WO2016009196A1 - Différenciation mésodermique in vitro - Google Patents

Différenciation mésodermique in vitro Download PDF

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WO2016009196A1
WO2016009196A1 PCT/GB2015/052039 GB2015052039W WO2016009196A1 WO 2016009196 A1 WO2016009196 A1 WO 2016009196A1 GB 2015052039 W GB2015052039 W GB 2015052039W WO 2016009196 A1 WO2016009196 A1 WO 2016009196A1
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cells
population
fgf
mesoderm
cardiac
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Roger Pedersen
Sasha MENDJAN
Victoria MASCETTI
Daniel ORTMANN
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Cambridge Enterprise Limited
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • This invention relates to the in vitro differentiation of pluripotent stem cells into mesodermal lineages, in particular cardiac and chondrogenic lineages.
  • 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).
  • PS primitive streak
  • 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.
  • 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).
  • 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.
  • extra-embryonic and (pre)somitic mesoderm consisting of presomitic and somitic mesoderm
  • hPSCs pluripotent stem cells
  • 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).
  • BRA early pan-mesodermal marker gene
  • PS primitive streak
  • An aspect of the invention provides a method for producing a population of mesodermal cells of a defined lineage which comprises:
  • 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,
  • FGF fibroblast growth factor
  • BMP bone morphogenetic protein
  • PI3K inhibitor a PI3K inhibitor and activin
  • Another aspect of the invention provides a method for producing a population of mesodermal cells of cardiac lineage which comprises:
  • anterior PS induction medium comprises FGF, BMP, a PI3K inhibitor and activin
  • Mesodermal cells of cardiac lineage may include anterior primitive streak (PS) mesoderm cells, anterior lateral mesoderm cells, cardiac mesoderm cells, cardiomyocyte precursors and cardiomyocytes.
  • PS anterior primitive streak
  • Another aspect of the invention provides a method for producing a population of cardiomyocytes which comprises:
  • 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:
  • 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:
  • 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.
  • the pluripotent stem cells used in the methods described above are human.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pluripotent cell may lack markers associated with mesodermal fates.
  • 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).
  • ESCs embryonic stem cells
  • IPCs induced pluripotent stem cells
  • 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.
  • 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.
  • repositories e.g. NIH Human Embryonic Stem Cell Registry
  • hESCs may be obtained without either destroying a human embryo or using a human embryo for any industrial or commercial purpose.
  • 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)
  • 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.
  • Klf genes such as Klf-1, -2, and -5
  • Myc genes such as L-Myc and N-Myc
  • Nanog and Lin28
  • 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.
  • 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.
  • multiple populations of IPSCs may be obtained from a population of individuals and used to produce a panel of mesodermal populations as described herein.
  • the iPSCs may be derived from somatic cells or other antecedent cells obtained from an individual with a distinct genetic background.
  • 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).
  • 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.
  • Pluripotent stem cells for use in the present methods may be grown in defined conditions or on feeder cells.
  • 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.
  • feeder cells such as irradiated mouse embryonic fibroblasts (MEF)
  • an appropriate density e.g. 10 5 to 10 6 cells/60mm dish
  • 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 ⁇ -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.
  • KO-DMEM Knockout Dulbecco's Modified Eagle's Medium
  • KS Knockout
  • lmM ⁇ -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 ⁇ nonessential amino acids, 100 ⁇ 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin.
  • KSR knockout serum replacement
  • MeproTech 6 ng/ml FGF2
  • ImM L-Gln 100 ⁇ nonessential amino acids
  • 100 ⁇ 2-mercaptoethanol 50 U/ml penicillin and 50 mg/ml streptomycin.
  • a population of pluripotent stem cells for use in the present methods may be cultured in a chemically defined medium (CDM).
  • CDM chemically defined medium
  • 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.
  • 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:
  • 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).
  • CDM chemically defined medium
  • 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-1TM, chemically defined lipids, albumin, 1 -thiolglycerol, polyvinyl alcohol, insulin and transferrin.
  • Suitable chemically defined basal media include Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12, Advanced Dulbecco's modified eagle medium (DMEM) (Price et al Focus (2003), 25 3-6), and RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508).
  • IMDM Iscove's Modified Dulbecco's Medium
  • DMEM Advanced Dulbecco's modified eagle medium
  • 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.
  • 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.
  • 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 ⁇ , preferably 10 ⁇ ).
  • Activin 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 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.
  • 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
  • fibroblast growth factor is FGF2.
  • Fibroblast growth factors such as FGF2, FGF7 and FGF10
  • 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 bind to Bone Morphogenic Protein Receptors (BMPRs) and stimulate intracellular signalling through pathways mediated by SMAD1, SMAD5 and SMAD9.
  • BMPRs Bone Morphogenic Protein Receptors
  • Suitable Bone Morphogenic Proteins include any member of the BMP family, for example BMP2, BMP3, BMP4, BMP5, BMP6 or BMP7.
  • the second TGF ligand is BMP2 (NCBI GenelD: 650, nucleic acid sequence NM_001200.2 GI:
  • Suitable BMPs include BMP4.
  • 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
  • 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.
  • 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.
  • the anterior PS-like cells may be anterior PS cells.
  • 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-1TM, chemically defined lipids, albumin, 1 -thiolglycerol, polyvinyl alcohol, insulin and transferrin.
  • additional defined components such as L- glutamine or substitutes, such as GlutaMAX-1TM, 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
  • 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.
  • 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 (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-l-yl)nona-2,4,6,8-tetraenoic acid
  • RA retinoic acid
  • RAR retinoic acid receptor
  • all-trans retinoic acid is employed in media described herein.
  • the concentration of retinoic acid (RA) in a medium may be 1 to 10 ⁇ , preferably about 1 ⁇ .
  • 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.
  • 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 10 ⁇ , or 0.5 to 2 ⁇ , preferably ⁇ ) and retinoic acid (0.1 to 10 ⁇ or 0.02 to 2 ⁇ , preferably ⁇ ⁇ ).
  • 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.
  • 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.
  • a method for producing a population of cardiomyocytes may comprise:
  • 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.
  • 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.
  • 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.
  • CDM chemically defined medium
  • 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 3 ⁇ (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.
  • the late PS induction medium may contain 1 to 80 ⁇ of a GSK3p inhibitor, such as
  • CHIR99021 preferably about 8 ⁇ .
  • 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.
  • 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 40 ⁇ , preferably 8 ⁇ ) 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.
  • 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.
  • late PS-like cells may be late PS cells.
  • 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.
  • 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).
  • the somitic induction medium may contain 0.02 to 2 ⁇ of a BMP inhibitor, such as LDN193189, preferably about 0.2 ⁇ .
  • Sonic hedgehog (SHH) pathway agonists stimulate signalling through the SHH pathway, which includes PTCH1, SMO and the transcription factors Glil, Gli2 and Gli3.
  • 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.
  • 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 10 ⁇ , preferably ⁇ ⁇ ) and optionally LDN193189 (0.02-2 ⁇ ) and/or purmorphamine (200nM - 20 ⁇ , preferably 2 ⁇ ),
  • 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.
  • 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.
  • 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.
  • somitic mesoderm markers such as TCF15, MEOX1, MYF5, PAX1, ZO- 1, SOX9 and PAX3
  • 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.
  • 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.
  • CDM-PVA and CDM-BA chemically defined medium
  • the populations of cells are cultured in 2 dimensional cell culture in all of the steps of the methods described herein.
  • 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 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.
  • 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).
  • DMEM Advanced Dulbecco's Modified Eagle Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • RPMI-1640 Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508; see Table 1
  • 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.
  • 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.
  • CDM-PVA medium may consist of: 50% Iscove's Modified Dulbecco's Medium (IMDM) plus 50% Ham's F12 with GlutaMAX-1TM or 50% F12 NUT-MIX (Gibco, supplemented with 1 % chemically defined lipid concentrate, 450 ⁇ 1-thiolglycerol, 15 ⁇ g/ml transferrin, 1 mg/ml polyvinyl alcohol, 7 ⁇ g/ml Insulin.
  • IMDM Iscove's Modified Dulbecco's Medium
  • F12 NUT-MIX F12 NUT-MIX
  • 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.
  • hESC maintenance medium which is identical to the CDM-PVA described above with the replacement of PVA with 5 mg/ml BSA
  • RPMI basal medium supplemented with B27 and Activin (for example at least 50ng/ml).
  • 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.
  • differentiation factors examples 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.
  • 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.
  • 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.
  • 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.
  • MCS or FACS fluorescence activated cell sorting
  • 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).
  • a tissue-specific promoter i.e. a cardiac or chondrogenic specific promoter.
  • FACS fluorescence activated cell sorting
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a pharmaceutically acceptable excipient such as a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients, such as buffer, preservative, stabiliser or anti -oxidant.
  • buffer, preservative, stabiliser or anti -oxidant 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.
  • 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.
  • 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.
  • a “prophylactic ally effective amount” or a “therapeutically effective amount” as the case may be, although prophylaxis may be considered therapy
  • 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.
  • 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.
  • Another aspect of the invention provides a method of screening a compound comprising;
  • 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.
  • 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
  • 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.
  • 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
  • kits 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.
  • FGF fibroblast growth factor
  • BMP bone morphogenetic protein
  • 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.
  • 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
  • the one or more media may be a lx formulation or a more concentrated formulation, e.g. a 2x to 250x concentrated medium formulation.
  • a lx formulation each ingredient in the medium is at the concentration intended for cell culture, for example a concentration set out above.
  • 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.
  • the media in the kit may be supplied in constituent parts for formulation by the user.
  • 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.
  • 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 ;
  • kits 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.
  • 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.
  • 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.
  • PSC differentiation was initiated by AnteriorPS conditions (50ng/ml of ACTIVIN A) for 36h followed by 4 days of FGF2+BMP4+IWR1 ( ⁇ , Tocris) + retinoic acid (Ra, ⁇ , 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.
  • 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.
  • FGF2 8ng/ml
  • BMP4 lOng/ml
  • Alcian Blue staining cells were fixed in 4% paraformaldehyde, washed with PBS and stained in 0.025% Alcian solution pH2.0.
  • PD0325901 ⁇ (Selleckchem), SU5402 ⁇ (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 ⁇ (Tocris), IWP2 2 ⁇ (Tocris), XAV936 5 ⁇ (Selleckchem), SP600125 10 ⁇ (Tocris), SB202190 10 ⁇ (Selleckchem),
  • 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).
  • F F
  • ACTIVIN A A
  • B B
  • FGF2 and BMP4 induce posterior PS-like cells
  • 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).
  • 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).
  • ACTIVIN/BMP signalling and GSK3-P inhibition could act independently of each other to induce expression of pan-mesodermal marker genes.
  • ⁇ - 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.
  • a short lkb fragment containing the proximal promoter was sufficient to strongly stimulate BRA expression by PosteriorPS+Ch.
  • 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.
  • CDX2 expression levels similar to BRA, depended on Ch dosage (3 uM vs. 8 ⁇ ) and that both markers strongly co-localised.
  • BRA+ PS mesoderm progenitors AnteriorPS vs. PosteriorPS vs. LatePS, Figure 4
  • the strengths of this in vitro model enabled a rigorous analysis of the mechanism whereby cells exit pluripotency and become specified into mesodermal subtypes.
  • 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).
  • SMCs smooth muscle cells
  • 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.
  • 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.
  • LatePS and PosteriorPS Cells are Committed and Cannot Undergo Patterning into Cardiac Lineage
  • 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.
  • 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.
  • SMCs can be Generated after AnteriorPS, PosteriorPS and LatePS Mesoderm Induction
  • SMCs Smooth muscle cells
  • FGF2+BMP4 lateral plate patterning treatment
  • PDGF-B B+TGB - ⁇ functional SMC differentiation
  • NANOG protein co-localised with EOMES and was co-expressed with MESP1 during the first 24 to 48 hours in AnteriorPS (Figure 10).
  • 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 ⁇ -CATENIN relocation to the cytoplasm and down- regulation of CDX2.
  • transcriptional activation by inducible nuclear ⁇ -CATENIN was not sufficient to down-regulate NANOG or to up regulate CDX2 suggesting alternative regulation.
  • NANOG is Required for AnteriorPS-induced and CDXl/2 is Required for LatePS-induced Mesodermal Patterning
  • hPSCs stably expressing small hairpin RNAs (shRNA) directed against NANOG (NANOG-KD).
  • shRNA small hairpin RNAs
  • 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
  • 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.
  • 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
  • NANOG and CDX2 were sufficient to act as determinants of these lineages.
  • NANOG-OE hPSCs
  • NANOG-OE clones differentiated normally in AnteriorPS but in PosteriorPS and LatePS (showing increased apoptosis) CDX2 upregulation was blocked depending on ACTIVIN signalling.
  • 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.
  • 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.
  • 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.
  • UCSC/ENCODE putative regulatory regions
  • 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.
  • ⁇ -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.
  • hPSCs hPSCs as an experimental model to determine whether and how PS induction affects subsequent mesoderm patterning.
  • 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).
  • SMC smooth muscle cells
  • mesoderm patterning is defined by different exit mechanisms from pluripotency that depend on NANOG down-regulation dynamics and its mutual repression by CDX2.
  • 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).
  • PS mesoderm is instead a collection of specified mesodermal tissues with distinct identities and plasticities.
  • PS 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.

Abstract

La présente invention concerne des méthodes de production de populations de cellules mésodermiques de différentes lignées, y compris les lignées de cellules cardiaques et chondrogènes, en cultivant des cellules souches pluripotentes dans un milieu d'induction de lignée primitive (PS) comprenant le facteur de croissance des fibroblastes (FGF), la protéine morphogénétique osseuse (PMO), un inhibiteur de la PI3K et l'activine pour produire une population de cellules de type PS antérieures, ou en cultivant les cellules souches pluripotentes dans un milieu d'induction de lignée primitive (PS) comprenant le FGF et un inhibiteur de GSK3β pour produire une population de cellules de type PS tardives. Les cellules de type PS antérieures ou les cellules de type PS tardives sont ensuite différenciées pour produire une population de cellules mésodermiques de lignée définie, par exemple une lignée de cellules cardiaques ou de chondrocytes. L'invention concerne également des méthodes de différenciation et des kits destinés à être utilisés avec ces méthodes, ainsi que des populations de cellules et leurs méthodes d'utilisation.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10240123B2 (en) 2011-08-29 2019-03-26 Inserm (Institut National De La Sante Et De La Recherche Medicale) Method for preparing induced paraxial mesoderm progenitor (IPAM) cells and their use
EP3882341A1 (fr) * 2020-03-20 2021-09-22 IMBA-Institut für Molekulare Biotechnologie GmbH Modèle de tissu cardiaque
WO2022094628A3 (fr) * 2020-11-02 2022-07-21 Conception Biosciences, Inc. Dérivation in vitro de cellules somatiques gonadiques

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008056166A2 (fr) * 2006-11-09 2008-05-15 Cambridge Enterprise Limited Différenciation de cellules pluripotentes en cellules progénitrices de couches germinales primaires
WO2009120762A2 (fr) * 2008-03-27 2009-10-01 Mount Sinai School Of Medicine Of New York University Cellules progénitrices cardio-vasculaires humaines
WO2013056072A1 (fr) * 2011-10-13 2013-04-18 Wisconsin Alumni Research Foundation Génération de cardiomyocytes à partir de cellules souches pluripotentes humaines
WO2013063305A2 (fr) * 2011-10-25 2013-05-02 Mount Sinai School Of Medicine Différenciation dirigée en cardiomyocytes de cellules souches

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008056166A2 (fr) * 2006-11-09 2008-05-15 Cambridge Enterprise Limited Différenciation de cellules pluripotentes en cellules progénitrices de couches germinales primaires
WO2009120762A2 (fr) * 2008-03-27 2009-10-01 Mount Sinai School Of Medicine Of New York University Cellules progénitrices cardio-vasculaires humaines
WO2013056072A1 (fr) * 2011-10-13 2013-04-18 Wisconsin Alumni Research Foundation Génération de cardiomyocytes à partir de cellules souches pluripotentes humaines
WO2013063305A2 (fr) * 2011-10-25 2013-05-02 Mount Sinai School Of Medicine Différenciation dirigée en cardiomyocytes de cellules souches

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDREIAS BERNARDO ET AL: "BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages", CELL STEM CELL, ELSEVIER, CELL PRESS, AMSTERDAM, NL, vol. 9, no. 2, 30 June 2011 (2011-06-30), pages 144 - 155, XP028265575, ISSN: 1934-5909, [retrieved on 20110707], DOI: 10.1016/J.STEM.2011.06.015 *
JAMES HUDSON ET AL: "Primitive Cardiac Cells from Human Embryonic Stem Cells", STEM CELLS AND DEVELOPMENT, vol. 21, no. 9, 10 June 2012 (2012-06-10), pages 1513 - 1523, XP055157300, ISSN: 1547-3287, DOI: 10.1089/scd.2011.0254 *
MENDJAN SASHA ET AL: "NANOG and CDX2 Pattern Distinct Subtypes of Human Mesoderm during Exit from Pluripotency", CELL STEM CELL, vol. 15, no. 3, September 2014 (2014-09-01), pages 310 - 325, XP002744227 *
SUMI TOMOYUKI ET AL: "Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD, GB, vol. 135, no. 17, 1 September 2008 (2008-09-01), pages 2969 - 2979, XP009165629, ISSN: 0950-1991, [retrieved on 20080730] *
YAMAUCHI KAORI ET AL: "Cardiomyocytes develop from anterior primitive streak cells induced by beta-catenin activation and the blockage of BMP signaling in hESCs", GENES TO CELLS, vol. 15, no. 12, December 2010 (2010-12-01), pages 1216 - 1227, XP002744226, ISSN: 1356-9597 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10240123B2 (en) 2011-08-29 2019-03-26 Inserm (Institut National De La Sante Et De La Recherche Medicale) Method for preparing induced paraxial mesoderm progenitor (IPAM) cells and their use
EP3882341A1 (fr) * 2020-03-20 2021-09-22 IMBA-Institut für Molekulare Biotechnologie GmbH Modèle de tissu cardiaque
WO2021186044A1 (fr) * 2020-03-20 2021-09-23 Imba - Institut Für Molekulare Biotechnologie Gmbh Modèle de tissu cardiaque
WO2022094628A3 (fr) * 2020-11-02 2022-07-21 Conception Biosciences, Inc. Dérivation in vitro de cellules somatiques gonadiques

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