FIELD

This invention relates to the in vitro differentiation of pluripotent stem cells into mesodermal lineages, in particular cardiac and chondrogenic lineages.

BACKGROUND

Organ development in vertebrates begins with induction of the primary embryonic tissue layers ectoderm, mesoderm and endoderm, and their subsequent patterning into specific tissue subtypes. The induction of mesoderm from pluripotent stem cells marks the onset of this process, evidenced by primitive streak (PS) formation (Arnold and Robertson, 2009; Stern et al., 2006; Tam and Loebel, 2007). The specification of mesoderm in the vertebrate embryo is initiated and driven by dynamic BMP, NODAL/ACTIVIN, FGF and WNT signalling gradients along the anterior-posterior axis of the embryo and the PS. The same signalling activities are also essential for subsequent spatial and temporal allocation (or patterning) of mesodermal tissue subtypes after PS induction.

Fate-mapping experiments in vertebrates show that mesoderm patterning into subtypes strictly correlates with the place and time of mesoderm induction in the PS (Lawson et al., 1991 ; Tam et al., 1997). For instance, anterior- specific subtypes include anterior lateral plate and cardiac mesoderm, while extra-embryonic and (pre)somitic mesoderm (consisting of presomitic and somitic mesoderm) are exclusively posterior. However, it remains unclear how mesoderm induction and its patterning into organ precursors depends on diverse and changing signals during initial differentiation.

Human mesoderm and its subtypes can be generated in vitro from pluripotent stem cells (hPSCs), which provide a model of early human development and a major source of therapeutically relevant cell types (Murry and Keller, 2008; Nishikawa et al., 2007). Mesoderm can be induced from hPSCs by a remarkably wide range of conditions, which include different signals that are present during PS formation (Kattman et al., 2011 ; Mae et al., 2013). Regardless of induction method, a common feature is expression of the early pan-mesodermal marker gene, BRACHYURY (BRA) (Martin and Kimelman, 2010). However, there is no comprehensive human mesoderm induction and patterning model to test how different BRA and mesoderm induction signals direct efficient generation of distinct mesodermal subtypes and tissues.

SUMMARY

The present inventors have recognised that primitive streak (PS) progenitors of different sub-types have different mesodermal patterning potentials and can be used for the efficient production of populations of mesodermal cells of specific subtypes, including cardiac and chondrogenic lineages.

An aspect of the invention provides a method for producing a population of mesodermal cells of a defined lineage which comprises:

(i) providing a population of pluripotent stem cells; and,

(ii) culturing the population in a primitive streak (PS) induction medium to produce a population of PS-like cells of a defined sub-type, wherein the PS-like cells of a defined sub-type are anterior PS-like cells and the PS induction medium comprises fibroblast growth factor (FGF), bone morphogenetic protein (BMP), a PI3K inhibitor and activin, or wherein the PS-like cells of a defined sub-type are late PS-like cells and the PS induction medium comprises FGF and a GSK3p inhibitor,

(iii) further differentiating the PS-like cells of a defined identity to produce a population of mesodermal cells of defined lineage.

Another aspect of the invention provides a method for producing a population of mesodermal cells of cardiac lineage which comprises:

(i) providing a population of pluripotent stem cells; and,

(ii) culturing the population in an anterior PS induction medium to produce a population of anterior PS-like cells, wherein the anterior PS induction medium comprises FGF, BMP, a PI3K inhibitor and activin, and

(iii) further differentiating the anterior PS-like cells to produce a population of mesodermal cells of cardiac lineage.

Mesodermal cells of cardiac lineage may include anterior primitive streak (PS) mesoderm cells, anterior lateral mesoderm cells, cardiac mesoderm cells, cardiomyocyte precursors and cardiomyocytes.

Another aspect of the invention provides a method for producing a population of cardiomyocytes which comprises:

(i) providing a population of pluripotent stem cells;

(ii) culturing the population in an anterior PS induction medium comprising FGF, BMP, a PI3K Inhibitor and activin to produce a population of anterior PS-like cells,

(iii) culturing the population of anterior PS-like cells in a first cardiac induction medium comprising FGF, BMP, a Wnt signalling inhibitor and retinoic acid, to produce a population of cardiac mesoderm cells, and

(iv) culturing the population of cardiac mesoderm cells in an second cardiac induction

medium comprising FGF and BMP to produce a population of cardiomyocytes.

The population of cardiomyocytes produced by said culture may be maintained in a maintenance medium that is not supplemented with differentiation factors.

Another aspect of the invention provides a method for producing a population of mesodermal cells of a somitic lineage which comprises:

(i) providing a population of pluripotent stem cells; and,

(ii) culturing the population in a late PS induction medium comprising FGF and a GSK3p inhibitor to produce a population of late PS cells,

(iii) further differentiating the late PS -like cells to produce a population of mesodermal cells of somitic lineage.

Mesodermal cells of somitic lineage may late primitive streak mesoderm cells, pre-somitic mesoderm cells, somitic mesoderm cells, chondrocyte precursors, and chondrocytes. Another aspect of the invention provides a method for producing a population of chondrocytes which comprises:

(i) providing a population of pluripotent stem cells;

(ii) ) culturing the population in a late PS induction medium comprising FGF and a GSK3p inhibitor to produce a population of late PS -like cells,

(iii) culturing the population of late PS-like cells in a somitic induction medium comprising FGF and retinoic acid to produce a population of somitic cells, and

(iv) culturing the population of somitic cells in a chondrogenic induction medium comprising FGF and BMP to produce a population of chondrocytes.

The somitic induction medium may further comprise a BMP inhibitor and/or an SHH agonist.

The population of chondrocytes may be maintained by culturing them in a maintenance medium that is not supplemented with differentiation factors.

Preferably, the pluripotent stem cells used in the methods described above are human.

Other aspects of the invention provide populations of cells produced by the methods set out herein, and uses and applications of these populations of cells.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows RT-QPCR analysis of transcript levels for the general PS markers BRA and TBX6 in H9 hESCs grown in pluripotency conditions (Pluri, FGF+ACTIVIN) or FGF2+LY294002 (FGF+Ly), AnteriorPS (AntPS, FGF+ACTIVIN+BMP4+Ly), PosteriorPS (PostPS, FGF+BMP4+Ly), or with the GSK3-P inhibitor CHIR99021 (Ch).

Figure 2 shows that GSK3-P inhibition independently induces BRA and TBX6. RT-QPCR analysis of transcript levels of BRA and TBX6 with indicated combinations of Ch and FGF2 (F), ACTIVIN A (A), BMP4 (B),

Dorsomorphin (Do), SU5402 (Su), or SB431542 (Sb).

Figure 3 shows RT-QPCR analysis of indicated early mesodermal transcripts in H9 hESCs grown in mesoderm inducing conditions (B) FGF2 plus inhibition of GSK3-P (LatePS) induce the late PS marker CDX1, which co- localises with BRA and is not up-regulated by PosteriorPS conditions. Representative confocal images of H9 hESCs.

Figure 4 shows a human in vitro PS induction model.

Figure 5 shows retinoic acid (RA) treatment and inhibition of canonical WNT signalling by IWR1 (Iwr) promote the expression of cardiac transcription factors TBX5 and NKX2-5 as well as cardiomyocyte structural proteins MYL7 and MYH6 during lateral plate mesoderm (LPM, FGF2+BMP4, 4 days) differentiation in H9 hESCs Figure 6 shows that GSK3-P inhibition blocks expression of lateral plate mesoderm markers in H9 hESCs induced by PosteriorPS (D1/D2) and followed by FGF2+BMP4 (D3/D4).

Figure 7 shows LatePS promotes differentiation of cells expressing markers of presomitic (MESP2, TCF15) and somitic (MEOX1, MYF5) mesoderm. Daily transcript levels in H9 hESCs induced by 2-day PS treatments followed by 2-day treatment in FGF2+RA.

Figure 8 shows chondrocytes emerge from somitic treatment followed by 10 days of FGF2+BMP4. Proteoglycan production was quantified by Alcian blue staining and release.

Figure 9 shows a human in vitro PS induction and patterning model.

Figure 10 shows an inhibition of ACTIVIN signalling by SB431542 in PosteriorPS up-regulates CDX2 and inhibits EOMES and MESP1. Treatment protocol and transcript levels in H9 hESCs undergoing LP mesoderm

differentiation.

Figure 11 shows that Sh-RNA-mediated knock-downs (KD1 , 2) of CDXl/2 do not affect NKX2-5 and TROPO-T expression during cardiac differentiation.

Figure 12 shows that Sh-RNA-mediated knock-downs (KD1 , 2) of CDXi/2repress PAX3 and SOX9 up-regulation after somitic treatment and prevent Alcian blue+ chondrocyte differentiation.

Figure 13 shows RT-QPCR analysis of somite marker (PAXl and ZO-1) expression after PS induction and 3 days of somitic differentiation and chondrocyte markers COL2A and ACAN after chondrogenic differentiation following different PS induction treatments.

DETAILED DESCRIPTION

The experiments herein show that different mechanisms of primitive streak mesoderm induction in pluripotent stem cells lead to distinct mesodermal subtypes. For example, populations of anterior PS -like cells produced using FGF signalling in combination with activin and BMP may be efficiently differentiated into homogeneous populations of anterior lateral mesoderm cells, cardiomyocyte precursors and cardiomyocytes but not somitic mesodermal lineages and populations of late PS-like cells produced using FGF and GSK3- inhibition may be efficiently differentiated into homogeneous populations of pre-somitic mesoderm cells, somitic mesoderm cells, chondrocyte precursors, and chondrocytes, but not cardiac mesodermal lineages.

Cells are cultured in the methods described herein in a 2-dimensional format without the formation of embryoid bodies. 2-dimensional culture methods offer significant advantages over suspension culture methods.

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

Pluripotent stem 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 pluripotent cell may lack markers associated with specific differentiative fates, such as Bra, Soxl7, FoxA2, oFP, Soxl, NCAM, GATA6, GATA4, Handl and CDX2. In particular, a pluripotent cell may lack markers associated with mesodermal fates.

Preferably, the pluripotent stem cells are human pluripotent stem cells.

Pluripotent stem 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). In some embodiments, the pluripotent stem cells may be other than human ESCs (hESCs) or other than human embryonic cells.

ESCs for use in some embodiments may be obtained using conventional techniques. For example, ESCs 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, RUES l to RUES3, HUES 1 to HUES28, HUES45, HUES48, HUES49, HUES53, HUES62 to HUES66, WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14), NYUES 1 to NYUES7, MFS5, and UCLA1 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).

In some embodiments, hESCs may be obtained without either destroying a human embryo or using a human embryo for any industrial or commercial purpose. For example, hESCs may be obtained by blastomere biopsy techniques (see for example Klimanskaya (2013) Semin Reprod Med. 31(l):49-55; Klimanskaya et al Nature (2006)

444(7118)481-5; Chung et al Cell Stem Cell. 2008 Feb 7; 2(2): 113-117). iPSCs are pluripotent stem cells which are derived from non-pluripotent, fully differentiated ancestor or antecedent 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, KLF4 and c-Myc 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 Klf genes, such as Klf-1, -2, and -5; Myc genes such as 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 Nov 27(11):2655-66).

Preferably, the pluripotent stem cells are iPSCs, more preferably human iPSCs (hiPSCs). 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 mesoderm cells, such as cardiomyocytes and chondrocytes with a normal (i.e. non- disease associated) genotype. These mesoderm cells may be useful in therapy, modelling or other applications.

The IPSCs may be obtained from an individual. The IPSCs may be used, for example, to produce autologous mesodermal cells, such as cardiomyocytes or chondrocytes as described herein for use in the treatment of the individual. In some embodiments, multiple populations of IPSCs may be obtained from a population of individuals and used to produce a panel of mesodermal populations as described herein.

In some embodiments, the iPSCs may be derived from somatic cells or other antecedent 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. Disease conditions may include disorders associated with mesodermal tissue e.g. a cardiac disease or dysfunction or chondrogenic disease or dysfunction. iPSCs may be also produced from cells from an individual having a genetic background that confers increased resistance or sensitivity to a pharmaceutical compound or class of compounds, (e.g., antihistamines or other medicines). Mesodermal 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 cardiac or chondrogenic conditions, mechanisms of drug resistance and sensitivity, 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 mesodermal tissue, such as cardiac or chondrogenic disorders or dysfunction. 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 mesodermal cells which have the genotype of the genetic disorder. These mesodermal cells may be further differentiated into cardiac, chondrogenic or other mesodermal lineages which possess the disease genotype. These mesodermal cells may be useful, for example, in modelling the genetic disorder.

Conventional techniques may be employed for the culture and maintenance of human and non-human pluripotent stem 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 stem cells for use in the present methods may be grown in defined conditions or on feeder cells. For example, pluripotent stem 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. 10 ⁵ to 10 ⁶ cells/60mm dish), or on an appropriate substrate with feeder conditioned or defined medium. Pluripotent stem cells for use in the present methods may be passaged by enzymatic or mechanical means.

Suitable culture media for pluripotent stem 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, O. lmM 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, O. lmM 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), ImM L-Gln, 100 μπι nonessential amino acids, 100 μΜ 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin.

In preferred embodiments, a population of pluripotent stem cells for use in the present methods may be cultured in a chemically defined medium (CDM). Chemically defined media are described in more detail below. For example, pluripotent stem cells may be maintained in CDM supplemented with Activin and FGF, before differentiation. A suitable CDM may consist of a chemically defined basal medium supplemented with 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).

A population of pluripotent stem cells suitable for use in the present methods may be heterogeneous or may be substantially free from one or more other cell types (i.e. homogenous). Pluripotent stem 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.

Pluripotent stem cells may be differentiated into primitive streak-like (PS-like) cells by culturing in a suitable primitive streak induction medium, depending on the identity of the desired primitive streak cells.

In some embodiments, pluripotent stem cells may be differentiated into anterior primitive streak-like cells by culturing in a suitable anterior PS induction medium.

A method for producing a population of anterior PS-like cells may comprise:

providing a population of pluripotent stem cells; and

culturing the population in an anterior induction medium comprising FGF, BMP, a PI3K inhibitor and activin to produce the population of anterior PS-like cells.

The anterior PS induction medium is preferably a chemically defined medium (CDM). The anterior PS induction medium may be a chemically defined nutrient medium comprising a chemically defined basal medium supplemented with one or more additional defined components, such as L-glutamine or substitutes, such as GlutaMAX-1™, chemically defined lipids, albumin, 1 -thiolglycerol, polyvinyl alcohol, insulin and transferrin. Suitable chemically defined basal media are described below and 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).

Preferred chemically defined nutrient media for use in the anterior PS induction medium include CDM-PVA and CDM-BA as described below.

The anterior PS induction medium may comprise a chemically defined nutrient medium and a set of differentiation factors consisting of activin, FGF, bone morphogenetic protein (BMP) and a PI3K inhibitor. The anterior PS induction medium may be devoid of differentiation factors other than the activin, FGF, bone morphogenetic protein (BMP) and a PI3K inhibitor.

In some embodiments, the anterior PS induction medium may consist of a chemically defined nutrient medium, such as CDM-PVA, supplemented with an effective amount of activin, FGF, bone morphogenetic protein (BMP) and a PI3K inhibitor. For example, the anterior PS induction medium may consist of a chemically defined nutrient medium, such as CDM-PVA, supplemented with activin, FGF, BMP and LY294002.

A preferred anterior PS induction medium may consist of CDM-PVA, Activin- A (lOng/mL to lOOng/mL or 25 ng/mL to 75ng/ml, preferably 50ng/mL), BMP4 (1 to 100 ng/mL, preferably lOng/mL), FGF (2 to 200 ng/ml preferably 20ng/mL) and LY294002 (1 to ΙΟΟμΜ, preferably 10μΜ).

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).

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 FGF1 to FGF14 and FGF15 to FGF23.

Preferably, the FGF is FGF2 (also known as bFGF, 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 FGF10 (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). Bone morphogenetic proteins (BMPs) bind to Bone Morphogenic Protein Receptors (BMPRs) and stimulate intracellular signalling through pathways mediated by SMAD1, SMAD5 and SMAD9. Suitable Bone Morphogenic Proteins include any member of the BMP family, for example BMP2, BMP3, BMP4, BMP5, BMP6 or BMP7. Preferably the second TGF ligand is BMP2 (NCBI GenelD: 650, nucleic acid sequence NM_001200.2 GI:

80861484; amino acid sequence NP_001191.1 GI: 4557369) or BMP4 (NCBI GenelD: 652, nucleic acid sequence NM_001202.3 GI: 157276592; amino acid sequence NP_001193.2 GI: 157276593). Suitable BMPs include BMP4. Conveniently, the concentration of a Bone Morphogenic Protein, such as BMP2 or BMP4 in a medium described herein may be from 1 to 500ng/ml, preferably about lOng/ml.

Bone Morphogenic Proteins may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g. R&D, Minneapolis, USA, Stemgent Inc, USA).

PI3K inhibitors inhibit the activity of phosphatidylinositol 3-kinases, such as phosphatidylinositol-4, 5-bisphosphate 3-kinase (EC2.7.1.153).

Suitable PI3K inhibitors include wortmannin; LY301497 (17-b-hydroxywortmannin); LY294002 (2-morpholin-4- yl-8-phenylchromen-4-one: Maclean et al (2007) Stem Cells 25 29-38); CLB 1309 (KN309: (+)-2-({ l-[7-methyl- 2-(morpholin-4-yl)-4-oxo-pyrido[ 1 ,2-a]pyrimidin-9-yi]ethyl } amino)benzoic acid); PX-866

((lE,4S,4aR,5R,6aS,9aR)- 5-(Acetyloxy)-l-[(di-2-propen-l-ylamino)methylene]-4,4a,5,6, 6a,8,9,9a-octahydro-l 1- hydroxy-4-(methoxymethyl)-4a,6a-dimethylcyclopenta [5,6]naphtho[l,2-c]pyran-2,7,10(lH)-trione); IC87114 (quinolone pyrrolopyrimidine) ; GDC-0941 (2-( 1 H-Indazol-4-yl)-6-[ [4-(methylsulfonyl)- 1 -piperazinyl] methyl] -4- (4-morpholinyl)-thieno[3,2- djpyrimidine); TGX-221 (7-methyl-2-(4-morpholinyl)-9-[l-(phenylamino)ethyl]-4//- pyrido[l,2-fl]pyrimidin-4-one), quercetin; BEZ235; XL147; X1765; PX-866; ZSTK474 (2-(2- difluoromethylbenzimidazol-l-yl)4,6-dimorpholino-l,3,5-triaz ine); and SF1126 (2-[2-methoxyethylamino]-8- phenyl-4//-l-benzopyran-4-one). Other PI3K inhibitors are available in the art.

In some preferred embodiments, the PI3K inhibitor is LY294002.

Suitable PI3K inhibitors may be obtained from commercial suppliers (e.g. Calbiochem CA USA).

The pluripotent stem cells may be cultured in the anterior PS induction medium for 1 to 2 days, preferably about 36 hours, to produce the population of anterior PS-like cells.

In some embodiments, the population of anterior PS-like cells may be a homogeneous or substantially homogeneous population. For example, 80% or more, 90% or more, 95% or more, 98% or more or most preferably all of the cells in the cell population may be anterior PS -like cells.

Anterior PS-like cells exhibit one or more characteristics of anterior PS cells and express anterior PS markers, such as NANOG, BRA, EOMES, MESP1, MIXL1 and GSC. In some embodiments, the anterior PS-like cells may be anterior PS cells. Following culturing in the medium as described above, the population of anterior PS-like cells produced by the methods described above may be isolated and/or removed from the medium and/or purified. A population of anterior PS-like cells produced by the methods described above may be stored, cultured, matured, maintained or expanded.

A population of anterior PS-like cells produced as described above is suitable for differentiation into mesoderm cells of the cardiac lineage, such as cardiac mesoderm cells, cardiomyocyte progenitors and cardiomyocytes.

The population may be further differentiated into cardiac mesoderm cells. A method may comprise culturing the population of anterior PS-like cells in a first cardiac mesoderm induction medium comprising FGF, BMP, a Wnt signalling inhibitor and retinoic acid (RA) to produce a population of cardiac mesoderm cells.

The first cardiac mesoderm induction medium is preferably a chemically defined medium (CDM).

The first cardiac mesoderm induction medium may comprise a chemically defined nutrient medium comprising a chemically defined basal medium supplemented with one or more additional defined components, such as L- glutamine or substitutes, such as GlutaMAX-1™, chemically defined lipids, albumin, 1 -thiolglycerol, polyvinyl alcohol, insulin and transferrin. Suitable chemically defined basal media are described below and 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). Preferred chemically defined nutrient media for use in the first cardiac mesoderm induction medium include CDM-PVA and CDM-BA as described below.

The first cardiac mesoderm induction medium may comprise a chemically defined nutrient medium and the differentiation factors FGF, BMP, a Wnt signalling inhibitor and RA.

Suitable Wnt inhibitors inhibit canonical WNT signalling and may include Frizzled inhibitors, such as niclosamide (Chen et al Biochemistry. 2009 Nov 3;48(43): 10267-74), vacuolar ATPase inhibitors, such as apicularen and bafilomycin (Cruciat et al Science. 2010 Jan 22;327(5964):459-63), porcupine inhibitors such as IWP2, LGK974, and C59 (Proffitt et al Cancer Res. 2013 Jan 15;73(2):502-7), CK1 inhibitors, such as pyrvinium (Thorne et al Nat Chem Biol. 2010 Nov;6(l l):829-36), Dsh inhibitors, such as NSC668036 (Shan et al Biochemistry. 2005 Nov 29;44(47): 15495-503), TCF/beta-catenin inhibitors, such as 2,4-diamino-quinazoline, quercetin and PKF115-584 (Chen et al Bioorg Med Chem Lett. 2009 Sep 1 ; 19(17):4980; Park et al Biochem Biophys Res Commun. 2005 Mar 4;328(l):227-34; Lepourcelet et al Cancer Cell. 2004 Jan;5(l):91-102) and AXIN stabilisers, such as IWR1 and XAV939 (Huang et al Nature. 2009 Oct 1 ;461(7264):614-20).

Preferred Wnt inhibitors include AXIN stabilisers, such as IWR1 and XAV939.

Retinoic acid (RA) (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-l-yl )nona-2,4,6,8-tetraenoic acid) is a metabolite of vitamin A that modulates transcription through binding to the retinoic acid receptor (RAR) and modulates differentiation in a range of cell types. Preferably all-trans retinoic acid is employed in media described herein.

Conveniently, the concentration of retinoic acid (RA) in a medium may be 1 to 10 μΜ, preferably about 1 μΜ.

Retinoic acid is available from commercial suppliers (e.g. Sigma Aldrich, USA; Stemgent Inc, USA).

The first cardiac mesoderm induction medium may be devoid of differentiation factors other than FGF, BMP, Wnt signalling inhibitor and retinoic acid.

The first cardiac mesoderm induction medium may consist of a chemically defined nutrient medium, such as CDM- PVA, supplemented with an effective amount of FGF, BMP, Wnt signalling inhibitor and RA. For example, the first cardiac mesoderm induction medium may consist of a chemically defined nutrient medium, such as CDM-PVA, FGF, BMP, Wnt signalling inhibitor and RA.

A preferred first cardiac mesoderm induction medium may consist of CDM-PVA, BMP4 (1 to 100 ng/mL or 5 to 20ng/mL, preferably lOng/mL), FGF2 (1 to 80 ng/ml or 4 to 16 ng/mL, preferably 8ng/mL), IWR1 (0.1 to 10μΜ, or 0.5 to 2 μΜ, preferably ΙμΜ) and retinoic acid (0.1 to 10μΜ or 0.02 to 2 μΜ, preferably Ι μΜ).

The cells may be cultured in the first cardiac induction medium for 3 to 5 days, preferably about 4 days to produce the population of cardiac mesoderm cells.

The cardiac mesoderm cells may express cardiac markers, such as TBX5, HANDl, NKX2-5, GATA4, GATA6 and ISL1.

The population of cardiac mesoderm cells may be further differentiated into cardiomyocytes. A method may further comprise culturing the population of cardiac mesoderm cells in a second cardiac induction medium comprising FGF and BMP to produce a population of cardiomyocytes.

The second cardiac mesoderm induction medium is preferably a chemically defined medium (CDM).

The second cardiac mesoderm induction medium may comprise a chemically defined nutrient medium that comprises a chemically defined basal medium supplemented with one or more additional defined components, as described above. Preferred chemically defined media for use in the second cardiac mesoderm induction medium include CDM-PVA and CDM-BA as described below.

The second cardiac mesoderm induction medium may comprise a chemically defined nutrient medium and the differentiation factors; FGF and BMP. The second cardiac mesoderm induction medium may be devoid of differentiation factors other than FGF and BMP. The second cardiac mesoderm induction medium may consist of a chemically defined nutrient medium, such as CDM-PVA, supplemented with effective amounts of FGF and BMP. For example, the second cardiac mesoderm induction medium may consist of CDM-PVA supplemented with FGF and BMP.

A preferred second cardiac mesoderm induction medium may consist of CDM-PVA, BMP4 (1 to 100 ng/mL, preferably lOng/mL), and FGF2 (1 to 40 ng/ml preferably 8ng/mL).

The cells are cultured in said medium for 1 to 4 days or 2 to 4 days, for example about 2 days to produce the population of cardiomyocytes.

Cardiomyocytes may express the markers MYL7, TROPO-T, a-ACTININ, NKX2-5, HANDl, MYH6, TBX5 and GATA6

A method may further comprise culturing the population of cardiomyocytes in a maintenance medium that is not supplemented with differentiation factors.

The cardiomyocytes may be cultured in the maintenance medium for 1 to 10 days.

The maintenance medium is preferably a chemically defined medium (CDM). The maintenance medium may comprise or consist of a chemically defined basal medium supplemented with one or more additional defined components, as described above. Preferred chemically defined basal media for use in the maintenance medium include CDM-PVA and CDM-BA as described below.

The maintenance medium may be devoid of differentiation factors.

Cardiomyocytes produced as described above may have one or more functional properties of cardiomyocytes, including the formation of beating cellular clusters or sheets, active Ca ²⁺ signalling and the generation of action potentials.

Cardiomyocyte function may be determined in the cardiomyocyte populations by conventional techniques, such as Ca ²⁺ imaging and patch clamp electrophysiology.

In some preferred embodiments, a method for producing a population of cardiomyocytes may comprise:

(i) providing a population of pluripotent stem cells;

(ii) culturing the population in an anterior PS induction medium comprising FGF, BMP, activin and a PI3K Inhibitor to produce a population of anterior PS-like cells,

(iii) culturing the population of anterior PS-like cells in a first cardiac induction medium comprising FGF, BMP, a Wnt signalling inhibitor and retinoic acid to produce a population of cardiac mesoderm cells, and,

(iv) culturing the population of cardiac mesoderm cells in an second cardiac induction medium comprising FGF and BMP to produce a population of cardiomyocytes. The population of cardiomyocytes may be further cultured in a maintenance medium that lacks differentiation factors. The population of cardiomyocytes may form beating clusters or sheets in the maintenance medium.

In other embodiments, pluripotent stem cells may be differentiated into late primitive streak-like cells by culturing in a late PS induction medium. A method for producing a population of late primitive streak-like (PS -like) cells may comprise culturing a population of pluripotent stem cells in a late PS induction medium comprising FGF and a GSK3 inhibitor.

Optionally, the late PS medium may further comprise a BMP inhibitor.

The late PS induction medium is preferably a chemically defined medium (CDM). Chemically defined nutrient media are described above. In preferred embodiments, the late PS induction medium may lack insulin.

The late PS induction medium may comprise a chemically defined nutrient medium that comprises a chemically defined basal medium supplemented with one or more additional defined components, as described above. Preferred chemically defined media for use in the late PS induction medium include CDM-PVA and CDM-BA as described below.

The late PS induction medium may comprise a chemically defined nutrient medium supplemented with FGF and a GSK3p inhibitor and optionally a BMP inhibitor. The late PS induction medium may be devoid of differentiation factors other than FGF, the GSK3p inhibitor and optional BMP inhibitor.

GSK3 inhibitors inhibit the activity of glycogen synthase kinase 3β (Gene ID 2932: EC2.7.11.26). Suitable inhibitors include CHIR99021 (6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl )pyrimidin-2- yl)amino)ethyl)amino)nicotinonitrile; Ring D. B. et al., Diabetes, 52:588-595 (2003)) alsterpaullone, kenpaullone, SB216763 (3-(2,4-dichlorophenyl)-4-( 1 -methyl- 1 H-indol-3-yl)- 1 H-pyrrole-2,5-dione), BIO (6-bromoindirubin-3 '- oxime) and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-lH-py rrole-2,5-dione). Suitable GSK3p inhibitors are available from commercial suppliers (e.g. Stemgent Inc. MA USA; Cayman Chemical Co. MI USA). For example, the late PS induction medium may contain 1 to 80μΜ of a GSK3p inhibitor, such as

CHIR99021, preferably about 8 μΜ.

The late PS induction medium may consist of a chemically defined nutrient medium, such as CDM-PVA, supplemented with an effective amount of FGF and a GSK3p inhibitor and optionally a BMP inhibitor. For example, the late PS induction medium may consist of CDM-PVA, FGF, CHIR99021 and optionally LDN193189.

A preferred late PS induction medium may consist of CDM-PVA as described below, FGF (2 to 200 ng/ml preferably 20ng/mL) and CHIR99021 (1 to 40μΜ, preferably 8μΜ) and optionally LDN193189 (5nM to 500nM, preferably 50nM.)

The pluripotent stem cells may be cultured in the late PS induction medium for 1 to 2 days, preferably about 36 hours, to produce a population of late PS-like cells. In some embodiments, the population of late PS-like cells may be homogeneous or substantially homogeneous. For example, 80% or more, 90% or more, 95% or more, 98% or more or most preferably all of the cells in the cell population may be late PS-like cells.

Late PS-like cells exhibit one or more characteristics of late PS cells and express late PS and presomitic markers, such as BRA, TBX6, CDX1, CDX2, MSGN1 and CDX4. In some embodiments, late PS-like cells may be late PS cells.

Following culturing in the medium as described above, the population of late PS-like cells produced by the methods described above may be isolated and/or removed from the medium and/or purified. A population of late PS-like cells produced by the methods described above may be stored, cultured, matured, maintained or expanded.

Late PS-like cells as described herein are suitable for differentiation into mesoderm cells of the somitic lineage, such as chondrogenic progenitors, and chondrocytes.

The population of late PS-like cells may be further differentiated into somitic mesoderm cells. A method may comprise culturing the population of late PS-like cells in a somitic induction medium comprising FGF and RA and optionally a BMP inhibitor and/or a sonic hedgehog (SHH) pathway agonist.

BMP inhibitors inhibit signalling pathways activated by BMP, including Smad 1/5/8 mediated pathways and pathways mediated by MAPK and Akt. For example, a BMP inhibitor may inhibit Smadl/5/8 phosphorylation by ALK [ , ALK2, ALK3 or ALK6. Suitable inhibitors include LDN193189 (4-(6-(4-(piperazin-l- yl)phenyl)pyrazolo[l,5-a]pyrimidin-3-yl)quinoline), dorsomorphin (DM) and K02288 ((3-[6-amino-5-(3,4,5- triniethoxy-pher]yl)-pyridin-3-yl] -phenol). For example, the somitic induction medium may contain 0.02 to 2μΜ of a BMP inhibitor, such as LDN193189, preferably about 0.2 μΜ.

Sonic hedgehog (SHH) pathway agonists stimulate signalling through the SHH pathway, which includes PTCH1, SMO and the transcription factors Glil, Gli2 and Gli3. For example, an SHH agonist may inhibit PTCH1 or activate SMO. Suitable agonists include purmorphamine (9-cyclohexyl-N-(4-morpholinophenyl)-2-(naphthalen-l-yloxy)- 9H-purin-6-amine; Sinha et al Nature Chemical Biol 2 (1) 29-30), Hh-Agl . l (Frank-Kamenetsky J Biol. 2002; 1(2): 10), and SAG (N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophe ne-2-carbonyl)-l,4- diaminocyclohexane; Chen et al PNAS 2002, 99: 14071-1407)

The somitic induction medium is preferably a chemically defined medium (CDM). Chemically defined media are described below.

The somitic induction medium may comprise a chemically defined nutrient medium that comprises a chemically defined basal medium supplemented with one or more additional defined components, as described above. Preferred chemically defined media for use in the somitic induction medium include CDM-PVA and CDM-BA as described below. The somitic induction medium may comprise a chemically defined nutrient medium and the set of differentiation factors consisting of FGF and RA and optionally a BMP inhibitor, such as LDN193189, and/or an SHH agonist. The somitic induction medium may be devoid of differentiation factors other than FGF and RA and optionally a BMP inhibitor and/or an SHH inhibitor.

The somitic induction medium may consist of CDM-PVA supplemented with effective amounts of FGF and RA and optionally, one or both of a BMP inhibitor and an SHH inhibitor. A preferred somitic induction medium may consist of CDM-PVA, FGF (0.4 to 40 ng/ml preferably 4ng/mL) and RA (0.1 to 10μΜ, preferably Ι Μ) and optionally LDN193189 (0.02-2 μΜ) and/or purmorphamine (200nM - 20 μΜ, preferably 2μΜ),

The late PS-like cells may be cultured in the somitic induction medium for 3 to 4 days, to produce a population of somitic mesoderm cells.

In some embodiments, the late PS-like cells may be cultured in the somitic induction medium for 36 to 48 hours to produce a population of pre-somitic mesoderm cells and the pre somitic mesoderm cells may be cultured in fresh somitic induction medium for 36 to 48 hours to produce the population of somitic mesoderm cells.

The late PS-like cells differentiate in the somitic induction medium into a cell population that comprises or consists of somitic mesoderm cells. In some embodiments, the cell population may be a homogeneous or substantially homogeneous population of somitic mesoderm cells. For example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or most preferably all of the cells in the cell population may be somitic mesoderm cells.

The somitic mesoderm cells may express somitic mesoderm markers, such as TCF15, MEOX1, MYF5, PAX1, ZO- 1, SOX9 and PAX3 and/or presomitic markers, such as MSGN1, MESP2, TCF15 and PAX3.

The population of somitic mesoderm cells may be further differentiated into chondrocytes. A method may further comprise culturing the population of somitic mesoderm cells in chondrogenic induction medium comprising FGF and BMP to produce the population of chondrocytes.

The chondrogenic induction medium is preferably a chemically defined medium (CDM). Chemically defined nutrient media are described above.

The chondrogenic induction medium may comprise a chemically defined nutrient medium that comprises a chemically defined basal medium supplemented with one or more additional defined components, as described above. Preferred chemically defined media for use in the chondrogenic induction medium include CDM-PVA and CDM-BA as described below.

The chondrogenic induction medium may comprise a chemically defined nutrient medium and the set of differentiation factors consisting of FGF and BMP. The chondrogenic induction medium may be devoid of differentiation factors other than FGF and BMP. The chondrogenic induction medium may be devoid of differentiation factors other than FGF and BMP.

The chondrogenic induction medium may consist of a chemically defined nutrient medium supplemented with effective amounts of FGF and BMP. For example, the chondrogenic induction medium may consist of CDM-PVA, FGF and BMP.

A preferred chondrogenic induction medium may consist of CDM-PVA as described below, BMP4 (1 to 100 ng/mL, preferably 20ng/mL), and FGF2 (1 to 80 ng/ml preferably 8ng/mL).

The cells are cultured in the chondrogenic induction medium for 8 to 15 days, for example about 10 days, to produce the population of chondrocytes.

The population of chondrocytes may be homogeneous or substantially homogeneous. For example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or most preferably all of the cells in the cell population may be chondrocytes.

The chondrocytes may express COL2A1 and AC AN. SOX5 may also be expressed at the first stages in chondrogenic induction medium.

The population of chondrocytes may display a characteristic cobblestone morphology and produce proteoglycans or mucins that are stained by Alcian blue.

The population of chondrocytes may be cultured or maintained in a maintenance medium. A method may further comprise culturing the population of chondrocytes in a maintenance medium that is not supplemented with differentiation factors.

The maintenance medium is preferably a chemically defined medium (CDM), such as CDM-PVA and CDM-BA as described below.

Preferably, the populations of cells are cultured in 2 dimensional cell culture in all of the steps of the methods described herein. For example, the cells may be cultured in a monolayer (i.e. a layer of cells that is one cell thick) on a surface of a tissue culture vessel, such as a dish, plate or well. The cell populations in the methods described herein do not form embryoid bodies or other three dimensional structures that are characteristic of suspension culture. Culture in 2 dimensional formats increases the reproducibility, scalability and flexibility of the methods described herein and allows the use of any tissue culture format, including multiwall tissue culture plates.

Methods of the invention may be performed in any appropriate format, for example in tissue culture vessels, such as dishes, single well plates or 6, 12, 24, 96 or 384 well plates. Methods of the invention employ chemically defined media. This allows the production of cells in accordance with GMP standards and may be advantageous in many clinical and drug development applications.

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; see Table 1) are known in the art and available from commercial sources (e.g. Sigma-Aldrich MI USA; Life Technologies USA).

In some preferred embodiments, a chemically defined medium may comprise a chemically defined basal medium that is supplemented with a serum-free media supplement and/or 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.

Suitable chemically defined 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, CDM-PVA medium may consist of: 50% Iscove's Modified Dulbecco's Medium (IMDM) plus 50% Ham's F12 with GlutaMAX-1™ or 50% F12 NUT-MIX (Gibco, supplemented with 1 % chemically defined lipid concentrate, 450μΜ 1-thiolglycerol, 15μg/ml transferrin, 1 mg/ml polyvinyl alcohol, 7μg/ml Insulin.

CDM-PVA medium is 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. (201 1 ) 25: 238-250 and Peterson & Losing 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).

The chemically defined nutrient media in the methods described herein may be supplemented with one or more defined differentiation factors. In some preferred embodiments, a medium may comprise CDM-PVA supplemented with one or more defined differentiation factors, as described above. The medium may be devoid of differentiation factors other than the factors specified above for the medium. 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, TGF ligands, such as Activin, TGF or Nodal, GDFs, LIF, IL, activin and phosphatidylinositol 3-kinase (PI3K) inhibitors. A differentiation factor may be present in a medium described herein in an amount that is effective to modulate a signalling pathway in cells cultured in the medium.

Populations of cells may be cultured according to techniques that 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 & Loriog (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.

The extent of differentiation of the cell populations described above 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. Cell markers for each cell population are described above. For example, an increase in the expression of markers characteristic of cardiac or chondrogenic lineages or a decrease in the expression of markers characteristic of pluripotency or primitive streak cells may be determined.

A method described above may further comprise monitoring and/or detecting the presence of one or more markers of mature cardiomyocytes or chondrocytes and/or the absence of one or more markers of mesodermal progenitors, in the population of cells.

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

The methods described above may further comprise identifying one or more cells in the population as

cardiomyocytes or chondrocytes, for example from the presence of expression of one or more cardiomyocyte or chondrocyte markers. Differentiation of PS -like cells of defined sub-type as described herein may produce a population of cardiomyocytes or chondrocytes which is substantially free from other cell types. For example, the population may contain 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more cardiomyocytes or chondrocytes, following culture as described above

The population of cardiomyocytes or chondrocytes may be sufficiently free of other cell types that no purification is required. If required, the cardiomyocytes or chondrocytes 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.

In some embodiments, the pluripotent stem cells may comprise a reporter, preferably a fluorescent reporter, which is operably linked to a tissue-specific promoter (i.e. a cardiac or chondrogenic specific promoter). Following differentiation into cardiomyocytes or chondrocytes as described herein, cells which express the reporter may be isolated and/or purified from other cell types, for example by fluorescence activated cell sorting (FACS).

Following culturing in the medium as described above, the population of cardiomyocytes or chondrocytes produced by the methods described above may be isolated and/or removed from the medium and/or purified.

A population of cardiomyocytes or chondrocytes produced by the methods described above may be cultured, matured, maintained or expanded. Standard cell culture techniques may be employed.

A population of cardiomyocytes or chondrocytes produced by the methods described above may be stored, for example by freezing using conventional cell storage techniques.

Further aspects of the invention provide a population of isolated anterior PS-like cells, late PS-like cells, cardiomyocytes or chondrocytes obtained or obtainable by a method described herein. Preferably the cells in the population are human.

"Isolated" indicates that the isolated cells exist in an environment which is distinct from the environment in which they occur in nature. For example, a population of isolated anterior PS-like cells, late PS-like cells, cardiomyocytes or chondrocytes may be substantially isolated with respect to the tissue environment in which the cells naturally occur. The population may be more homogeneous than natural populations and may be devoid or substantially devoid of other cell types or extracellular molecules that naturally occur with anterior PS cells, late PS cells, cardiomyocytes or chondrocytes, respectively.

Further aspects of the invention provide an in vitro cell culture comprising a population of isolated anterior PS-like cells, late PS-like cells, cardiomyocytes or chondrocytes obtained or obtainable by a method described herein in a culture medium. The culture medium containing the population may be contained in a culture vessel, such as a single or multiwell tissue culture plate. The population of isolated cardiomyocytes or chondrocytes may have a normal (i.e. non-disease associated) genotype, a disease associated genotype, or a distinct genetic background, for example a genetic background that is associated a high risk of a disease condition or a low risk of a disease condition, as described above.

A population of isolated cardiomyocytes or chondrocytes may be used in a method of treatment of an individual, such a human or other mammal, for example for the repair or replacement of damaged or diseased tissue in an individual. Cardiomyocytes or chondrocytes may be useful for the replacement, enhancement and/or stimulation of tissue in an individual. Tissue replacement involves the replacement of dead or dysfunctional cells in an individual by transplanting appropriately cardiomyocytes or chondrocytes to region of cell death. Examples include the transplant of new cardiomyocytes to an ischemic heart, and the transplant of new chondrocytes to a damaged cartilage. Tissue enhancement involves transplanting cells to add extra tissue bulk when tissue loss has occurred in an individual, for example in cartilage. Tissue stimulation involves stimulating the body to repair itself or enhance the immune system or other endogenous repair mechanisms.

Aspects of the invention also extend a pharmaceutical composition, medicament, drug or other composition comprising a population of chondrocytes or cardiomyocytes produced as described herein, a method comprising administration of such a population or composition to a patient, e.g. for treatment (which may include preventative treatment) of damaged, diseased or dysfunctional cardiac tissue or cartilage, as described above, a population or composition for use in a method of treatment, e.g. a method of treatment of damaged, diseased or dysfunctional cardiac tissue or cartilage, use of a population or composition in the manufacture of a medicament for use in the treatment of damaged, diseased or dysfunctional cardiac tissue or cartilage, and a method of making a

pharmaceutical composition comprising admixing such a population of chondrocytes or cardiomyocytes with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients, such as buffer, preservative, stabiliser or anti -oxidant. Such materials should be non-toxic and should not interfere with the viability of the chondrocytes or cardiomyocytes. The precise nature of the carrier or other material will depend on the route of administration.

Liquid compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. The composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection.

In some embodiments, the chondrocytes or cardiomyocytes may be provided in a scaffold or matrix to facilitate tissue repair or supplementation, for example as an artificial cardiac graft or cartilage for implantation. Suitable scaffolds may be composed of artificial polymers such as polyglycolic acid (PGA) or biological matrix components, such as collagen.

Administration of a composition in accordance with the present invention is preferably in a "prophylactic ally effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition comprising chondrocytes or cardiomyocytes may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

A population of isolated cardiomyocytes or chondrocytes produced as described herein may be useful in screening. Screening may include toxicology and drug safety screening. For example, the isolated cardiomyocytes or chondrocytes may be contacted with a test compound at various concentrations that mimic abnormal/normal concentrations in vivo. The effect of the test compound on the cardiomyocytes or chondrocytes may be determined and toxic effects identified. Toxicology screening is well known in the art (see for example Barbaric I et al.

Biochem Soc Trans. 2010 Aug; 38(4): 1046-50).

Another aspect of the invention provides a method of screening a compound comprising;

contacting a population of isolated late PS-like cells, anterior PS-like cells, cardiomyocytes or chondrocytes described herein with a test compound, and;

determining the effect of the test compound on said late PS-like cells, anterior PS-like cells,

cardiomyocytes or chondrocytes and/or the effect of said cardiomyocytes or chondrocytes on the test compound.

The proliferation, growth, maturation, specification or viability of cells or their ability to differentiate or perform one or more cell functions may be determined in the presence relative to the absence of the test compound. A decrease in differentiation, proliferation, growth, viability or ability to perform one or more cell functions, such as cardiomyocyte beating, is indicative that the compound has a toxic effect and an increase in growth, viability or ability to perform one or more cell functions is indicative that the compound has an ameliorative effect.

In some embodiments, the effect of the test compound on cardiomyocytes may be determined by electrophysiology and the effect of the test compound on said chondrocytes may be determined by measuring mucin production for example using Alcian staining

In some embodiments, gene expression in the cells may be determined in the presence relative to the absence of the test compound and the effect of the test compound on gene expression determined. For example, the effect of the test compound on the activation of the expression of foetal and cardiac hypertrophy-related genes in cardiomyocytes may be determined, for example to identify compounds useful in the development of treatments for

cardiomyopathies.

Compounds identified by the methods may useful in the development of cardiac or chondrogenic drugs. Another aspect of the invention provides a kit for production of cardiomyocytes comprising; an anterior PS induction medium as described above, for example a CDM medium consisting of a chemically defined nutrient medium supplemented with fibroblast growth factor (FGF), bone morphogenetic protein (BMP), a PI3K Inhibitor and activin.

The kit may further comprise a first cardiac induction medium as described above, for example a medium consisting of a chemically defined nutrient medium supplemented with FGF, BMP, a Wnt signalling inhibitor and RA.

The kit may further comprise a second cardiac induction medium as described above, for example a medium that consists of a chemically defined nutrient medium supplemented with FGF and BMP.

The kit may further comprise a maintenance medium as described above, for example a medium that consists of a chemically defined nutrient medium that is not supplemented with differentiation factors.

Another aspect of the invention provides a kit for production of chondrocytes comprising;

a late PS induction medium described above, for example a medium that consists of a chemically defined nutrient medium supplemented with an effective amount of a FGF and a GSK3p inhibitor.

The kit may further comprise a somitic induction medium as described above, for example a medium consisting of a chemically defined nutrient medium supplemented with FGF and RA.

The kit may further comprise a chondrogenic induction medium as described above, for example a medium consisting of a chemically defined nutrient medium supplemented with a FGF and BMP.

The kit may further comprise a maintenance medium as described above, for example a medium that consists of a chemically defined nutrient medium that is not supplemented with differentiation factors.

Suitable culture media are described above.

The one or more culture media in a kit for the production of chondrocytes or cardiomyocytes may be formulated in deionized, distilled water. The one or more media will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. The one or more media may be frozen (e.g. at -20°C or - 80°C) for storage or transport. The one or more media may contain one or more antibiotics to prevent

contamination.

The one or more media 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 are 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. In some embodiments, the media in the kit may be supplied in constituent parts for formulation by the user. For example, the kit may comprise a chemically defined basal medium, a supplement comprising one or more additional defined medium components, and a set of differentiation factors. Basal medium, supplement and differentiation factors may be formulated by the user to produce a culture medium, as described herein.

The one or more media and reagents in the kit may be contained in hermetically-sealed vessels. 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.

A kit may further comprise one, two, three, four, five or more antibodies for the characterisation of the cells produced by the methods described above. Each of the antibodies in the kit may bind specifically to a cell marker described above. For example, each of the antibodies in the kit may bind specifically to a marker selected from pluripotent cell markers POU5fl, NANOG and SOX2; anterior PS cell markers NANOG, BRA, EOMES, MESP1, MIXL1 and GSC; cardiac mesoderm markers TBX5, HAND1, NKX2-5, GATA4, GATA6 and ISL1 ;

cardiomyocyte markers MYL7, TROPO-T, a-ACTININ, NKX2-5, HAND1, MYH6, TBX5 and GATA6; late PS markers BRA, TBX6, CDX1, CDX2, MSGN1 and CDX4; somitic mesoderm markers TCF15, MEOX1, MYF5, PAX1, ZO-1, SOX9 and PAX3; and chondrocyte markers COL2A1 and AC AN.

In some embodiments, a kit may further comprise Alcian blue solution for the characterisation of mucin production in chondrocytes.

A kit may further comprise instructions for use in a method described above.

Another aspect of the invention provides the use of an anterior PS induction medium, and first and second cardiac induction media as described herein in the in vitro differentiation of pluripotent stem cells into cardiomyocytes.

Another aspect of the invention provides the use of a late PS induction medium, somitic mesoderm induction medium and chondrogenic induction medium as described herein in the in vitro differentiation of pluripotent stem cells into chondrocytes.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

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 above.

EXPERIMENTS

Methods

Human PSCs differentiation into PS mesoderm and derivatives

Human ESCs (H9, H7, HES 3 -NKX2-5 -GFP) and induced PSCs (BOB) were grown in chemically defined medium (CDM) with ACTIVIN A (lOng/ml and FGF2 (12ng/ml). All differentiations were performed in CDM. PosteriorPS was induced by FGF2 (20ng/ml), LY294002 (Ly 10 uM, Sigma) and BMP4 (lOng/ml, R&D) typically for 36 hours. AnteriorPS was induced as PosteriorPS plus ACTIVIN A at either 50ng/ml for cardiac or lOOng/ml for DE differentiation. Full DE differentiation required 3 days. LatePS was induced by FGF2 (20ng/ml) and CHIR99021 (Ch 8uM, Tocris) in CDM without INSULIN. Lateral plate mesoderm treatment consisted of FGF2 (8ng/ml) and BMP4 (lOng/ml) for 2 or 4 days in CDM following PS induction. Presomitic mesoderm was initiated after LatePS induction by FGF2 (4ng/ml) and retinoic acid (RA, l uM, Sigma) for 36 hours and 3 days for somitic differentiation.

Extra-embryonic differentiation was initiated by BMP4 (20ng/ml) for 3 days following PosteriorPS induction. SMC differentiation was initiated by TGF-betal (2ng/ml) and PDGF-BB (lOng/ml) following PS induction and 4 days of lateral mesoderm treatment.

Cardiac differentiation

Cardiogenic differentiation was initiated after AnteriorPS induction by 4 days in FGF2 (8ng/ml), BMP4 (lOng/ml), IWR1 (l uM, Tocris) and retinoic acid (RA, l uM, Sigma) followed by 2 days in in CDM with FGF2+BMP4. Cells were afterwards maintained in CDM.

Chemically defined two-dimensional hPSC cardiac differentiation

One day after passaging, PSC differentiation was initiated by AnteriorPS conditions (50ng/ml of ACTIVIN A) for 36h followed by 4 days of FGF2+BMP4+IWR1 (ΙμΜ, Tocris) + retinoic acid (Ra, ΙμΜ, Sigma), and subsequent maintenance in FGF2+BMP4 (lOng/ml) with media (CDM_PVA) change every 2 days. Onset of beating was observed on day 7 or 8 of differentiation in clusters (low initial colony density) or sheets (high initial colony density). The method was also routinely performed on hESC lines H7 and HES3 as well as in several human iPSC lines.

Chondrogenic differentiation and Alcian blue staining

Chondrocyte differentiation was initiated after LatePS induction and 4 days somitic treatment by FGF2 (8ng/ml) and BMP4 (lOng/ml) in CDM for 10 days. For Alcian Blue staining, cells were fixed in 4% paraformaldehyde, washed with PBS and stained in 0.025% Alcian solution pH2.0.

Statistical Analysis

Statistical significance of quantitative data was determined by applying a two-tailed Student's test to raw values or to the average values obtained from independent experiments.

Chemically defined treatments ofhPSCs

All experiments were repeated at least three times on different passages of cells and key findings were repeated in H7 and HES3-NKX2-5-GFP (Elliott et al., 2011) hESC lines as well as in human iPSCs. The following additional reagents were used for various treatments: Dorsomorphin 5μΜ (Calbiochem), SB431542 10μΜ (Tocris),

PD0325901 ΙμΜ (Selleckchem), SU5402 ΙΟμΜ (Tocris), WNT3A 150ng/ml (R&D), R-SPONDIN-1 500ng/ml (R&D), 4-hydroxy-tamoxifen 500nM (Sigma), DKK1 150ng/ml (R&D), IWR-1 ΙμΜ (Tocris), IWP2 2μΜ (Tocris), XAV936 5 μΜ (Selleckchem), SP600125 10μΜ (Tocris), SB202190 10μΜ (Selleckchem),

Bisindolylmaleimide II 5μΜ (Tocris).

Results

ACTIVIN/BMP Signalling and GSKS-β Inhibition Can Independently Induce Mesoderm

Human PSCs can be differentiated into anterior PS-like cells (AnteriorPS) by combining FGF2 (F), ACTIVIN A (A) and BMP4 (B), whereas FGF2 and BMP4 induce posterior PS-like cells (PosteriorPS). We inhibited GSK3- using Ch in combination with either FGF2 alone, AnteriorPS, or PosteriorPS conditions. Cells induced to differentiate with Ch showed a distinctive, cobblestoned morphology and strong up-regulation the pan-PS markers BRA and TBX6 compared to PosteriorPS and AnteriorPS treatments (Figure 1). We confirmed this in live hPSCs carrying a fluorescent reporter of endogenous BRA transcription involving a histone 2B (H2B)-Venus fusion knock-in. We observed higher levels, more homogeneous population intensity peaks and widespread activation of BRA expression with Ch (Figure 1). An immunoflow analysis of BRA protein suggested more transient dynamics of BRA up- and down-regulation with AnteriorPS treatment as compared to FGF2+Ch. In conclusion, BRA and TBX6 were strongly induced by GSK3-P inhibition, while activation by ACTIVIN and BMP signalling was moderate. However, it remained unclear whether these different PS induction mechanisms were similar or distinct. Since GSK3- inhibition can markedly boost BRA expression, we asked whether it could do this independently of FGF, ACTIVIN or BMP signalling. Exogenous FGF and Ch were sufficient to induce the BRA-VENUS-H2B reporter, BRA transcription and protein expression (Figure 1). Moreover, when we blocked ACTIVIN (SB431542, SIS3), BMP (Dorsomorphin) and FGF/ERK pathways (SU5402, PD0325901) we still observed significant induction of BRA transcript and protein expression by GSK3-P inhibition alone (Figure 2). By contrast, PosteriorPS and AnteriorPS induction of BRA expression depended entirely on ACTIVIN/BMP combined with FGF signalling (Figure 1). We then asked the reciprocal question whether WNT and GSK3-P signalling were essential for ACTIVIN/BMP- dependent BRA induction. WNT Luciferase reporter activity and up-regulation of the WNT target AXIN2 in PosteriorPS was initially very low (after 18h) and increased only later (after 36h) compared to conditions containing WNT agonists (WNT3A, R-Spondin or Ch). This is in agreement with the identification of WNT3/3A as a targets of BRA (Evans et al., 2012; Martin and Kimelman, 2008). Furthermore, inhibition of WNT signalling in PosteriorPS (using DKK1 or IWR1) did not diminish induction of BRA or the mesoderm marker MESP1, which is consistent with the modest nuclear localisation of β-CATENIN in PosteriorPS. These results indicated that PosteriorPS induced BRA expression independently of endogenous canonical WNT signalling. We concluded that

ACTIVIN/BMP signalling and GSK3-P inhibition could act independently of each other to induce expression of pan-mesodermal marker genes.

ACTIVIN/BMP and GSKS-β Control BRA Expression via Distinct Transcriptional Regulatory Elements

To clarify how diverse mesoderm induction mechanisms intersect at the BRA locus, we performed chromatin immunoprecipitation followed by QPCR (ChlP-QPCR) for chromatin binding components of the ACTIVIN (SMAD2/3), BMP (SMAD1/5), and canonical WNT (β-CATENIN) pathways in pluripotency, AnteriorPS and AnteriorPS+Ch conditions. We observed SMAD2/3 and SMAD1/5 binding to two putative regulatory regions (proximal and distal) upstream of BRA in both differentiation conditions. SMAD2/3 bound both proximally and distally, but SMAD1/5 bound predominantly distally in a region with known DNase hypersensitivity. By contrast, β- CATENIN bound mainly in the AnteriorPS+Ch condition to the proximal promoter region. SMAD2/3 bound mostly to the proximal promoter region in pluripotent conditions. Luciferase assay analysis further confirmed that only a long 6kb fragment containing the distal SMAD1/5 site could drive BRA expression in PosteriorPS. By contrast, a short lkb fragment containing the proximal promoter was sufficient to strongly stimulate BRA expression by PosteriorPS+Ch. We concluded that induction of BRA is mediated by distinct transcriptional regulatory elements. Taken together, these results demonstrate how a limited set of signals can induce BRA and mesoderm differentiation by distinct mechanisms that have sufficient complexity to generate potentially different subtypes.

ACTIVIN/BMP Signalling or GSKS-β Inhibition Induce Distinct AnteriorPS, PosteriorPS and LatePS Mesodermal Identities

In contrast to BRA and TBX6, which are expressed throughout PS development, other early mesodermal markers show more localised expression in either the anterior or the posterior of the early PS or in the late PS at the onset of somitogenesis (Aulehla and Pourquie, 2010; Tam and Loebel, 2007). We therefore asked whether other key mesodermal markers were co-expressed with BRA in either ACTIVIN/BMP -dependent treatments or in conditions with GSK3" inhibition, using RT-QPCR, immunocytochemistry and immunoflow analysis. As previously shown, PosteriorPS induced early PS, extra-embryonic and lateral plate mesoderm transcripts (CDX2, HANDI, KDR and MESPI), while AnteriorPS induced mid and anterior PS markers (EOMES, GSC; Figure 3; Bernardo et al., 2011). By contrast, AnteriorPS+Ch and posteriorPS+Ch treatments both induced exclusively markers of late PS and presomitic mesoderm (MSGN1, TBX6, CDX1, CDX2 and CDX4; Figure 3). Because FGF2+Ch alone also activated all of these late PS mesoderm markers, we regarded this condition as inducing late PS-like differentiation (LatePS, Figure 4). Furthermore, we found that CDX2 expression levels, similar to BRA, depended on Ch dosage (3 uM vs. 8μΜ) and that both markers strongly co-localised. Importantly, the identification of distinct BRA+ PS mesoderm progenitors (AnteriorPS vs. PosteriorPS vs. LatePS, Figure 4) allowed us to test whether they are multipotent or restricted in their subsequent subtype specification potential. The strengths of this in vitro model enabled a rigorous analysis of the mechanism whereby cells exit pluripotency and become specified into mesodermal subtypes.

AnteriorPS but not PosteriorPS Induction Patterns hPSCs into Cardiac Mesoderm

We previously used PosteriorPS induction to subsequently pattern hPSCs into lateral plate mesoderm (by FGF2+BMP4) and to differentiate further into functional smooth muscle cells (SMCs) (Bernardo et al., 2011 ; Cheung et al., 2012). We now used this approach to ask whether PosteriorPS could be patterned into other lateral plate derivatives such as cardiac mesoderm and beating cardiomyocytes.

We combined RA and IWRl (AXIN stabiliser inhibiting canonical WNT signalling) during lateral plate treatment and boosted expression of cardiac markers NKX2-5, TBX5, MYH6 and MYL7 after 5 days of differentiation (Figure 5). However, further differentiation did not result in beating cardiomyocytes, and cardiac structural marker expression decreased. If mesoderm induction does affect later mesoderm patterning, PosteriorPS might not support formation of some mesoderm subtypes, including cardiac. Alternatively, the multipotent mesoderm precursor hypothesis predicts that all PS treatments should generate cardiac progenitors and beating cardiomyocytes.

We examined these possibilities using different PS treatments followed by a 6 day chemically defined, 2- dimensional cardiac differentiation protocol involving FGF2+BMP4 together with RA and IWRl . Strikingly, while AnteriorPS induced robust formation of beating clusters, PosteriorPS or PosteriorPS+Sb followed by identical treatment did not result in cardiac differentiation. Functional cardiomyocyte differentiation was achieved at a wide range of ACTIVIN A concentrations (20-100ng/ml) but most effectively at intermediate levels (50ng/ml). This method was successfully employed with different hESC lines and with induced hPSCs (HES3, H7, H9, BOB). When generated at lower initial seeding densities, we could quantify functional beating clusters, while higher seeding densities resulted in cluster networks or beating cardiomyocyte sheets.

AnteriorPS-induced cells up-regulated cardiac structural markers MYL7, TROPO-T and a-ACTININ as well as endogenous NKX2-5, as shown using the GFP reporter gene knock-in HES3 hPSC line. By contrast, PosteriorPS or PosteriorPS+Sb did not activate or maintain high expression of key cardiac transcription factors NKX2-5, HAND1 and GATA6. Our results show that the prerequisite for efficient cardiac specification occurs in the first 36 hours of mesoderm induction. These results do not support the hypothesis of a multipotent mesoderm PS precursor, which predicted equivalent capabilities for cardiomyocyte development following PosteriorPS or AnteriorPS induction. They favour instead the alternative hypothesis of early mesodermal subtype patterning via PS precursors of restricted differentiation potential.

GSKS-β Inhibition during AnteriorPS Induction Blocks Cardiac and Endoderm Specification

While ACTIVIN A and BMP4 were both required during AnteriorPS induction for efficient cardiac differentiation, the roles of WNT signalling and GSK3-P inhibition were unclear at this stage. Modest GSK3-P inhibition by Ch (3μΜ) or complete inhibition of upstream WNT signalling (both canonical and non-canonical) by IWP2 during AnteriorPS induction prevented specification into beating, a-ACTININ+ cardiomyocyte clusters. Consistent with this, Ch and IWP2 treatment during induction inhibited the up-regulation of the lateral plate mesoderm markers MESP1, NKX2-5, ISL1, GATA6 and HAND1 after lateral plate and cardiac differentiation (Figure 6). This inhibitory effect of Ch during PS induction was not due to nuclear β-CATENIN because Tam-induced translocation of stably transfected ΔΝ-β-CATENIN-ER did not block NKX2-5 up-regulation. Moreover, Ch did not inhibit cardiac differentiation when it was added after the initial induction step. In contrast to Ch or IWP2, inhibition of canonical WNT signalling by AXIN stabilisers IWR1 or XAV939 during AnteriorPS induction did not affect cardiac differentiation. These results suggested that additional, non-canonical WNT signalling (potentially JNK- or PKC-mediated) may be required during cardiogenic AnteriorPS induction. Inhibition of JNK activity abolished cardiac cluster differentiation while inhibition of PKC and p38 did not. The comparison of JNK phosphorylation levels during cardiogenic vs. non-cardiogenic induction further confirmed these results. This observation further supports the hypothesis that early PS induction is critical for later patterning of mesoderm into lateral plate and cardiac subtypes. We concluded that efficient cardiac differentiation required WNT-JNK signalling during mesoderm induction, while GSK3- -inhibition by Ch but not nuclear β-CATENIN blocked it.

Cardiac mesoderm and definitive endoderm (DE) share the requirement for the anterior PS marker EOMES, which has been shown to activate the pre-cardiac determinant MESP1 (Costello et al. 2011). We found that inhibition of GSK3- or inhibition of ACTIVIN signalling blocked MESP1 and EOMES up-regulation during AnteriorPS induction (Figure 3, 6). In agreement with this, hPSCs in DE+Ch condition did not up-regulate DE markers EOMES, SOX17 and FOXA2 suggesting that strong GSK3-P inhibition prevented endoderm differentiation.

Notably, in AnteriorPS +Ch, cells with high EOMES were distinct from those with high CDX2 protein levels (only 1.4% signal overlap). These effects were independent of nuclear β-CATENIN and depended instead on Ch dosage and exogenous FGF2 and BMP4. These findings might explain why protocols that use moderate concentrations of Ch nevertheless can induce DE or cardiac differentiation. In sum, derivatives of the early mid and anterior PS - DE, lateral plate and cardiac mesoderm are blocked by strong inhibition of GSK3- or inhibition of ACTIVIN signalling during PS induction. These signals lead instead to the upregulation of CDX2, a marker and determinant of posterior extra-embryonic mesoderm and the late PS (Lohnes, 2003).

LatePS and PosteriorPS Cells are Committed and Cannot Undergo Patterning into Cardiac Lineage

DE is known to have an inductive role in cardiac specification in vivo and in vitro (Mummery et al. 2007;

Schultheiss et al., 1995). A possible explanation for the negative effect of GSK3- inhibition on cardiac differentiation is that cardiogenic inductive signals between FOXA2+ DE and EOMES+ PS cells are absent. We therefore tested whether CDX2+ LatePS cells could differentiate efficiently into cardiac lineage when they were mixed with AnteriorPS cells during cardiac differentiation. We induced GFP -labeled and non-labeled hPSCs to differentiate in AnteriorPS and LatePS conditions for 36 hours. We then mixed GFP-AnteriorPS cells with unlabelled cells and subjected them to cardiac differentiation. Interestingly, we observed GFP labelled beating clusters only when mixtures contained GFP-labelled AnteriorPS -induced cells; GFP -labelled LatePS -induced cells could not generate beating cardiac clusters (despite the presence of AnteriorPS -derived unlabelled cells), suggesting that they are already committed to other fates. We observed the identical effect also with CDX2+ PosteriorPS cells. This result does not contradict the established role of DE during later cardiac specification but argues against a dominant instructive role during AnteriorPS induction. Accordingly, we concluded that PosteriorPS and LatePS conditions cannot generate cardiac mesoderm because they induce early lineage determinants that drive commitment to other mesodermal subtypes. LatePS Induction Promotes Patterning into (Pre)Somitic Mesoderm in Contrast to PosteriorPS and Ante riorPS According to our model in Figure 4, LatePS was sufficient to induce presomitic mesoderm markers that are expressed in the posterior region of the embryo after formation of DE, lateral plate and cardiac mesoderm. The hypothesis that mesoderm subtype patterning depends on PS induction predicts that further patterning treatments on LatePS cells would generate (pre)somatic (presomitic and somitic) mesoderm precursors of muscle and cartilage instead of lateral plate or cardiac mesoderm. We treated LatePS cells with RA and a lower dose of FGF2 thereby mimicking conditions during presomitic mesoderm differentiation in the vertebrate embryo (Aulehla and Pourquie, 2010). We observed the up-regulation of (pre)somitic markers (TCF15, MESP2, MEOX1, MYF5, PAX1, ZO-1, SOX9 and PAX3); conversely the PS marker TBX6 was efficiently down- regulated (Figure 7). Interestingly, up- regulation of PAX3 (together with PAX7, the key determinants of the skeletal muscle lineage) and SOX9 (early sclerotome/chondrogenic determinant) was completely blocked by BMP4 in LatePS and by PosteriorPS or AnteriorPS induction. Importantly, when we treated cells after presomitic differentiation with FGF2+BMP4 as we did during cardiac differentiation, we observed specific up-regulation of chondrogenic (cartilage) markers COL2A1 and ACAN instead of cardiac markers. To confirm that LatePS -derived presomitic cells differentiated into chondrogenic, extracellular matrix producing cells, we used Alcian Blue and found widespread proteoglycan staining (Figure 8). Moreover, PosteriorPS or AnteriorPS induction with identical subsequent treatments resulted in inefficient chondrogenic differentiation, confirming that GSK3-P inhibition used in LatePS condition is crucial for subsequent somite-like mesoderm specification. In conclusion, while PosteriorPS and AnteriorPS induction promotes subsequent patterning into lateral plate and cardiac mesoderm, LatePS conditions instead promote patterning into (pre)somitic fates (model in Figure 9).

SMCs can be Generated after AnteriorPS, PosteriorPS and LatePS Mesoderm Induction

We asked next, whether patterning of all mesoderm cell types depends on early PS induction. Smooth muscle cells (SMCs) are a particularly interesting case, as these are generated at all stages and from all segments of the PS and could be therefore less sensitive to PS mesoderm induction conditions (Majesky, 2007). Accordingly, we induced PS mesoderm with AnteriorPS, PosteriorPS, and LatePS conditions and then applied an identical lateral plate patterning treatment (FGF2+BMP4) followed by functional SMC differentiation (PDGF-B B+TGB -β) . These SMCs showed clear morphological differences suggesting that they were separate populations with distinct developmental origins as seen before (Cheung et al., 2012). After 9 days of SMC differentiation all three induction treatments resulted in the expression of key SMC markers CNN1, ACTA2, MYHll and TAGLN m ^' the absence of other mesodermal markers (NKX2.5, PAX3, BRA). Up-regulation of these markers was blocked by presomitic patterning treatment (FGF2+RA). Importantly, upon treatment with Carbachol (which induces SMC contractions), SMCs derived from all PS treatments showed contractile activity, thereby confirming their functional capabilities, while control PosteriorPS-derived extra-embryonic cells did not. Taken together, these findings support the finding that mesoderm subtype specification begins with PS induction, and they argue against a multipotent mesoderm progenitor. This effect is particularly clear with mesodermal subtypes that emerge at a defined time and place during PS formation. We therefore explored the molecular basis of these cell fate decisions and examined determinants capable of directing mesodermal patterning at the earliest stages of PS exit from pluripotency and ensuing differentiation. PosteriorPS, AnteriorPS and LatePS Pattern Mesoderm via NANOG and CDX2

Two linked processes occur during AnteriorPS-, PosteriorPS- and LatePS-induced differentiation: dynamic down- regulation of so-called pluripotency factors (e.g., OCT4, SOX2, NANOG); and the up-regulation of early lineage determinants (e.g., BRA, EOMES, CDX2). At first, we focused on NANOG because its expression differed most extensively between the distinct mesoderm induction conditions we employed here. While NANOG expression was rapidly down-regulated within 24 hours of GSK3-P inhibition and by reduced or blocked ACTIVIN signalling, it persisted in AnteriorPS. Moreover, NANOG protein co-localised with EOMES and was co-expressed with MESP1 during the first 24 to 48 hours in AnteriorPS (Figure 10). Conversely, CDX2 expression was strongly up-regulated in conditions where ACTIVIN signalling and NANOG expression was absent and either inhibition of GSK3-P or BMP4 was present. The inhibitory effect of Ch on NANOG and EOMES expression in AnteriorPS condition could be reversed by IWR1 treatment. This coincided with partial β-CATENIN relocation to the cytoplasm and down- regulation of CDX2. In contrast to induction of BRA, transcriptional activation by inducible nuclear β-CATENIN was not sufficient to down-regulate NANOG or to up regulate CDX2 suggesting alternative regulation. However, activation of CDX2 expression was still absolutely dependent on Ch. These observations strongly implicate additional mediators of GSK3-P signalling as being involved in mesoderm induction and patterning. We concluded that PosteriorPS, AnteriorPS and LatePS induction differed not only by differentiation marker expression but also in the exit mechanism from pluripotency.

NANOG is Required for AnteriorPS-induced and CDXl/2 is Required for LatePS-induced Mesodermal Patterning We explored the requirement of NANOG for mesoderm patterning by differentiating hPSCs stably expressing small hairpin RNAs (shRNA) directed against NANOG (NANOG-KD). NANOG-KD clones could be passaged normally, and they expressed similar levels of SOX2 and OCT4 as control cells; they showed moderately activated CDXl/2 but not TBX6 expression in pluripotency and AnteriorPS conditions. Strikingly, depletion of NANOG caused a total absence of beating structures and reduced the expression of TROPO-T but not of the lateral plate and

extraembryonic mesoderm marker HAND1. NANOG-KD clones could differentiate into somitic mesoderm, chondrocytes or SMCs, as seen by expression of PAX3 and SOX9, Alcian staining analysis and ACTA2 and CNNl, respectively (Figure 11). Taken together, these results show that NANOG is required for the differentiation of AnteriorPS -derived cardiac mesoderm but not for LatePS -derived somitic mesoderm or for ubiquitously derived mesodermal cell types, such as SMCs.

The requirement for NANOG in anterior (cardiac) mesoderm differentiation is complementary to the essential role of BRA and CDX factors in posterior (somitic and extra-embryonic) mesoderm development (van Rooijen et al., 2012). Accordingly, we tested whether CDX1 12 (which are highly redundant) knock-down clones differentiated into either cardiac or somitic lineages. While CDX1/2-KD clones up-regulated NKX2.5 during cardiac differentiation and formed beating structures similar to the control, they failed to up-regulate PAX3 and SOX9 during somitic differentiation and did not stain for Alcian blue after chondrogenic treatment (Figure 11, 12).

Furthermore, CDX1/2-KD clones had a proliferation defect and underwent increased apoptosis in LatePS and extraembryonic mesoderm differentiation. These results confirmed that CDXl/2 play a similarly essential roles during LatePS-induced differentiation as NANOG plays during AnteriorPS-induced differentiation. Reciprocal Inhibition of NANOG and CDX2 Directs Patterning into Mesodermal Subtypes

The requirement of NANOG for anterior PS-derived and requirement of CDXl/2 for posterior PS derived mesoderm led us to ask whether NANOG and CDX2 were sufficient to act as determinants of these lineages. We over-expressed NANOG in hPSCs (NANOG-OE) and placed the resulting clones into AnteriorPS, PosteriorPS and LatePS conditions. NANOG-OE clones differentiated normally in AnteriorPS but in PosteriorPS and LatePS (showing increased apoptosis) CDX2 upregulation was blocked depending on ACTIVIN signalling. Conversely, when we transduced hPSCs with a lentiviral vector expressing CDX2 to similar levels as in LatePS, we observed that CDX2 strongly repressed NANOG but not SOX2 expression. CDX2 over-expression was mutually exclusive of high EOMES and high SOX17 expression at the onset and during AnteriorPS -induced DE differentiation.

Furthermore, BRA was highly induced in CDX2 transduced cells in conditions with ACTIVIN signalling. This suggests a mutual positive regulation of CDX2 and BRA, with both acting as key determinants of posterior mesoderm development (Bernardo et al., 2011 ; Savory et al., 2009). Taken together, these results demonstrate a negative regulatory interaction between NANOG (which acts as early specifier of anterior mesoderm and DE) and CDX2 (which acts as a specifier of posterior mesoderm). Importantly, this negative interaction loop between NANOG and CDX2 provided a mechanistic explanation and support for the hypothesis of mesodermal subtype patterning by PS induction.

Finally, we explored in detail how ACTIVIN and GSK3-P signalling gradually establish the observed reciprocal inhibition between NANOG and CDX2 in pluripotency and at the onset of AnteriorPS and LatePS induction. First we performed a ChlP-QPCR analysis in AnteriorPS and LatePS using antibodies against SMAD2/3, NANOG, β- CATENIN, CDX2 and the transcriptional repressor TCF7L1 (orthologue of mouse TCF3). We chose TCF7L1 because it is essential for mesoderm development, it is a mediator of GSK3- signalling that is highly expressed in hPSCs, and it is a putative target of NANOG and ACTIVIN signalling (Brown et al., 2011; Merrill et al., 2004). NANOG, SMAD2/3 and in particular TCF7L1 bound putative regulatory regions (UCSC/ENCODE) in intron 1 of CDXl and CDX2 in pluripotency and in AnteriorPS. Moreover, TCF7L1 bound to a distal putative regulatory region of NANOG in LatePS after 2 hours but not after 12 hours of treatment, which is consistent with rapid NANOG down-regulation. Unlike TCF7L1, β-CATENIN remained bound at the CDXl and CDX2 Intron 1 after 12 hours of induction. Once induced, CDX2 bound close to a distal putative regulatory region of NANOG exclusively in LatePS. These results are consistent with reports on the direct regulation of CDX genes by canonical WNT signalling (Lohnes, 2003).

To conclude, we sought functional evidence for the observed transcription factor binding patterns at the NANOG locus. We used a Luciferase reporter assay to perform a detailed time-course analysis of different NANOG transcriptional regulatory regions in AnteriorPS and LatePS. Interestingly, a 2.2kb fragment upstream of NANOG containing several putative TCF binding sites inhibited reporter expression after 12h but not after 36h in LatePS. In contrast, a short fragment of the NANOG promoter containing only previously characterised SMAD2/3 binding sites was not able to mediate any repression by GSK3-P inhibition during LatePS. This argues against the possibility that GSK3" inhibition impacts on NANOG expression by interfering with SMAD2/3 signalling. Instead, these results are consistent with the model that GSK3-P signalling inhibits NANOG expression via TCF7L1, which can also interfere with β-CATENIN binding and function (Wu et al., 2012). In further support of this hypothesis, transcriptional activation by inducible nuclear Δ-Ν-β-CATENIN could activate CDX2 only in the absence of NANOG and TCF7L1 when ACTIVIN signalling was inhibited. This result finally explained why induced Δ-Ν-β- CATENIN alone did not recapitulate the negative effects of GSK3-P inhibition on cardiac and endoderm differentiation because these are most likely mediated by TCF7L1 and its repression of NANOG.

In conclusion, surprisingly early events during mesoderm induction in hPSCs have dramatic consequences for mesoderm patterning. The interplay between ACTIVIN/BMP and GSK3-P signalling during PS induction thereby culminates in the reciprocal repression of NANOG and CDX2, enabling direct mesoderm specification into anterior and posterior subtypes. We have used hPSCs as an experimental model to determine whether and how PS induction affects subsequent mesoderm patterning. We found evidence that BRA transcription and PS mesoderm can be induced independently by either ACTIVIN/BMP signalling or GSK3- inhibition, which are, as in other vertebrates, mediated by distinct BRA transcriptional regulatory elements (Harvey et al., 2010).

Our results indicate that these different mechanisms of PS induction produce cells of distinct identities (PosteriorPS, AnteriorPS and LatePS) that presage mesoderm patterning into lateral plate and cardiac or (pre)somitic subtypes. However, not all mesodermal cell types were restricted in their potential by PS induction, as seen with

differentiation of smooth muscle cells (SMC), which are generated throughout gastrulation from all segments of the PS (and from neural crest). In contrast to SMCs, cardiac and presomitic mesoderm emerge at a more defined time and place during gastrulation, which might explain their strong reliance on PS induction conditions.

Finally, by exploring the mechanistic basis of these observations, we demonstrated that mesoderm patterning is defined by different exit mechanisms from pluripotency that depend on NANOG down-regulation dynamics and its mutual repression by CDX2. Based on insights gained here, together with knowledge of vertebrate gastrulation, we propose a working model and gene regulatory network of human PS mesoderm induction and patterning. At the outset of PS induction, burgeoning FGF and BMP signals overcome NOD AL/ ACTIVIN signalling in the early posterior PS causing rapid down-regulation of NANOG and up-regulation of BRA and of CDX2 (major determinant of extra-embryonic mesoderm). In the early anterior PS, increased NODAL signalling, together with FGF and BMP, induces EOMES and ESP1. Sustained by NODAL signalling, NANOG, together with SMAD2/3 and its target TCF7L1, represses CDX factors that would otherwise block lateral plate, cardiac and DE differentiation. In the late PS, FGF and strong GSK3-P inhibition (with low ACTIVIN and BMP signalling) repress NANOG by stimulation of the TCF7L1 repressor. As NANOG and its putative target TCF7L1 become down-regulated, β-CATENIN is able to induce high levels of BRA and CDX factors, which enables late PS-derived (pre)somitic mesoderm specification. Finally, we propose that reciprocal inhibition between NANOG and CDX2 similar to that previously described for the trophoblast and ICM lineages (Chen et al., 2009) is harnessed during mesoderm development.

There are significant conceptual implications of these insights into human mesoderm and general tissue induction and patterning. We suggest that what is collectively called PS mesoderm is instead a collection of specified mesodermal tissues with distinct identities and plasticities. In the mouse embryo mesoderm formation begins at E6.25 and continues until the PS disappears almost 3 days later. In humans, this period is extended further to a week during which the embryo undergoes dramatic changes in size, morphogenesis, tissue composition and signalling. Accordingly, it should not be surprising that distinct subtypes of mesoderm emerge from hPSC differentiation depending on exposure to different BMP, ACTIVIN, FGF and GSK3- -mediated conditions. However, embryonic mesoderm might be more plastic and prone to compensation mechanisms and careful in vivo studies should be used to test the model presented here.

There are also important practical implications of this study for hPSC differentiation into mesoderm and its derivatives. Most significantly, mesodermal cell types that have been difficult to generate may be produced more efficiently by initiating their differentiation through correct PS mesoderm induction and patterning. While recent progress in the differentiation of some mesodermal lineages affirm this assessment (Kennedy et al., 2012; Mae et al., 2013; Xu et al., 2013), the current study provides the mechanistic underpinning and comprehensive explanations for it. Our insight that different PS mesoderm induction methods define human mesoderm subtype specification in hPSCs is therefore likely to have a major impact on the accessibility of key tissues for regenerative medicine.

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