Method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium

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 (iPS) 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.

1 .- 16 . (canceled) 
     
     
         17 . A myotube or myofiber formed from 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);   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 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, 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), and 90-110 ng/ml of FGF2 for at least 1 passage, to thereby provide a cell culture comprising a population of expanded C-Met+ and Hnk1− myogenic progenitor cells, wherein at least 50% of said population of expanded C-Met+ and Hnk1− myogenic progenitor cells are myogenesis marker MyoD positive and myogenesis marker Pax7 negative,   optionally on a scaffold.   
     
     
         18 . The myotube or myofiber according to  claim 17 , (i) having a fusion index of at least 60%, (ii) wherein the myotube or myofiber forms sarcomeres, (iii) wherein the myotube or myofiber expresses neuromuscular junctions, and/or (iv) wherein the myotube or myofiber shows spontaneous contraction in culture. 
     
     
         19 . An in vitro method of screening for a test compound that modulates the function of myogenic cells in 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);   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 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, 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), and 90-110 ng/ml of FGF2 for at least 1 passage, to thereby provide a cell culture comprising a population of expanded C-Met+ and Hnk1− myogenic progenitor cells, wherein at least 50% of said population of expanded C-Met+ and Hnk1− myogenic progenitor cells are myogenesis marker MyoD positive and myogenesis marker Pax7 negative,   or that modulates the function of a myotube or myofiber formed from said cell culture, comprising the step of   a) contacting a test compound with said cell culture or said myotube or myofiber;   b) observing a change in the function, phenotype, proteome, transcriptome, or interactome of said cells in said culture, or said myotube or myofiber, compared to a control compound, and optionally   c) selecting said test compound in the case a change is observed in step b) to thereby provide a candidate drug for treating skeletal muscle disorders.   
     
     
         20 .- 21 . (canceled) 
     
     
         22 . A method for the treatment of a muscle disorder comprising the administration of a myotube or myofiber according to  claim 17  to an individual in need thereof. 
     
     
         23 . The method according to  claim 22 , wherein the mode of administration is by transplantation. 
     
     
         24 . A synthetic culture medium for expanding myogenic progenitor cells, comprising a synthetic base medium, fetal bovine serum (FBS), and 90-110 ng/ml of FGF2. 
     
     
         25 . The medium of  claim 24 , wherein the synthetic culture medium is for expanding isolated myogenic progenitor cells, and wherein said culture medium further comprises a ROCK inhibitor. 
     
     
         26 .- 27 . (canceled) 
     
     
         28 . 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% knock-out serum replacement and/or 0.1-10 mg/ml, BSA. 
     
     
         29 . The myotube or myofiber of  claim 17 , wherein said PSC is an iPSC. 
     
     
         30 . The myotube or myofiber of  claim 17 , wherein the concentration of CHIR99021 in step b) is about 3.5 microM. 
     
     
         31 . The myotube or myofiber of  claim 17 , wherein the concentration FBS in step d) is about 10% (w/w). 
     
     
         32 . The myotube or myofiber of  claim 17 , wherein in step d) said at least one isolated C-Met+ and Hnk1− myogenic progenitor starting cell is expanded for at least 7 passages in step d). 
     
     
         33 . The myotube or myofiber of  claim 17 , wherein in step d) 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. 
     
     
         34 . The myotube or myofiber of  claim 18 , having a fusion index of at least 70%. 
     
     
         35 . The synthetic culture medium of  claim 24 , wherein the synthetic base medium is DMEM HG. 
     
     
         36 . The synthetic culture medium of  claim 24  comprising FBS in a concentration of about 10. 
     
     
         37 . The synthetic culture medium of  claim 24  comprising about 100 ng/ml FGF2. 
     
     
         38 . The medium of  claim 25 , wherein the synthetic culture medium is for expanding isolated myogenic progenitor cells isolated by FACS. 
     
     
         39 . The synthetic culture medium of  claim 28 , comprising about 1% knock-out serum replacement. 
     
     
         40 . The synthetic culture medium of  claim 28 , comprising about 0.5 mg/ml BSA.