WO2013159349A1 - Procédé de production de cardiomyocytes - Google Patents

Procédé de production de cardiomyocytes Download PDF

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Publication number
WO2013159349A1
WO2013159349A1 PCT/CN2012/074852 CN2012074852W WO2013159349A1 WO 2013159349 A1 WO2013159349 A1 WO 2013159349A1 CN 2012074852 W CN2012074852 W CN 2012074852W WO 2013159349 A1 WO2013159349 A1 WO 2013159349A1
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wnt
concentration
cells
cardiomyocytes
inhibitor
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PCT/CN2012/074852
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English (en)
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Songzhu An
Yong Wang
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Curegenix Inc.
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Priority to PCT/CN2012/074852 priority Critical patent/WO2013159349A1/fr
Publication of WO2013159349A1 publication Critical patent/WO2013159349A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • the present invention is generally related to the field of stem cells. More specifically, it concerns the production of cardiomyocytes from pluripotent stem cells.
  • cardiomyocytes Human adult cardiomyocytes are terminally differentiated. Although a small percentage of cardiomyocytes may have proliferative capacity, it is not sufficient to adequately replace injured or dead cardiac tissue during myocardial infarction after a coronary heart event. Loss of functional cardiomyocytes leads to chronic heart failure.
  • Pluripotent stem cells such as human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, are potential sources of cardiomyocytes. Differentiation of pluripotent stem cells into cardiomyocytes can be achieved upon induction in vitro.
  • cardiomyocytes are produced by technical challenges.
  • technical challenges remain for cardiomyocyte differentiation in order to obtain sufficient numbers and substantially enriched populations of
  • cardiomyocyte lineage cells from pluripotent stem cells The low efficiency and wide variation of differentiation methods are some of the most critical issues. Thus, there is a need to improve the differentiation efficiency of pluripotent stem cells into
  • cardiomyocytes especially for large-scale production.
  • EB embryoid body
  • Cardiomyocytes with spontaneous beating ability appear when these EBs are cultured in suspension. Cardiomyocytes prepared by the EB method vary in their purity and exhibit very similar properties to those of immature cardiomyocytes in fetal hearts. Methods of purifying only cardiomyocytes from a mixture of various kinds of cells from EBs include a method of density gradient separation and a method of labeling cardiomyocyte with a mitochondria staining dye and sorting through FACS. However, these methods are inefficient and time-consuming.
  • differentiation factors to modulate developmental signaling pathways. Examples include: 1) the addition of antagonists to reduce the total signal in certain pathways, 2) the addition of agonists to increase the total signal in certain pathways or 3) combinations of agonists and antagonists to optimize the signal.
  • Methods of increasing the efficiency of cardiogenesis include treating the differentiating cells with various cytokines or small molecule compounds. Such methods have various degrees of success with some of the most efficient methods derived from the biological principles guarding the development of cardiomyocytes during embryogenesis.
  • TGF signaling pathways regulate cardiac differentiation In particular, BMP and Activin are two exemplary TGF signaling pathways that can be optimized. Another pathway that governs cardiogenesis is the Wnt signaling pathway.
  • Wnt secretory proteins are widely found not only in vertebrate but also in invertebrate animals. There are 19 Wnt genes (Wnt-1 , 2, 2b/13, 3, 3a, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11, 16) in humans and mice. Wnt proteins bind to the seven-transmembrane Frizzled (abbreviated as Fzd) family of receptors present on the cell membrane. Such binding triggers signaling pathway involving accumulation and nuclear migration of ⁇ - catenin, which is called the "classical" Wnt pathway or the "canonical” Wnt signaling pathway. It is also known that activation of the canonical Wnt signaling pathway is induced by treatment with GSK-3 inhibitors of small molecules.
  • Wnt-1 the seven-transmembrane Frizzled family of receptors present on the cell membrane.
  • Fzd seven-transmembrane Frizzled
  • Wnt ligands are known to activate other signaling pathways through Fzd receptors.
  • Such signaling pathways include the planar cell polarity (PCP) pathway which activates J K (Jun N-terminal kinase).
  • PCP planar cell polarity
  • J K Jun N-terminal kinase
  • Wnt proteins are involved in a wide variety of biological functions during development, growth and differentiation of various cells and tissues. Cardiomyocytes develop from a part of the lateral plate mesoderm at the early stage of development, and then repeatedly divide and grow to form a heart. The presence or absence of Wnt signals plays an important role in this process.
  • the present invention provides a method for producing cardiomyocytes from pluripotent stem sells, especially, a method for increasing the efficiency of differentiation of pluripotent stem cells into cardiomyocytes by selectively activating or inhibiting the Wnt signaling pathways. Furthermore, the present invention provides the use of a compound in producing cardiomyocytes.
  • the present invention provides a method for producing cardiomyocytes, comprising the following steps:
  • step 2 Culturing the differentiating cells obtained from step 1 in a medium containing a Wnt inhibitor for 48-144 hrs, to induce cardiac mesoderm;
  • step 3 Culturing the differentiating cells obtained from step 2 in a medium containing a Wnt inhibitor for 48-120 hrs, to induce cardiac progenitor cells;
  • the present invention provides the use of a compound named CGX307 in producing cardiomyocytes as a Wnt inhibitor, wherein the compound is N-(3-methyl- 4-(2-methylpyridin-4-yl) benzyl)-6-(2-methylpyridin-4-yl)-2,7-naphthyridin- 1 -amine and the structure thereof is
  • the present invention provides the use of the compound in the preparation of a therapeutic drug for heart conditions.
  • Figure 1 shows that Wnt activators, recombinant Wnt-3a protein or small molecule compound CHIR99021, increase the expression of mesodermal marker Brachyury in differentiating human iPS cells.
  • Figure 2 shows that canonical Wnt pathway inhibitors, recombinant Wnt-5a protein or small molecule compounds XAV939 or CGX307, increase the expression of cardiac mesodermal marker Flk-l/KDR in differentiating human iPS cells.
  • Figure 3 shows that canonical Wnt pathway inhibitors, recombinant Wnt-5a protein or small molecule compounds XAV939 or CGX307, increase the expression of cardiac progenitor marker Isl-1 in differentiating human iPS cells.
  • Figure 4 shows that canonical Wnt pathway activator, recombinant Wnt-3a protein, increases the survival of differentiated cardiac cells in the presence of Thymosin ⁇ -4.
  • FIG 5 shows the enhancing effects of using Wnt activator and inhibitor (the groups labeled "Wnt") on the number of beating cells.
  • Figure 6 shows the increased expression cardiac-specific protein a-actinin after the entire procedure described above.
  • Left panel Control without Wnt activator and inhibitor, right panel: Wnt group.
  • BMP bone morphologic protein
  • TGF transforming growth factor
  • bFGF basic fibroblast growth factor
  • FACS fluorescence activated cell sorting
  • GSK glycogen stimulating kinase
  • iPS induced pluripotent stem
  • Pluripotent stem cells that can be used in the present invention include ES cells and iPS cells derived from mammals such as mice, monkeys and humans, as well as all pluripotent stem cells that are characteristically similar to ES cells.
  • the pluripotent stem cells which may be clonally derived from a single pluripotent stem cell, may comprise a substantial portion of cells clonally derived from a single cell, or may be a pool of multiple populations of cells, wherein each population of cells is clonally derived from a single cell.
  • An example for obtaining pluripotent stem cells from a single cell may comprise incubating a single pluripotent stem cell in medium comprising a ROCK inhibitor under conditions to promote cell growth, such as under adherent culture conditions.
  • certain chemicals such as ascorbic acid can be added at a concentration from 1 mg/L to 1 OOOmg/L to maintain pluripotency and increase cardiogenesis.
  • human iPS cells are obtained by transduction of human foreskin fibroblasts with retroviruses containing Oct4, Sox2 and Klf4 following published protocol (Takahashi et al., 2007). Transfected cells are plated on mouse fibroblast cells in medium PSGen (System Biosciences Inc., Mountain View, CA) supplemented with 100 ng/mL Wnt-3a (StemRD Inc., Burlingame, CA) for about 4 weeks. Colonies resembling ES cell colonies are picked and expanded in a commercially-available iPSC medium PSGro (StemPvD Inc., Burlingame, CA). The colonies are further characterized by immunocytochemical staining of Oct4 expression to ensure majority of the cells continue to express the pluoripotency marker.
  • the compound N-(3-methyl-4-(2-methylpyridin-4-yl) benzyl)-6-(2- methylpyridin-4-yl)-2,7-naphthyridin-l -amine is designated as CGX307 for
  • Wnt activator or “Wnt inhibitor”
  • Wnt inhibitor can be added to the medium during a certain stage of culture.
  • Specific examples of the substance that activates the canonical Wnt signaling pathway include various canonical Wnt proteins, small molecule GSK-3 inhibitors such as CHIR99021.
  • Specific examples of the substance that inhibits the canonical Wnt signaling pathway include various non-canonical Wnt proteins such as Wnt-5a, secreted Wnt antagonist such as Dkk-1 , and small molecule inhibitors such as inhibitors of Porcupine such as IWP-1 or CGX307 and Tankyrases inhibitor such as XAV939.
  • the pluripotent stem cells are transiently treated with a Wnt activator, including but not limited to recombinant Wnt proteins, for example, a recombinant protein obtained from Wnt gene expression.
  • a Wnt activator including but not limited to recombinant Wnt proteins, for example, a recombinant protein obtained from Wnt gene expression.
  • the concentration of the recombinant Wnt protein used is from 0.1 ng/mL to 1000 ng/mL, preferably 1 ng/mL to 200 ng/mL.
  • GSK-3 inhibitors are defined as substances that inhibit the kinase activity of GSK-3 protein (e.g., the ability to phosphorylate ⁇ -catenin).
  • Known examples include an indirubin derivative 6-bromoindirubin 3'-oxime (BIO), a maleimide derivative SB216763 (3-(2 ,4-dichlorophenyl)-4-( 1 -methyl- 1 H-indol-3 -yl)- 1 H-pyrrole-2 ,5 -dione) , or
  • the pluripotent stem cells are transiently treated with a Wnt inhibitor, including but not limited to small molecule inhibitors such as inhibitors of Porcupine or Tankyrases.
  • a Wnt inhibitor including but not limited to small molecule inhibitors such as inhibitors of Porcupine or Tankyrases.
  • concentrations of such inhibitors used vary from 0.001 nM to 1000 nM, preferably 1 nM to 100 nM.
  • the present invention provides a method for producing cardiomyocytes, comprising the steps of:
  • the Wnt activator is Wnt-3a or GSK-3 inhibitor 6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl- 1 H-imidazol-2-yl)pyrimidin-2- yl)amino)ethyl)amino) nicotinonitrile (also named CHIR99021).
  • the concentration of the Wnt activator is from 0.1 ng/mL to 1000 ng/mL, preferably 1 ng/mL to 200 ng/mL.
  • additional factors comprised in the medium for mesoderm differentiation include BMP-4 at a concentration from 0.1 ng/mL to 100 ng/mL, and Activin A at a concentration from 0.1 ng/mL to 100 ng/mL.
  • step 2 Culturing the differentiating cells obtained from step 1 in a medium containing a Wnt inhibitor for 48 to 144 hours, to induce cardiac mesoderm, wherein the Wnt inhibitor is Wnt-5a, XAV939 or CGX307.
  • the concentration of the inhibitor is from 0.001 nM to 1000 nM, preferably 1 nM to 100 nM.
  • Additional factors comprised in the medium for cardiac mesoderm differentiation include inhibitors of BMP signaling such as noggin at a concentration from 10 ng/mL to 1000 ng/mL, Apelin at a concentration from 0.1 nM to 100 nM, and a small molecule inhibitor for BMP receptor such as LDN-193189 or SB431542 at a concentration from 1 nM to 1000 nM.
  • inhibitors of BMP signaling such as noggin at a concentration from 10 ng/mL to 1000 ng/mL, Apelin at a concentration from 0.1 nM to 100 nM
  • a small molecule inhibitor for BMP receptor such as LDN-193189 or SB431542 at a concentration from 1 nM to 1000 nM.
  • a medium containing a Wnt inhibitor for 48 to 120 hours to induce cardiac progenitor cells, wherein the Wnt inhibitor is Wnt-5a, XAV939 or CGX307.
  • the concentration of the inhibitor is from 0.001 nM to 1000 nM, preferably 1 nM to 100 nM.
  • Additional factors comprised in the medium for cardiac progenitor differentiation include bFGF at a concentration from 0.1 ng/mL to 100 ng/mL and VEGF at 0.1 nM to 100 nM.
  • step 4 Culturing the differentiating cells obtained from step 3 in a medium containing a Wnt activator for 48 to 240 hours to maintain the survival of the cardiomyocytes, wherein the activator can be recombinant Wnt-3a or inhibitor CHIR99021.
  • the concentration of the Wnt activator is from 0.1 ng/mL to 1000 ng/mL, preferably 1 ng/mL to 200 ng/mL.
  • Additional factors comprised in the medium for cardiac progenitor differentiation is Thymosin ⁇ -4 at a concentration from 0.1 nM to 100 nM.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • the method further comprises measuring differentiation efficiency of each step.
  • the measurement of differentiation efficiency may comprise measuring expression of mesoderm markers at 5, 6, 7, 8, 9, 10 days, cardiac mesoderm markers at 11 to 14 days, cardiovascular progenitor markers at 11 to 21 days, and cardiomyocyte markers at 14 to 28 days after the initial differentiation by immunocytochemistry well known in the art.
  • markers are mesoderm marker Brachyury, cardiac mesoderm marker Flk- 1/KDR or Nkx2-5, cardiovascular progenitor marker Isl-1 , and cardiomyocyte-specific marker protein a-actinin.
  • cardiomyocytes derived from pluripotent stem cells by the aforementioned method can be further collected, isolated and purified by known methods to efficiently obtain large quantities of pure cardiomyocytes.
  • Cardiomyocytes prepared according to the present invention exhibit morphological, physiological and
  • cells prepared according to the present invention may express one or more markers specific to cardiomyocytes, for example, markers mentioned above.
  • cardiomyocytes prepared by the aforementioned method of the present invention can be used in methods of screening compounds which promote the development, differentiation, regeneration, survival of cardiomyocytes.
  • cardiomyocytes prepared by the aforementioned method can be used in methods for treating hearts suffering from cardiac disorders.
  • cardiac disorders include myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilative cardiomyopathy, myocarditis, chronic heart failure and the like.
  • the present invention provides the use of the compound CGX307 in producing cardiomyocytes.
  • CGX307 is used as a Wnt inhibitor, and acts to induce differentiation of stem cells into cardiomyocytes.
  • CGX307 can be used as a therapeutic drug for heart conditions including myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilative cardiomyopathy, myocarditis, chronic heart failure and the like. Examples
  • CGX307 is N-(3-methyl-4-(2-methylpyridin-4-yl) benzyl)-6-(2- methylpyridin-4-yl)-2,7-naphthyridin-l -amine, and the structure is listed as below. It is synthesized by the present inventor.
  • the synthesis process comprises the following steps.
  • 6-chloro-2,7-naphthyridin-l(2H)-one 200 mg, 1.10 mmol
  • 2-methylpyridin-4-yl-4- boronic acid 227.60 mg, 1.66 mmol
  • BuOH 5.0 mL
  • water 1.0 mL
  • K 3 PO 4 705.20 g, 3.32 mmol
  • Pd 2 (dba) 3 49.60 mg, 0.22 mmol
  • S-phos 9.00 mg, 0.11 mmol
  • 6-(2-methylpyridin-4-yl)-2,7-naphthyridin- l(2H)-one (150 mg, 0.63 mmol) is dissolved in POCI 3 (15.0 mL), the pressure tube is sealed and heated up to 160 ° C for 4 h. After cooling down the reaction to RT, excessive POCI 3 is removed under vacuum. Crushed ice is slowly added into the mixture, and then added into NaHC0 3 to neutralize until pH ⁇ 7.5. Extracted the solution by EA three times, the combined organic layer is washed with brine, dried over Na 2 SC>4, and concentrated under vacuum.
  • PBS phosphate-buffered saline
  • Accutase solution Millipore, SCR005
  • Induction of iPS cell differentiation is accomplished in the same manner as used in Example 2 in PSGro Basal Medium (StemRD Inc.) containing either 50 ng/mL recombinant Wnt-3a (StemRD Inc.), 100 ng/mL Wnt-5a (StemRD Inc.), or ⁇ CHIR99021.
  • the medium also contains 10 ng/mL Activin A (StemRD Inc.) and 10 ng/mL BMP-4 (StemRD Inc.). After 48 hrs to 120 hrs treatment, cells are harvested for the analysis of gene expression of mesodermal marker Brachyury by using qRT-PCR.
  • Wnt activator Wnt-3a or CHIR99021 As shown in Fig.l, after 48 hrs treatment, expression of Brachyury is significantly higher in groups treated with Wnt activator Wnt-3a or CHIR99021. These results indicate that Wnt activator significantly induces mesodermal differentiation from human iPS cells. This effect is observed as long as a Wnt activator is present for the first 48 hours. Optimal concentrations of the Wnt activators are determined. When the recombinant Wnt-3a protein is added at concentrations of 1 ng/mL to 100 ng/mL, optimal Brachyury expression is obtained at a concentration from 10 ng/mL to 50 ng/mL.
  • Induction of cardiac mesodermal cell differentiation is accomplished in the same manner as used in Example 2 in PSGro Basal Medium containing Wnt inhibitor either 100 ng/mL recombinant Wnt-5a (StemRD Inc.), 100 nM of XAV939, or 100 nM of CGX307.
  • the PSGro Basal Medium also contains 200 ng/mL Noggin (StemRD Inc.) or a small molecule inhibitor for BMP receptor such as LDN-193189 or SB431542 both at 1 nM to 1000 nM. In the example, the medium contains 500 nM of LDN-193189.
  • cells are harvested for the analysis of gene expression of cardiac mesodermal marker Flk-l/KDR by using qRT-PCR.
  • Differentiating cells are cultured in the medium containing an aforementioned Wnt inhibitor for 48 to 120 hours to induce cardiac progenitor cells.
  • the medium also contains factors known for cardiac progenitor differentiation such as bFGF at a concentration from 0.1 ng/mL to 100 ng/mL and VEGF at 0.1 nM to 100 nM. After treatment, cells are harvested for the analysis of gene expression of cardiovascular progenitor marker Isl-1.
  • differentiating cells are cultured in the PSGro Basal medium containing 100 ng/mL Wnt-5a, 100 nM of XAV939, or 100 nM CGX307 for 96 hours to induce cardiac progenitor cells.
  • the medium also contains bFGF at a concentration of 10 ng/mL and VEGF at 10 nM.
  • the resulting differentiated cells from aforementioned procedures are cultured in a medium containing a Wnt activator selected from aforementioned group for 48 to 240 hours to maintain the survival of the cardiomyocytes.
  • the medium also contains Thymosin "4, a factor known to increase the survival of cardiac cells, at 0.1 nM to 100 nM. Live cell numbers are measured by the CellTiter Glow assay (Promega).
  • the differentiated cells are cultured in the PSGro Basal medium containing 50 ng/mL Wnt-3a or CGX307 for 120 hours.
  • Two groups also contain Thymosin ⁇ -4 at 30 nM.
  • cardiomyocytes specific marker on protein level are specific marker on protein level.
  • cells in the Wnt activator- and inhibitor-treated group at 16 days after induction of differentiation increase in number for positive staining of cardiomyocyte- specific marker protein a-actinin.

Abstract

La présente invention concerne un procédé de production de cardiomyocytes humains dans un système de culture de manière sélective et efficace à partir de cellules souches pluripotentes au moyen d'activateurs et d'inhibiteurs de la voie de signalisation Wnt. La présente invention concerne également l'utilisation d'un inhibiteur dans la production.
PCT/CN2012/074852 2012-04-27 2012-04-27 Procédé de production de cardiomyocytes WO2013159349A1 (fr)

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Cited By (6)

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US20150299658A1 (en) * 2012-07-23 2015-10-22 Institute Of Biophysics, Chinese Academy Of Sciences Method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro
CN106867961A (zh) * 2017-03-27 2017-06-20 广州润虹医药科技有限公司 一种诱导多能干细胞形成中胚层细胞的诱导培养基及诱导方法
CN109609446A (zh) * 2018-11-14 2019-04-12 广西大学 一种用于分离培养兔胚胎干细胞的培养液和方法
WO2020023806A1 (fr) * 2018-07-25 2020-01-30 The Johns Hopkins University Compositions et procédés de génération de cellules progénitrices spécifiques d'un champ cardiaque
US10590386B2 (en) 2010-06-13 2020-03-17 Insitute Of Biophysics, Chinese Academy Of Sciences Methods and compositions for preparing cardiomyocytes from stem cells and uses thereof
WO2021250082A1 (fr) * 2020-06-09 2021-12-16 Genethon Traitement de cardiomyopathies dilatées

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US20040014209A1 (en) * 2002-01-23 2004-01-22 Lassar Andrew B. Compositions and methods for modulating cell differentiation
CN101365784A (zh) * 2005-10-24 2009-02-11 科学技术研究公司 确定中胚层、内胚层和中内胚层细胞命运的方法
CN101426902A (zh) * 2006-04-28 2009-05-06 阿斯比奥制药株式会社 将多能性干细胞分化诱导成心肌细胞的方法
CN101573442A (zh) * 2006-11-09 2009-11-04 J·大卫格莱斯顿学会 诱导心肌细胞形成的方法

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US20040014209A1 (en) * 2002-01-23 2004-01-22 Lassar Andrew B. Compositions and methods for modulating cell differentiation
CN101365784A (zh) * 2005-10-24 2009-02-11 科学技术研究公司 确定中胚层、内胚层和中内胚层细胞命运的方法
CN101426902A (zh) * 2006-04-28 2009-05-06 阿斯比奥制药株式会社 将多能性干细胞分化诱导成心肌细胞的方法
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10590386B2 (en) 2010-06-13 2020-03-17 Insitute Of Biophysics, Chinese Academy Of Sciences Methods and compositions for preparing cardiomyocytes from stem cells and uses thereof
US20150299658A1 (en) * 2012-07-23 2015-10-22 Institute Of Biophysics, Chinese Academy Of Sciences Method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro
US11339371B2 (en) * 2012-07-23 2022-05-24 Institute Of Biophysics, Chinese Academy Of Sciences Method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro
CN106867961A (zh) * 2017-03-27 2017-06-20 广州润虹医药科技有限公司 一种诱导多能干细胞形成中胚层细胞的诱导培养基及诱导方法
WO2020023806A1 (fr) * 2018-07-25 2020-01-30 The Johns Hopkins University Compositions et procédés de génération de cellules progénitrices spécifiques d'un champ cardiaque
CN109609446A (zh) * 2018-11-14 2019-04-12 广西大学 一种用于分离培养兔胚胎干细胞的培养液和方法
CN109609446B (zh) * 2018-11-14 2020-11-03 广西大学 一种用于分离培养兔胚胎干细胞的培养液和方法
WO2021250082A1 (fr) * 2020-06-09 2021-12-16 Genethon Traitement de cardiomyopathies dilatées

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