A method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium
AssigneeErasmus University Medical Center Rotterdam
InventorsWilhelmus Wenceslaus Matthias Pijnappel, Antje Tjitske van der Ploeg, Erik van der Wal
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Abstract
The invention is in the field of cell culturing. More specifically, it is in the field of generating and expanding myogenic cells from induced pluripotent stem (i PS) cells. The invention relates inter alia to cells generated and expanded via such a method, a growth medium specifically suited for the purpose of expanding isolated myogenic cells, and methods for screening compounds on cell structures such as myotubes and myofibers.
Claims28
A method for producing a cell culture comprising a population of expanded myogenic progenitor cells in a synthetic culture medium, comprising the steps of:
- a) providing a pluripotent stem cell (PSC), preferably an iPSC;
- b) culturing said PSC in a synthetic culture medium supporting differentiation of said PSC towards a myogenic cell lineage for (i) a first period of 3-8 days in the presence of between 2-5 microM, preferably about 3.5 microM, of CHIR99021, (ii) a second period of 5-20 days in the presence of 10-30 ng/ml of FGF2;
- and, optionally, (iii) a third period of 10-20 days in the presence of insulin-transferrin-selenium-ethanolamine (ITS-X), to thereby provide a cell culture of pre-differentiated PSCs comprising myogenic progenitors cells;
- c) isolating from said cell culture comprising myogenic progenitors cells at least one C-Met+ and Hnk1− myogenic progenitor starting cell, preferably by FACS, to thereby provide a purified myogenic cell lineage;
- d) expanding said at least one isolated C-Met+ and Hnk1− myogenic progenitor starting cell in a synthetic culture medium comprising fetal bovine serum (FBS), preferably in a concentration of about 10% (w/w), and 90-110 ng/ml of FGF2 for at least 1 passage, preferably at least 7 passages, to thereby provide a cell culture comprising a population of expanded C-Met+ and Hnk1− myogenic progenitor cells, wherein at least 50%, preferably at least 90% of said population of expanded C-Met+ and Hnk1− myogenic progenitor cells are myogenesis marker MyoD positive and myogenesis marker Pax7 negative.
A cell culture comprising a population of expanded myogenic progenitor cells in a synthetic culture medium, obtainable by a method comprising the steps of:
- a) providing a pluripotent stem cell (PSC), preferably an iPSC;
- b) culturing said PSC in a synthetic culture medium supporting differentiation of said PSC towards a myogenic cell lineage for (i) a first period of 3-8 days in the presence of between 2-5 microM, preferably about 3.5 microM, of CHIR99021, (ii) a second period of 5-20 days in the presence of 10-30 ng/ml of FGF2;
- and, optionally, (iii) a third period of 10-20 days in the presence of insulin-transferrin-selenium-ethanolamine (ITS-X), to thereby provide a cell culture of pre-differentiated PSCs comprising myogenic progenitors cells;
- c) isolating from said cell culture comprising myogenic progenitors cells at least one C-Met+ and Hnk1− myogenic progenitor starting cell, preferably by FACS, to thereby provide a purified myogenic cell lineage;
- d) expanding said at least one isolated C-Met+ and Hnk1− myogenic progenitor starting cell in a synthetic culture medium comprising fetal bovine serum (FBS), preferably in a concentration of about 10% (w/w), and 90-110 ng/ml of FGF2 for at least 1 passage, preferably at least 7 passages, to thereby provide a cell culture comprising a population of expanded C-Met+ and Hnk1− myogenic progenitor cells, wherein at least 50%, preferably at least 90% of said population of expanded C-Met+ and Hnk1− myogenic progenitor cells are myogenesis marker MyoD positive and myogenesis marker Pax7 negative.
An in vitro cell culture comprising a population of expanded myogenic progenitor cells, wherein at least 90% of the cells in said population is positive for myogenesis marker MyoD, wherein at least 50% of the cells in said population is negative for myogenesis marker Pax7, and wherein the cells in said culture do not express one or more, preferably at least 5, more preferably at least 10, most preferably all of the genes selected from the group of ARX, CD34, CITED1, FGF4, FGF5, FGF6, FGF9, FOXC1, ITGA9, LAMA4, MEOX1, SEMA3D, SEMA4G, SMAD6, TCF15, and UNCX and wherein said cell culture further comprising a synthetic culture medium.
The cell according to claim 15, the myotube or myofiber according to claim 175 or claim 18, or a myogenic cell or cell structure intermediate to said cell and said myotube or myofiber in terms of differentiation status, for use in the treatment of a muscle disorder.
A synthetic culture medium for expanding myogenic progenitor cells, comprising a synthetic base medium, preferably DMEM HG;
- fetal bovine serum (FBS), preferably in a concentration of about 10%;
- and 90-110 ng/ml, more preferably about 100 ng/ml, of FGF2.
A synthetic culture medium for differentiating expanded myogenic progenitor cells into myotubes and/or myofibers, comprising, or consisting of (i) DMEM high glucose, DMEM low glucose or DMEM/F12, (ii) about 1% of ITS-X and (iii) 0.5%-5%, preferably about 1%, of knock-out serum replacement and/or 0.1-10 mg/ml, preferably about 0.5 mg/ml, of BSA.
Dependencies are inferred from the claim text.