inhibition in hESCs causes endoderm differentiation. Figure 17 shows CDKi-produced endoderm can give rise to pancreatic and hepatic cells. H9 hESCs were differentiated into endoderm with 0.75 μΜ PD0332991 for 6 days and then the resulting cells were grown in culture conditions inducing pancreatic and hepatic differentiation for 12 and 22 days respectively. Marker expression was analysed by Q-PCR. Conventional endoderm

differentiation protocol was used as a positive control for

pancreatic and hepatic differentiation. Data shown as mean ±s.d. (n=3) . Figures 18-21 show that cyclin Dl TI56A mutant increases endoderm/mesoderm and decreases neuroectoderm differentiation of hESCs. Figure 18 shows Cyclin Dl TI56A overexpressing cells were analysed by Q-PCR for marker expression. Figure 19 shows

neuroectoderm differentiation. Cyclin Dl T156A cells were differentiated into neuroectoderm and then expression of neuroectodermal markers was analysed by Q-PCR. Figure 20 shows endoderm differentiation. Cyclin Dl TI56A cells were differentiated into endoderm and then expression of endoderm markers was analysed by Q-PCR. Figure 21 shows mesoderm, differentiation. Cyclin Dl T156A cells were differentiated into mesoderm and then expression of mesoderm markers was analysed by Q-PCR Data shown as meanls.d.

(n=3) .

Figures 22 to 24 show that CDK4/6 inhibition in hiPSCs causes endoderm differentiation. Figure 22 shows CDK4/6 inhibition in the hiPSC IPS40 cell line. IPS40 cells were cultured in the presence of 0.75 μΜ PD0332991 for 6 days and analysed by Q-PCR and

immunostaining (not shown) . Figure 23 shows CDK4/6 inhibition in the hiPSC cell line A1ATD1. AlATDl cells were cultured in the presence of 0.75 μΜ PD0332991 for 6 days and analysed by Q-PCR or

immunostaining. Figure 24 shows CDK4/6 inhibition in hiPSC BBHX8 cell line. BBHX8 cells were cultured in the presence of 0.75 μΜ PD0332991 for 6 days and analysed by Q-PCR or immunostaining (not shown) . Data shown as mean+s.d. (n=3) . Scale bar, ΙΟΟμπι.

Figure 25 shows that hiPSC CDKi-produced endoderm cells can give rise to pancreatic cells. IPS40, AlATDl and BBHX8 cells were differentiated into endoderm with 0.75 μΜ PD0332991 for 6 days and then further into pancreatic cells. Cells were analysed by Q-PCR or by immunofluorescence microscopy (not shown) at day 18 (day 12 for Pdxl) in IPS40, AlATDJ or BBHX8. Conventional endoderm

differentiation protocol was used as a positive control. Data shown as mean+s.d. (n=3) . Scale bar, ΙΟΟμπι.

Figure 26 shows that hiPSC CDKi-produced endoderm cells can give rise to hepatic cells. Figures 9a-d show hepatic differentiation of endoderm cells generated from WPSCs using CDKi . IPS40, AlATDl and BBHX8 cells were differentiated into endoderm with 0.75 μΜ PD0332991 for 6 days and then further into hepatocytes. Cells were analysed by Q-PCR (a) or by immunofluorescence microscopy at day 25 in IPS40 (b) , AlATDl (c) or BBHX8 (d) . Conventional hepatic differentiation protocol was used as a positive control. Data shown as meanls.d. (n=3) . Scale bar, ΙΟΟμπι.

Experiments

1. Methods

1.1 Cell Culture

hESCs (H9 from WiCell) were used for all the experiments otherwise stated. H9 cells were grown in defined culture conditions as described previously (Brons, I. G. et al. Nature 448, 191-195

(2007)) . H9 cells were passaged weekly using collagenase IV and maintained in chemically defined medium (CDM) supplemented with Activin A (10 ng/ml) and FGF2 (12 ng/ml) . hIPSCs (IPS40 and BBHX824; A1ATD125) were grown in culture conditions as described before (Rashid, S. T. et al. J Clin Invest 120, 3127-3136 (2010)), mESCs were grown on irradiated mouse embryonic fibroblasts in the presence of Leukemia Inhibitory Factor (LIF) or in 2i system as described previously (Ying, Q. L. et al. Nature 453, 519-523 (2008)) .

1.2 Differentiation of hESCs and IPSCs

hESCs were differentiated into neuroectoderm, endoderm and mesoderm as described previously (Valier et al PLoS One 4, e6082, (2009) .

Briefly, cells were cultured in CDM supplemented with SB-431542 (10 μΜ; Tocris) and FGF2 (12 ng/ml) for neuroectoderm, in CDM+PVA supplemented with Activin A (100 ng/ml), FGF2 (20 ng/ml), BMP4 (10 ng/ml), Ly294002 (10 μΜ; Promega) and CHIR99021 (3μΜ; Selleck) for mesoderm and in CDM-PVA supplemented with Activin A (100 ng/ml) , FGF2 20 ng/ml), BMP4 (10 ng/ml) and Ly294002 (10 μΜ; Promega) for endoderm. hIPSCs were differentiated into endoderm and to hepatocytes as described before (Touboul et al Hepatology 51, 1754-1765 (2010)) . Pancreatic differentiation of hESCs and hIPSCs was carried out as follows. Daily media changes were made during the entire

differentiation protocol. After endoderm differentiation, cells were cultured in Advanced DMEM (Invitrogen) supplemented with SB-431542 (10 μΜ; Tocris), FGF10 (50 ng/ml; AutogenBioclear) , all-trans retinoic acid (RA, 2 μΜ; Sigma) and Noggin (50 ng/ml; R&D Systems) for 3 days. Cells were then cultured in Advanced DMEM + human FGF10 (50 ng/ml; AutogenBioclear ) , all-trans retinoic acid (RA, 2 μΜ;

Sigma), KAAD-cyclopamine (0.25 μΜ; Toronto Research Chemicals) and Noggin (50 ng/ml; R&D Systems) for 3 days. Next, cells were cultured in human KGF (50 ng/ml; R&D Systems) for 3 days. For maturation of pancreatic progenitors, cells were grown in Advanced DMEM + 1% vol/vol B27 and DAPT (1 mM) for 3 days and for 3 additional days in Advanced DMEM + 1% vol/vol B27. 1.3. Endoderm differentiation of hESCs with CDK4/6 inhibition.

hESCs were maintained in CDMA supplemented with Activin A (10 ng/ml) + FGF2 (12 ng/ml; produced in-house) and splitted two days prior to differentiation. Maintenance media was changed 24 hours prior to starting the differentiation. CDKi-mediated endoderm differentiation of hESCs was carried out for up to 6 days in CDM-PVA media

supplemented with a low dose of Activin A (10 ng/ml) , FGF2 (12 ng/ml) and CDK4/6 inhibitor PD0332991 (Selleck, 0.75 μΜ) . Media was changed every day. CDMA media was prepared as follows: 250 ml IMDM (Gibco, Cat. No. 21980), 250 ml F12+GlutaMAX-l (Gibco, Cat. No. 31756), 5 ml CD Concentrated Lipids (Gibco, Cat. No. 11905), 20 μΐ 1-Thioglycerol (Sigma, Cat. No. M6145) , 350 μΐ Insulin (Roche, Cat. No. 1376497), 250 μΐ Transferrin (Roche, Cat. No. 652202), 5 ml Penicillin/

Streptomycin (Invitrogen, Cat. No. 15140122), 2.5 g BSA (PPA

Laboratories, Cat. No. K41-001) . CDMA media was filtered prior to use .

CDM PVA-insulin media was prepared as follows: 250 ml IMDM (Gibco, Cat. No. 21980), 250 ml F12+GlutaMAX-l (Gibco, Cat. No. 31756), 5 ml CD Concentrated Lipids (Gibco, Cat. No. 11905), 20 μΐ 1-Thioglycerol (Sigma, Cat. No. M6145) , 250 μΐ Transferrin (Roche, Cat. No.652202), 5 ml Penicillin/Streptomycin (Invitrogen, Cat. No .15140122 ) , 0.5 g PVA (Sigma, Cat. No P8136) . CDM PVA-insulin medium was filtered prior to use. 1.4. Endoderm differentiation of hIPSCs with CDK4/6 inhibition

The CDM PVA media was prepared as follows: 250 ml IMDM (Gibco, Cat. No. 21980), 250 ml F12+GlutaMAX-l (Gibco, Cat. No. 31756), 5 ml CD Concentrated Lipids (Gibco, Cat. No. 11905) , 20 μΐ 1-Thioglycerol (Sigma, Cat. No. M6145) , 350 μΐ Insulin, 250 μΐ Transferrin (Roche, Cat. No. 652202), 5 ml Penicillin/Streptomycin (Invitrogen, Cat. No. 15140122), 0.5 g PVA (Sigma, Cat. No P8136) . CDM PVA media was filtered prior to use. hIPSCs were maintained in CDM PVA supplemented with Activin A (15 ng/ml) + FGF2 (12 ng/ml) and splitted two days prior to

differentiation. Maintenance media was changed 24 hours prior to starting the differentiation. CDKi-mediated differentiation of hIPSCs was carried out for up to 6 days in CDM PVA media

supplemented with a low dose of Activin A (10 ng/ml), FGF2 (12 ng/ml) and CDK4/6 inhibitor PD0332991 (Selleck, 0.75 μΜ) . Media was changed every day.

1.5. Q-PCR and immunostaining

Methods for Q-PCR and immunostaining have been described previously (Valier et al 2009) . Q-PCR data are presented as the mean of three independent experiments and error bars indicate standard deviations.

1.6. Generating Fucci-hESCs

Human mK02-Cdtl and mAG-Geminin fusion sequences were inserted into the pTP6 plasmid, so that mK02-Cdtl construct contained a selection marker for G418 and mAG-Geminin for puromycin. Constructs were verified by sequencing. Stable H9 Fucci-hESC lines were then generated with mK02-Cdtl, mAG-Geminin or with both mK02-Cdtl /mAG- Geminin as follows. Constructs were transfected (a simultaneous transfection with both constructs for double mK02-Cdtl /mAG-Geminin cell line) into H9 hESCs with lipofectamine as described previously and grown for 3 days. Cells were then cultured in the presence of appropriate antibiotics (0.2 mg/ml G418 for and lmM for puromycin) until the emergence of resistant colonies. Clones were individually picked and further characterised for the expression of the Fucci reporter proteins. Fucci-hESCs were identified in early Gl, late Gl, Gl/S transition and S/G2/M.

1.7. Generating Cyclin D single knockdown, double knockdown and triple knockdown cells

For Cyclin D single knockdown, previously validated shRNA expression vectors (Open Biosystems, Cat no. RHS4533-NM053056, RHS4533- NM001759, RHS4533-NM001136017) directed against Cyclin Dl, D2 or D3 were transfected into H9 hESCs with lipofectamine (Vallier, L. et al. Stem Cells 22 (1), 2 (2004)) and grown for 3 days. Cells were then cultured in the presence of puromycin until antibiotic

resistant colonies appeared. These were picked and characterised for knockdown efficiency. For Cyclin D double knockdown, single

knockdown sublines were stably transfected with a second shRNA expression vector directed against a different Cyclin D and

containing a hygromycin resistance gene. Double knockdown cells were cultured in the presence of puromycin and hygromycin until colonies appeared. These were picked and characterised for knockdown

efficiency. For Cyclin D triple knockdown, Cyclin D double knockdown cells were transitorily transfected with an shRNA expression vector directed against the third Cyclin D family member and containing eGFP as a marker for transfected (Cyclin D triple knockdown) cells. eGFP cells were either sorted for Q-PCR, western blot or analysed directly by immunostaining .

1.8. Generating Cyclin D overexpressing cells and Cyclin Dl mutant cells

cDNA of Cyclin Dl, D2, D3, Dl T156A and Dl T286A was cloned into the pTP6 vector (Pratt, T. et al. Dev Biol 228 (1), 19 (2000)) with an N-terminal FLAG-HA tag, under the regulation of CAG promoter. The inserts were confirmed by sequencing. Vectors were transfected into H9 hESCs by lipofection7 and grown for 3 days. Thereafter, cells with a stable integration were selected by continuous presence of puromycin. Individual clones were picked, propagated and analysed for subsequent analyses. 1.9. Cell sorting by FACS

FACS was performed as described before9. In sum, hESCs were washed with PBS and detached_from the plate by incubating them for 10 min at 37°C in Cell Dissociation Buffer (Gibco) . Cells were washed with cold PBS and then subjected to FACS with a BeckmanCoulter MoFlo MLS high-speed cell sorter, using parameters described previously

(Sakaue-Sawano, A. et al., Cell 132 (3), 487 (2008)) .

1.10. Luciferase assay

Cells were transfected with a Smad2/3 reporter construct (SBE4- luciferase) and Renilla luciferase at a ratio of 10:1, using

Lipofectamine 2000 (Invitrogen) (Vallier, L. et al., Development 136 (8) , 1339 (2009) ) . Luciferase activity was measured with the dual luciferase assay kit following (Promega) manufacturer instructions. Firefly luciferase activity was normalized to Renilla luciferase activity for cell numbers and transfection efficiency. Samples were analysed on a Glomax Luminometer and software.

1.11. Time-lapse imaging

Cells were grown in Chambered 1.0 Borosilicate Cover Glass System (Lab-TEK) . Time-lapse imaging of cells was carried out with Leica SP5 invert + live cell chamber x2 confocal microscope, using parameters as described previously (Valier et al (2009) ) . Cells were maintained in the presence of C02 at 37 °C during microscopy.

1.12. Chromatin immunoprecipitation (ChIP)

hESCs were washed with PBS and detached from the plate by incubating them for 10 min at 37 °C in Cell Dissociation Buffer (Gibco) . ChIP was carried out as described before (Brown, S. et al. Stem Cells 29 (8), 1176 (2011); Teo, A. K. et al . Genes Dev 25 (3), 238 (2011)), except that crosslinking was performed in solution in PBS, if samples were sorted by FACS.

1.13. Cell fractionations

Cells were harvested with trypsin and washed twice with cold PBS. For cytoplasmic lysis, cells were suspended in 5 times packed cell volume (1 ul PCV = 106 cells) equivalent of Isotonic Lysis Buffer (10 mM Tris HC1, pH 7.5, 3 mM CaCl, 2 mM MgC12, 0.32 M Sucrose, Complete protease inhibitors and phosphatase inhibitors), and incubated for 12 min on ice. Triton X-100 was added to a final concentration of 0.3% and incubated for 3 min. The suspension was centrifuged for 5 min at 1,500 rpm at 4 °C and the supernatant (cytoplasmic fraction) transferred to a fresh chilled tube. For nuclear lysis, nuclear pellets were resuspended in 2 x PCV Nuclear Lysis Buffer+Triton X-100 (50 mM Tris HC1, pH 7.5, 100 mM NaCl, 50 mM KC1, 2 mM MgC12, 1 mM EDTA, 10% Glycerol, 0.3% Triton X-100, Complete protease inhibitors and phosphatase inhibitors) and dounce homogenized. The samples were incubated with gentle agitation for 30 min at 4 °C and then centrifuged with a Ti 70.1 rotor at 22,000 rpm for 30 min at 4 °C or with a Ti 45 rotor for 30 min at 20,000 rpm at 4 °C. The chromatin pellets were dounce homogenized in 2 x PCV Nuclear Lysis Buffer+Triton X-100 and Benzonase until the pellets gave much less resistance. The samples were incubated at RT for 30 min and centrifuged with either a Ti 70.1 rotor for 30 min at 22,000 rpm at 4 °C or with a Ti 45 rotor for 30 min at 20,000 rpm at 4 °C.

1.14 Protein co-immunoprecipitation

Antibodies were cross-linked to Protein G-Agarose beads (Roche, 1 ug of antibody per 5 ul of beads) with dimethyl pimelimidate (Sigma) using standard biochemical techniques, prior to performing

immunoprecipitations . Samples were incubated with 5 ug of cross- linked antibodies for 12h at 4 °C. Beads were washed five times with ten bead volumes of Nuclear Lysis Buffer and eluted in SDS western blotting buffer (30 mM Tris pH 6.8, 10% Glycerol, 2% SDS, 0.36 M beta-mercaptoethanol (Sigma), 0.02% bromophenol blue) by heating at 90 °C for 5 min. Samples were analysed by standard western blotting techniques .

1.15 Flow cytometry

Flow cytometry was carried out with a BD MoFlo flow cytometer and analysed by FloJo software. Cell cycle distribution was analysed by Click-It EdU incorporation Kit (Invitrogen) according to

manufacturer's guidelines. Marker expression was analysed at various timepoxnts during differentiation by first dissociating cells into single cells with Cell Dissociation Buffer (Gibco) and fixing in 4% PFA for 20 min at 4°C. This was followed by permeabilisation and blocking with 10% serum + 0.1% Triton X-100 in PBS for 30 min at RT and incubation with primary antibody in 1% serum + 0.1% Triton X-100 for 2h at 4°C. After washing the samples three times with PBS, they were incubated with a secondary antibody for 2h at 4°C, washed three times with PBS and analysed by flow cytometry.

2. Results

2.1 Differentiation capacity of hESCs varies during cell cycle. We examined the responsiveness of hESCs to differentiation signals at different phases of the cell cycle. However, analysis of cell cycle specific events in pluripotent cells is challenging since cell cycle synchronization using chemicals induces their differentiation. To overcome this challenge, we adapted the Fucci reporter system (Sakaue-Sawano, A. et al. n. Ceil 132, 487-498 (2008)) to hESCs, which allowed us to sort cells in Gl, late Gl, Gl/S and S/G2/M without modifying their culture conditions. Sorted Fucci-hESCs were then grown in culture conditions driving differentiation toward the three germ layers (neuroectoderm, mesoderm, and endoderm) (Vallier, L. et al. PLoS One 4, e6082 (2009)) . Real time PCR analyses

confirmed that only cells in Gl could induce differentiation markers but also revealed that cells in early Gl specifically expressed endoderm markers while cells in late Gl induced neuroectoderm markers (Fig. 2). These results were reinforced by FACS analyses showing that cells sorted in early Gl were able to differentiate more rapidly and more homogenously into endoderm whereas the same observations was made for neuroectoderm with cells sorted in late Gl (Fig. 3) . Thus, hESCs respond to specific differentiation stimuli during different stages of the Gl phase thereby providing indication of the existence of permissive and restrictive cell fate choice mechanisms during cell cycle progression. Furthermore, cell cycle analyses also revealed that differentiation of hESCs into the three germ layers is associated with different length of Gl phase. Indeed, cells differentiating into neuroectoderm display a Gl phase almost similar to hESCs while mesoderm and especially endoderm cells display an extended Gl phase providing indication that Gl duration could determine cell fate choice in hESCs . To uncover the molecular circuitry responsible for these observations, we decided to define the transcriptional activity of the Activin/Nodal signalling during cell cycle progression of hESCs. This pathway is particularly interesting since it plays an essential role in endoderm

specification by controlling the expression of key regulators (Brown et al Stem Cells 29, 1176-1185 (2011) ) . We first performed Smad2/3 chromatin immunoprecipitation (ChIP) on sorted Fucci-hESCs and observed that Smad2/3 bound specifically to endoderm genes in early Gl (Mixll, GSC and EOMES, Fig. 1) . Then, transfection of Fucci-hESCs with a luciferase reporter for Smad2/3 transcriptional activity confirmed that the Activin/Nodal-Smad2 /3 pathway is less potent during the late Gl phase and Gl/S transition. These results show that Smad2/3 can only access endoderm genes during early Gl phase and that the activity of key differentiation signalling pathways such as Activin/Nodal could be cell cycle dependent in hESCs.

2.2 Cyclin Ds are necessary for pluripotency

Since the Gl phase seemed to be central for cell fate choice, we hypothesized that factors specifically expressed at this cell cycle phase could control the binding of Smad2/3 on endoderm promoters. Among the master regulators of Gl phase progression are Cyclin D proteins, which coordinate the cell cycle in conjunction with their catalytic partners CDK4/6 (Orford et al Nat Rev Genet 9 115-118 (2008) ; Musgrove et al Nat Rev Cancer (2011) 11 558-572) . Analysis of FACS sorted Fucci-hESCs indicated that Cyclin D proteins are indeed specifically expressed in late Gl and during Gl/S transition of hESCs. Furthermore, Western blot and Q-PCR analyses showed that differentiation of hESCs toward neuroectoderm resulted in a rapid induction of all three Cyclin D genes at both rtiRNA and protein levels (Fig. 4) while endoderm differentiation (Fig. 4) was

accompanied by a decrease in Cyclin Dl expression and low expression of Cyclin D2 / D3. Mesoderm differentiation showed an up-regulation of Cyclin D2 , whereas Cyclin Dl and D3 exhibited a minor decrease (Fig. 4) . Therefore, Cyclin D protein expression is lineage specific reinforcing our initial hypothesis for a function in cell fate specification. Interestingly, Cyclin Ds are highly expressed during lineage specifications that require Activin/Nodal signalling inhibition (neuroectoderm) (Orford et al 2008) whereas their expression is lower during specification of lineages requiring Activin/Nodal signalling activity (endoderm and mesoderm) . To further define their function in pluripotency, we performed loss of function experiments by stably knocking down the expression of the three Cyclin Ds in all possible combinations using overexpression of ShRNA in hESCs . Single knockdown hESC lines (ShDl-, ShD2- and ShD3- hESCs) were able to self-renew, although we observed a moderate increase in expression of differentiation markers, especially mesoderm/endoderm genes. Double knockdown hESCs (ShDlD2, ShD2D3 and ShDlD3-hESCs) showed a stronger propensity for spontaneous

differentiation into cells expressing endoderm markers while pluripotency markers and neuroectoderm marker expression was systematically diminished (Figs. 5 & 6) . Furthermore, double knockdown hESCs displayed a diminished capacity to differentiate into neuroectoderm and an increased capacity to differentiate into endoderm/mesoderm. Finally, triple knockdown hESCs ( shDlD2D3-hESCs ) could not be expanded in vitro, providing indication that an essential function for Cyclin D in pluripotency and/or self-renewal. To bypass this limitation, we performed transitory knockdown experiments using Cyclin Dl/3 double knockdown cells transfected with a Cyclin D2 shRNA construct expressing . GFP . Transfectants were analysed for GFP, Oct4, Nanog and Sox2 expression by

immunofluorescence microscopy. These experiments showed that decreased expression of all three Cyclin Ds resulted in the loss of pluripotency markers while inducing differentiation into endoderm ( Figs . 5 and 6 ) . Taken together these data demonstrate that Cyclin Ds are necessary to maintain pluripotency in hESCs by limiting their capacity to differentiate into endoderm.

2.3 Cyclin Ds promote neuroectoderm and block endoderm

To further validate these results, we performed gain of function experiments by stably overexpressing Cyclin Ds in hESCs. The resulting cells (OED-hESCs) maintained self-renewal and pluripotency, but showed an increase in neuroectoderm marker expression. Cyclin OED-hESCs also display an enhanced capacity to express neuroectoderm markers when grown in culture conditions inductive for this lineage. On the other hand, they have a limited capacity to differentiate into mesoderm/endoderm. We also generated stable hESC lines expressing a mutant form of Cyclin D containing a Threonine to Alanine substitution at position 286 (CycDl-Mut) . This Cyclin Dl mutant is resistant to phosphorylation by Θ3Κ3 and thus accumulates in the nucleus due to inhibition of its nuclear export (Alt et al Genes Dev 14, 3102-3114 (2000)) (Figs. 7 and 8) . hESCs overexpressing CycDl-Mut showed a pronounced loss of pluripotency genes (Oct4, Nanog) and an increase in neuroectodermal genes (Sox2, Soxl, Pax6, Nestin) , accompanied by a decrease in their capacity to differentiate into mesoderm/endoderm. Collectively, these gain of function experiments show that Cyclin D can promote neuroectoderm differentiation while being able to inhibit endoderm differentiation induced by Activin/Nodal signalling.

2.4 Cyclin Ds regulate differentiation genes

The results described above show that a mutant form of Cyclin Dl accumulating in the nucleus can be more potent to induce

neuroectoderm differentiation than its wild type form, providing indication that Cyclin D could have a nuclear function. Accordingly, western blot analyses revealed that Cyclin D can be found in the chromatin fraction of hESCs and their neuronal derivatives but not of endoderm/mesoderm cells. We tested if Cyclin D proteins could direct transcriptional regulation of key factors involved in early differentiation. Chromatin immunoprecipitation analyses performed on Cyclin Dl, D2 and D3 in sorted Fucci-hESCs showed that Cyclin D proteins bind to promoters of early developmental regulators in a cell cycle dependent manner. This occurred almost exclusively during late Gl phase and at Gl/S transition (Figs. 10 & 11) . Furthermore, Cyclin D proteins share a considerable degree of overlap in their target loci, helping to explain their redundant functions in hESCs.

Among the genomic regions bound by Cyclin D were various regulators of neuroectoderm differentiation such as Soxl Pax6, and Sox2 (Fig. 10) . Interestingly, Cyclin D proteins also bound promoters of factors that are essential for endoderm differentiation (Fig. 11), including EOMES and Soxl7. Combined with the results generated by our functional studies, these data suggest that Cyclin D proteins could directly control the transcriptional activation of

neuroectoderm genes and the transcriptional repression of key factors for endoderm/mesoderm specification. To further investigate this mechanism, we performed biochemical analyses to determine if Cyclin Ds interact with histone modifying enzymes. We observed that they could be found in protein complexes containing the

transcriptional co-activator p30013 or the transcriptional co- repressor HDAC114 (Fig. 12) . Furthermore, ChIP analyses showed that p300 recruitment to neuroectodermal loci (Sox2, Soxl and Pax6) was impaired in hESCs knocked down for the expression of two Cyclin Ds whereas the presence of p300 at endoderm loci (EOMES, Soxl7) or other germ layers (CDX2) were unaffected (Fig. 13) . These changes in p300 binding to neuroectoderm loci were accompanied by a loss of the positive histone mark H3K4me3 (Fig. 13) . On the other hand, similar ChIP analyses revealed that HDAC1 binding to endoderm loci (EOMES, Soxl7) is strongly decreased in Cyclin D double knockdown cells and the negative histone mark H3K27me3 (Fig. 13) was lost. Considered together, these data demonstrate that Cyclin D proteins promote neuroectoderm specification and repress endoderm differentiation by recruiting key transcriptional regulators to the promoters of genes directing these early cell fate decisions during the late Gl phase of hESCs.

2.5 Endoderm production by CDK4/6 inhibition.

Importantly, we observed that knockdown in Cyclin D expression resulted in an increase in Smad2/3 proteins localized on chromatin, while overexpression of Cyclin D had the opposite effect (Fig. 6 and Fig. 8) . Therefore, the level of Cyclin D could modulate the quantity of Smad2/3 on chromatin especially in late Gl when the protein level of Cyclin D is at its peak. Co-immunoprecipitation analyses showed that Cyclin D1/D2/D3 interact with Smad2/3 in hESCs (Figs. 14a and 14b) suggesting that Smad2/3 cellular localization could be controlled by this interaction. To further investigate this possibility in hESCs, we inhibited CDK4/6 using a small molecule (PD0332991) 16 and observed an increase in Smad2/3 localisation to the chromatin fraction (Fig. 14c) and increase in the Smad2/3 dependent transcription in late Gl and Gl/S transition. Considered together, these results show that Cyclin D-CDK4/6 regulates the subcellular localisation of Smad2/3 in hPSCs, thereby preventing its positive effect on endoderm gene expression.

Cells were grown for 6 days in the presence of CDKi (0.75mM

PD0332991) and then analysed for the expression of endoderm markers by immunofluorescence microscopy. Interestingly, hESCs grown in the presence of PD0332991 gradually differentiated as shown by the decrease in pluripotency marker expression and by the specific increase in endoderm markers, and to a lesser extent, in mesoderm marker expression (Fig. 15) . On the other hand, neuroectoderm markers were never expressed (Fig. 16) . CDKi produced endoderm was grown for 25 days in culture conditions for hepatic differentiation and then the expression of hepatocyte markers was analysed using immunostaining . The endoderm cells generated by CDKi gave rise to cells expressing hepatic markers. CDKi produced endoderm was also grown for 18 days in culture conditions for pancreatic

differentiation and then the expression of pancreatic markers was analysed using immunostaining. The endoderm generated by CDKi gave rise to pancreatic cells. The endoderm cells generated using

PD0332991 were therefore able to differentiate further into hepatic and pancreatic progenitors when grown in culture condition inductive for these lineages (Figs. 17 to 21) .

Similar results were obtained with three different hIPSC lines demonstrating that our observations are relevant for other

pluripotent cells (Figs. 22 to 26) . Therefore, modulating the cell cycle progression is sufficient to drive differentiation of hPSCs into endoderm derivatives providing indication that directed manipulation of cell cycle regulators could be used for producing cells with potential therapeutic applications from a broad number of pluripotent stem cells. The above results demonstrate that cell fate decisions of human pluripotent cells are directly regulated by the cell cycle machinery and provide novel insights into the mechanisms by which the cell cycle can control the activity of developmental signalling pathways. These reveal new parameters in the regulation of early germ layer specification, providing indication that cell cycle state and the extracellular milieu actively collaborate in directing cell fate choice. Such mechanisms are essential for early development since synchronisation of differentiation, morphogenetic movement and proliferation ultimately determines the number of cells generated in each primary germ layer (Tarn et al Mechanisms of development 68, 3- 25 (1997) ) .

Species divergence could be responsible for the lack of Cyclin D function in early mouse embryo. Indeed, there is growing evidence that mechanisms controlling early development are different in human and in mouse. For instance, FGF signalling which plays an essential function in specification of extraembryonic tissue in mouse

blastocyst does not seem to be important in human (Roode, M. et al. Dev Biol 361, 358-363 (2012)) .

Importantly, our results are supported by studies on adult stem cells. Indeed, a cell cycle function for differentiation of these cells has been independently proposed by laboratories working on neural, skin, gut and hematopoietic stem cells (Fuchs, E. Cell 137, 811-819 (2009),-Li, L. et al Science 327 542-545 (2010) ) . Functional studies performed in the cortex and retina have demonstrated that loss of function of Cyclin D / CDK results in the lengthening of Gl phase and is always accompanied by increased differentiation of Neuronal Stem Cells specifically into neurons (Lange et al Cell Cycle 9, 1893-1900 (2010) ) . Similarly, absence of Cyclin Ds or CDK4/6 results in premature differentiation of Hematopoietic Stem Cells. Cyclin D activity and cell cycle dependent regulation of differentiation signals could therefore define the capacity of multipotent stem cells to differentiate in vivo. Of note, these mechanisms could also be relevant to pathological situations since Cyclin Ds are well known oncogenes and their function in cell fate choice could be involved in the occurrence and maintenance of cancer stem cells.

This study also provides a novel approach for directing

differentiation of pluripotent stem cells by simply manipulating the cell cycle. This approach could be more effective than a

conventional method relying on extra-cellular growth factors alone. Accordingly, cell cycle synchronization enables the coordination and acceleration of differentiation of hPSCs toward a homogenous population of differentiated cells. Therefore, the use of small molecules controlling cell cycle regulators could allow the

development of a universal method of differentiation. In summary, our findings have uncovered mechanisms interconnecting the cell cycle with cell fate decisions in hPSCs . These results are not only important for a better understanding of the spatio-temporal

regulation of early embryonic development but could also be relevant for similar mechanisms that control the proliferation and

differentiation of stem cells in developing organs.

Claims

1. A method for producing human progenitor cells of a specific germ layer comprising;

providing a population of human pluripotent cells,

modulating CyclinD-CDK4 /6 activity in said cells, and;

allowing said cells to differentiate into progenitor cells.

2. A method according to claim 1 wherein the germ layer is definitive endoderm and method comprises inhibiting CyclinD-CDK4/6 activity in the pluripotent cells.

3. A method for producing human definitive endoderm cells comprising;

providing a population of human pluripotent cells,

inhibiting CyclinD-CDK4 /6 activity in said cells and

allowing said cells to differentiate into definitive endoderm cells .

4. A method according to claim 2 or claim 3 wherein CyclinD- CDK4/6 activity is inhibited by contacting the pluripotent cells with a CyclinD-CDK4/6 inhibitor.

5. A method according to claim 3 or claim 4 wherein the CyclinD- CDK4/6 inhibitor is an inhibitor of CDK4 and/or CDK6.

6. A method according to claim 4 or claim 5 wherein the CyclinD- CDK4/6 inhibitor is a small chemical molecule. 7. A method according to claim 6 wherein the CyclinD-CDK4 /6 inhibitor is selected from the group consisting of PD0332991 (6- Acetyl-8-cyclopentyl-5-methyl-2- ( 5-piperazin-l-yl-pyridin-2- ylamino) -8H-pyrido [2, 3-d] pyrimidin-7-one hydrochloride, Ro09-3003, roscovitine (Seliciclib: 6-Benzylamino-2 [ (R) - ( 1 ' -ethyl-2 ' - hydroxyethylamino) ] -9-isopropylpurine) , olomoucine ( 6-Benylamino-2- (2-hydroxyethylamino) -9-methyl- purine); butyrolactone

(dihydrofuran-2 (3H) -one) , flavopiridol (2- (2-Chlorophenyl ) -5, 7- dihydroxy-8- [ (3S, 4R) -3-hydroxy-l-methyl-4-piperidyl ] chromen-4-one ) and purvalanol ( ( 2R) -2- [ [ 6- [ ( 3-Chlorophenyl ) amino ] -9- ( 1- methylethyl ) -9ff-purin-2-yl] amino] -3-methyl-l-butanol ) . AT-7519 (4- (2, 6-Dichloro-benzoylamino) -lH-pyrazole-3-carboxylic acid piperidin- 4-ylamide methanesulfonic acid) , P276-00 (2-Aromatic-Substituted- 5 ,

7-dihydroxy-8- (2- (hydroxymethyl ) -l-methylpyrrolidin-3-yl ) - 4H- chromen-4-one) , SNS-032 (BMS 387032; N- (5- ( ( (5- (1, 1-dimethylethyl } - 2-oxazolyl)methyl) thio) -2-thiazolyl) -4-piperidinecarboxamide ) , ZK 304709, R-547 (Ro-458 820 ; [4-Amino-2- [ ( l-methylsulfonylpiperidin-4- yl) amino] yrimidin-5-yl ] (2, 3-difluoro-6-methoxyphenyl ) methanone) and AG-24322. P1446A-05 SCH 727965 (dinaciclib; (S) -3- ( ( (3-ethyl-5- (2- (2-hydroxyethyl ) piperidin-l-yl )pyrazolo[l,5-a] pyrimidin-7- yl) amino) methyl) pyridine 1-oxide), BAY1000394, CCI-779 ((1R,2R,4S)- 4-{ (2R) -2- [ (3S, 6R, 7E, 9R, 10R, 12R, 14S, 15E, 17E, 19E, 2 IS, 23S,26R,27R, 34aS) -9, 27-dihydroxy-10, 21-dimethoxy-6, 8, 12, 14 , 20 , 26-hexamethyl- 1,5, 11, 28, 29-pentaoxo-l, 4, 5, 6, 9, 10, 11, 12, 13, 14, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 34a-tetracosahydro-3H-23, 27-epoxypyrido [2, 1- c] [1,4] oxazacyclohentriacontin-3-yl ] ropyl } -2-methoxycyclohexyl 3- hydroxy-2- (hydroxymethyl ) -2-methylpropanoate ) , LY2835219 and

Terameprocol (EM-1421; meso-1, 4-Bis (3, 4-dimethoxyphenyl)

dimethylbutane ) .

8. A method according to any one of claims 4 to 7 wherein the CyclinD-CDK4/6 inhibitor is PD0332991 ( 6-Acetyl-8 -cyclopentyl-5- methyl-2- (5-piperazin-l-yl-pyridin-2-ylamino) -8H-pyrido [2, 3- d] pyrimidin-7-one hydrochloride.

9. A method according to claim 2 or claim 3 wherein CyclinD- CDK4/6 activity is inhibited by transfecting the pluripotent cells with an exogenous nucleic molecule which suppresses the expression of one or more of Cyclin Dl to D3, CDK4 and CDK6.

10. A method according to claim 2 or claim 3 wherein CyclinD- CDK4/6 activity is inhibited by expressing in the population of human pluripotent cells a heterologous nucleic acid which encodes either i) a protein factor which inhibits CyclinD-Cdk4/6 activity or ii) a nucleic acid molecule which suppresses CyclinD-Cdk4 /6

activity .

11. A method according to claim 10 wherein the protein factor is pl6.

12. A method according to claim 9 or claim 10 wherein the nucleic molecule is an siRNA or shRNA.

13. A method according to any one of claims 2 to 12 wherein the pluripotent cells are differentiated in a DE differentiation medium, wherein the DE differentiation medium is a chemically defined medium (CDM) .

14. A method according to claim 13 wherein the DE differentiation medium comprises a chemically defined nutrient medium.

15. A method according to claim 14 wherein the chemically defined nutrient medium comprises a basal medium supplemented with polyvinyl alcohol, 1-thioglycerol , insulin, transferrin and defined lipids.

16. A method according to claim 15 wherein the DE differentiation medium comprises 50% IMDM 50% F12; 7μg/ml insulin; 15μg/ml

transferrin; 1 mg/ml polyvinyl alcohol (PVA; 1% chemically defined lipid concentrate ( Invitrogen) ; and 450μΜ 1-thiolglycerol .

17. A method according to any one of claims 13 to 16 wherein the DE differentiation medium is devoid of differentiation factors.

18. A method according to any one of claims 13 to 16 wherein the DE differentiation medium further comprises FGF and Activin A.

19. A method according to claim 18 wherein the DE differentiation medium is devoid of differentiation factors other than FGF and Activin A.

20. A method according to claim 18 or claim 19 wherein the DE differentiation medium comprises less than 50ng/ml Activin A.

21. A method according to any one of claims 13 to 20 wherein the DE differentiation medium is supplemented with the CyclinD-CDK4 / 6 inhibitor .

22. A method according to claim 21 wherein the DE differentiation medium consists of a chemically defined nutrient medium supplemented with the CyclinD-CDK4/6 inhibitor.

23. A method according to claim 21 wherein the DE differentiation medium consists of a chemically defined nutrient medium supplemented with Activin, FGF and the CyclinD-CDK4/6 inhibitor.

24. A method according to any one of claims 13 to 23 wherein the human pluripotent cells are cultured in the definitive endoderm (DE) medium for 3 to 6 days.

25. A method according to any one of claims 2 to 24 wherein the human pluripotent cells are hESCs.

26. A method according to any one of claims 2 to 24 wherein the human pluripotent cells are not hESCs.

27. A method according to any one of claims 2 to 24 wherein the human pluripotent cells are iPSCs.

28. A method according to any one of claims 2 to 27 wherein the human pluripotent cells express one or more of the following pluripotency associated markers: 0ct4, Sox2, Alkaline Phosphatase, POU5fl, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc.

29. A method according to any one of claims 2 to 28 wherein the definitive endoderm progenitors express one or more of the following endoderm associated markers: Soxl7, foxA2, GSC, Mixll, Lhxl, CXCR4, GATA4, eomesodermin (EOMES) , Mixll, HNF-3 beta, Cerberus, OTX4, goosecoid, C-kit, CD99, and Hex

30. A method according to any one of claims 2 to 29 wherein at least 90% of the cells in the population differentiate into DE progenitor cells following inhibition of CyclinD-CDK4/6 activity.

31. A method according to any one of claims 2 to 30 comprising monitoring and/or detecting the expression of one or more definitive endoderm markers in the population of cells.

32. A method according to any one of claims 2 to 31 comprising monitoring and/or detecting the expression of one or more

pluripotency markers in the population of cells.

33. A method according to any one of claims 2 to 32 comprising identifying one or more cells in the population cells as DE

progenitor cells.

34. A method according to any one of claims 2 to 33 comprising expanding the population of DE progenitor cells.

35. A method according to any one of claims 2 to 34 comprising culturing or maintaining the population of DE progenitor cells.

36. A method according to any one of claims 2 to 35 comprising storing the population of DE progenitor cells.

37. A method according to any one of claims 2 to 36 comprising further differentiating the DE progenitor cells.

38. A method according to any one of claims 2 to 37 comprising differentiating the DE progenitor cells into hepatic progenitor cells or hepatocytes.

39. A method according to any one of claims 2 to 37 comprising differentiating the DE progenitor cells into pancreatic progenitor cells or pancreatic endocrine cells.

40. A definitive endoderm (DE) differentiation medium comprising; a chemically defined cell nutrient medium and;

a CyclinD-CDK4/6 inhibitor.

41. A medium according to claim 40 wherein the CyclinD-CDK4 /6 inhibitor is a small chemical molecule.

42. A medium according to claim 40 or claim 41 wherein the

CyclinD-CDK4/6 inhibitor is an inhibitor of CDK4 and/or CDK6.

43. A medium according to any one of claims 40 to 42 wherein

CyclinD-CDK4/6 inhibitor is selected from the group consisting of PD0332991 ( 6-Acetyl-8-cyclopentyl-5-methyl-2- ( 5-piperazin-l-yl- pyridin-2-ylamino) -8H-pyrido [2, 3-d] pyrimidin-7-one hydrochloride, Ro09-3003, roscovitine (Seliciclib: 6-Benzylamino-2 [ (R) - ( 1 ' -ethyl-

2 ' -hydroxyethylamino) ] -9-isopropylpurine ) , olomoucine ( 6-Benylamino- 2- (2-hydroxyethylamino) -9-methyl- purine) ; butyrolactone

(dihydrofuran-2 ( 3H) -one ) , flavopiridol (2- (2-Chlorophenyl ) -5, 7- dihydroxy-8- [ (3S, 4R) -3-hydroxy-l-methyl-4-piperidyl] chromen-4-one) and purvalanol ( {2R) -2- [ [ 6- [ ( 3-Chlorophenyl ) amino] -9- ( 1- methylethyl) -9H-purin-2-yl] amino] -3-methyl-l-butanol ) . AT-7519 (4- (2, 6-Dichloro-benzoylamino ) -lH-pyrazole-3-carboxylic acid piperidin- 4-ylamide methanesulfonic acid) , P276-00 ( 2-Aromatic-Substituted- 5, 7-dihydroxy-8- (2- (hydroxymethyl ) -l-methylpyrrolidin-3-yl ) -4H- chromen-4-one) , SNS-032 (BMS 387032; N- (5- ( ( (5- (1, 1-dimethylethyl ) - 2-oxazolyl) methyl) thio) -2-thiazolyl ) -4-piperidinecarboxamide ) , ZK 304709, R-547 (Ro-4584820; [ -Amino-2- [ ( 1-methylsulfonylpiperidin-4- yl) amino] pyrimidin-5-yl ] (2, 3-difluoro-6-methoxyphenyl ) methanone) and AG-24322. P1446A-05 SCH 727965 (dinaciclib; (S ) -3- ( ( ( 3-ethyl-5- (2- ( 2 -hydroxyethyl ) piperidin-l-yl ) pyrazolo [ 1 , 5-a] pyrimidin-7- yl) amino) methyl) yridine 1-oxide) , BAY1000394, CCI-779 ((1R,2R,4S)- 4-{ (2R) -2- [ (3S, 6R, 7E, 9R, 10R, 12R, 14S, 15E, 17E, 19E,21S, 23S, 26R,27R, 34aS) -9,27-dihydroxy-10, 21-dimethoxy-6, 8, 12 , 14 , 20 , 26-hexamethyl- l,5,ll,28,29-pentaoxo-l,4,5,6,9,10,ll,12,13,14,21,22,23,24,25,26,27, 28, 29, 31, 32, 33, 34, 34a-tetracosahydro-3H-23, 27-epoxypyrido [2, 1- c] [1,4] oxazacyclohentriacontin-3-yl ] propyl } -2-methoxycyclohexyl 3- hydroxy-2- (hydroxymethyl ) -2-methylpropanoate ) , LY2835219 and Terameprocol (EM-1421; meso-1, 4-Bis (3, 4- dimethoxyphenyl ) dimethyIbutane ) .

44. A medium according to any one of claims 40 to 43 wherein the CyclinD-CDK4/6 inhibitor is PD0332991 ( 6-Acetyl-8-cyclopentyl-5- methyl-2- (5-piperazin-l-yl-pyridin-2-ylamino) -8H-pyrido [2, 3- d] pyrimidin-7-one hydrochloride) .

45. A medium according to any one of claims 40 to 44 wherein the chemically defined nutrient medium comprises a chemically defined basal medium.

46. A medium according to any one of claims 40 to 45 wherein the chemically defined nutrient medium further comprises polyvinyl alcohol, insulin, transferrin and defined lipids.

47. A medium according to any one of claims 40 to 46 which is devoid of differentiation factors.

48. A medium according to claim 47 wherein the DE differentiation medium consists of a chemically defined nutrient medium supplemented with the CyclinD-CDK4/6 inhibitor.

49. A medium according to any one of claims 40 to 46 which further comprises FGF and Activin A.

50. A medium according to claim 49 wherein the DE differentiation medium is devoid of differentiation factors other than FGF and Activin A.

51. A medium according to claim 50 wherein the DE differentiation medium consists of a chemically defined nutrient medium supplemented with Activin, FGF and the CyclinD-CDK4/6 inhibitor.

52. A medium according to any one of claims 49 to 51 wherein the DE differentiation medium comprises less than 50ng/ml Activin A.

53. A cell culture comprising a definitive endoderm (DE)

differentiation medium according to any one of claims 40 to 52 and a population of human cells.

54. A cell culture according to claim 53 wherein the population of human cells are pluripotent cells, DE progenitor cells or a mixture thereof .

55. A culture medium supplement for formulation of a DE differentiation medium comprising;

a cyclin D-CDK4/6 inhibitor,

one or more of polyvinyl alcohol, insulin, transferrin and defined lipids, and optionally;

FGF and Activin.

56. A kit for the preparation of a DE differentiation medium comprising;

a chemically defined cell nutrient medium and;

a CyclinD-CDK4/6 inhibitor.

57. Use of a CyclinD-CDK4/6 inhibitor in a method for the in vitro differentiation of human pluripotent cells into definitive endoderm cells .

58. Use of a DE differentiation medium according to any one of claims 40 to 52 in a method for the in vitro differentiation of human pluripotent cells into definitive endoderm cells.

59. A method according to claim 1 wherein method comprises increasing CyclinD-CDK /6 activity in the pluripotent cells and the pluripotent cells differentiate into neuroectoderm cells.

60. A method for producing human neuroectoderm progenitor cells comprising;

providing a population of human pluripotent cells,

increasing CyclinD-CDK4 /6 activity in said cells and

allowing said cells to differentiate into neuroectoderm cells.

61. A method according to claim 60 wherein the neuroectoderm cells are neuroectoderm progenitor cells 62. A method according to any one of claims 59 to 61 wherein

CyclinD-CDK4 /6 activity is increased by expressing in the population of human pluripotent cells a heterologous nucleic acid which encodes one or more of Cyclin Dl to D3, CDK4 and CDK6.

63. A method according to any one of claims 59 to 62 wherein

CyclinD-CDK4/6 activity is increased by transfecting the pluripotent cells with an exogenous nucleic molecule which increases the expression of one or more of Cyclin Dl to D3, CDK4 and CDK6.

64. A method according to any one of claims 59 to 63 wherein the pluripotent cells are differentiated in a neuroectoderm

differentiation medium, wherein the neuroectoderm differentiation medium is a chemically defined medium (CDM) .

65. A method according to claim 64 wherein the neuroectoderm differentiation medium comprises a chemically defined nutrient medium.

66. A method according to claim 65 wherein the chemically defined nutrient medium comprises a basal medium supplemented with Serum

Albumin, preferably bovine Serum Albumin, 1-thioglycerol , insulin, transferrin and defined lipids.

67. A method according to any one of claims 64 to 66 wherein the neuroectoderm differentiation medium comprises 50% IMDM 50% F12;

7μg/ml insulin; 15μg/ml transferrin; 5mg/ml Bovine Serum Albumin (BSA) ; 1% chemically defined lipid concentrate (Invitrogen) ; and 450μΜ l-thiolglycerol .

68. A method according to any one of claims 59 to 67 wherein the neuroectoderm differentiation medium is devoid of differentiation factors .

69. A method according to any one of claims 59 to 67 wherein the neuroectoderm differentiation medium further comprises FGF and an activin/TGF antagonist.

70. A method according to claim 69 wherein the neuroectoderm differentiation medium is devoid of differentiation factors other than FGF and activin/TGF antagonist.

71. A method according to claim 69 or claim 70 wherein the activin/TGF antagonist is SB-431542.

72. A method according to claim 64 wherein the neuroectoderm differentiation medium consists of a chemically defined nutrient medium.

73. A method according to any one of claims 64 to 72 wherein the human pluripotent cells are cultured in the neuroectoderm medium for 3 to 6 days .

74. A method according to any one of claims 59 to 73 wherein the human pluripotent cells are hESCs.

75. A method according to any one of claims 59 to 73 wherein the human pluripotent cells are not hESCs.

76. A method according to any one of claims 59 to 73 wherein the human pluripotent cells are iPSCs.

77. A method according to any one of claims 59 to 76 wherein the human pluripotent cells express one or more of the following pluripotency associated markers: Oct4, Sox2, Alkaline Phosphatase, POU5fl, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc.

78. A method according to any one of claims 59 to 77 wherein the neuroectoderm progenitors express one or more of the following neuroectoderm associated markers: Sox2, Soxl, Pax6 and Nestin.

79. A method according to any one of claims 59 to 78 wherein at least 90% of the cells in the population differentiate into neuroectoderm progenitor cells following inhibition of CyclinD- CD 4/6 activity.

80. A method according to any one of claims 59 to 79 comprising monitoring and/or detecting the expression of one or more

neuroectoderm markers in the population of cells.

81. A method according to any one of claims 59 to 80 comprising monitoring and/or detecting the expression of one or more

pluripotency markers in the population of cells.

82. A method according to any one of claims 59 to 81 comprising identifying one or more cells in the population cells as

neuroectoderm progenitor cells.

83. A method according to any one of claims 59 to 82 comprising expanding the population of neuroectoderm progenitor cells.

84. A method according to any one of claims 59 to 83 comprising culturing or maintaining the population of neuroectoderm progenitor cells .

85. A method according to any one of claims 59 to 84 comprising storing the population of neuroectoderm progenitor cells.

86. A method according to any one of claims 59 to 85 comprising further differentiating the neuroectoderm progenitor cells.