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J Cell Biol. 1994 Jun 2; 125(6): 1275–1287.
PMCID: PMC2290930

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy


The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Barr E, Leiden JM. Systemic delivery of recombinant proteins by genetically modified myoblasts. Science. 1991 Dec 6;254(5037):1507–1509. [PubMed]
  • Blau HM, Webster C. Isolation and characterization of human muscle cells. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5623–5627. [PMC free article] [PubMed]
  • Blau HM, Chiu CP, Webster C. Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell. 1983 Apr;32(4):1171–1180. [PubMed]
  • Braun T, Rudnicki MA, Arnold HH, Jaenisch R. Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell. 1992 Oct 30;71(3):369–382. [PubMed]
  • Butcher GW, Howard JC. Genetic control of transplant rejection. Transplantation. 1982 Oct;34(4):161–166. [PubMed]
  • Campion DR. The muscle satellite cell: a review. Int Rev Cytol. 1984;87:225–251. [PubMed]
  • Chaudhari N, Delay R, Beam KG. Restoration of normal function in genetically defective myotubes by spontaneous fusion with fibroblasts. Nature. 1989 Oct 5;341(6241):445–447. [PubMed]
  • Chen M, Li HJ, Fang Q, Goodwin TG, Florendo JA, Law PK. Dystrophin cytochemistry in mdx mouse muscles injected with labeled normal myoblasts. Cell Transplant. 1992;1(1):17–22. [PubMed]
  • Clegg CH, Linkhart TA, Olwin BB, Hauschka SD. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol. 1987 Aug;105(2):949–956. [PMC free article] [PubMed]
  • Dai Y, Roman M, Naviaux RK, Verma IM. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10892–10895. [PMC free article] [PubMed]
  • Dhawan J, Pan LC, Pavlath GK, Travis MA, Lanctot AM, Blau HM. Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science. 1991 Dec 6;254(5037):1509–1512. [PubMed]
  • Gussoni E, Pavlath GK, Lanctot AM, Sharma KR, Miller RG, Steinman L, Blau HM. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature. 1992 Apr 2;356(6368):435–438. [PubMed]
  • Hardiman O, Sklar RM, Brown RH., Jr Direct effects of cyclosporin A and cyclophosphamide on differentiation of normal human myoblasts in culture. Neurology. 1993 Jul;43(7):1432–1434. [PubMed]
  • Jiao S, Gurevich V, Wolff JA. Long-term correction of rat model of Parkinson's disease by gene therapy. Nature. 1993 Apr 1;362(6419):450–453. [PubMed]
  • Jones GE, Murphy SJ, Watt DJ. Segregation of the myogenic cell lineage in mouse muscle development. J Cell Sci. 1990 Dec;97(Pt 4):659–667. [PubMed]
  • Kaleko M, Garcia JV, Osborne WR, Miller AD. Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow. Blood. 1990 Apr 15;75(8):1733–1741. [PubMed]
  • Karpati G, Ajdukovic D, Arnold D, Gledhill RB, Guttmann R, Holland P, Koch PA, Shoubridge E, Spence D, Vanasse M, et al. Myoblast transfer in Duchenne muscular dystrophy. Ann Neurol. 1993 Jul;34(1):8–17. [PubMed]
  • Karpati G, Pouliot Y, Carpenter S. Expression of immunoreactive major histocompatibility complex products in human skeletal muscles. Ann Neurol. 1988 Jan;23(1):64–72. [PubMed]
  • Karpati G, Pouliot Y, Zubrzycka-Gaarn E, Carpenter S, Ray PN, Worton RG, Holland P. Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation. Am J Pathol. 1989 Jul;135(1):27–32. [PMC free article] [PubMed]
  • Kaufman SJ, Foster RF. Replicating myoblasts express a muscle-specific phenotype. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9606–9610. [PMC free article] [PubMed]
  • KONIGSBERG IR. Cellular differentiation in colonies derived from single cells platings of freshly isolated chick embryo muscle cells. Proc Natl Acad Sci U S A. 1961 Nov 15;47:1868–1872. [PMC free article] [PubMed]
  • Labrecque C, Roy R, Tremblay JP. Immune reactions after myoblast transplantation in mouse muscles. Transplant Proc. 1992 Dec;24(6):2889–2892. [PubMed]
  • Law PK. Beneficial effects of transplanting normal limb-bud mesenchyme into dystrophic mouse muscles. Muscle Nerve. 1982 Oct;5(8):619–627. [PubMed]
  • Law PK, Goodwin TG, Li HJ. Histoincompatible myoblast injection improves muscle structure and function of dystrophic mice. Transplant Proc. 1988 Jun;20(3 Suppl 3):1114–1119. [PubMed]
  • Law PK, Goodwin TG, Wang MG. Normal myoblast injections provide genetic treatment for murine dystrophy. Muscle Nerve. 1988 Jun;11(6):525–533. [PubMed]
  • Le Quintrec JS, Le Quintrec JL. Drug-induced myopathies. Baillieres Clin Rheumatol. 1991 Apr;5(1):21–38. [PubMed]
  • Morgan JE. Myogenicity in vitro and in vivo of mouse muscle cells separated on discontinuous Percoll gradients. J Neurol Sci. 1988 Jun;85(2):197–207. [PubMed]
  • Morgan JE, Hoffman EP, Partridge TA. Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse. J Cell Biol. 1990 Dec;111(6 Pt 1):2437–2449. [PMC free article] [PubMed]
  • Morgan JE, Moore SE, Walsh FS, Partridge TA. Formation of skeletal muscle in vivo from the mouse C2 cell line. J Cell Sci. 1992 Aug;102(Pt 4):779–787. [PubMed]
  • Morgan JE, Pagel CN, Sherratt T, Partridge TA. Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice. J Neurol Sci. 1993 Apr;115(2):191–200. [PubMed]
  • Nolan GP, Fiering S, Nicolas JF, Herzenberg LA. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2603–2607. [PMC free article] [PubMed]
  • Palmer TD, Rosman GJ, Osborne WR, Miller AD. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1330–1334. [PMC free article] [PubMed]
  • Partridge TA. Invited review: myoblast transfer: a possible therapy for inherited myopathies? Muscle Nerve. 1991 Mar;14(3):197–212. [PubMed]
  • Partridge TA, Grounds M, Sloper JC. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature. 1978 May 25;273(5660):306–308. [PubMed]
  • Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature. 1989 Jan 12;337(6203):176–179. [PubMed]
  • Phillips GD, Hoffman JR, Knighton DR. Migration of myogenic cells in the rat extensor digitorum longus muscle studied with a split autograft model. Cell Tissue Res. 1990 Oct;262(1):81–88. [PubMed]
  • Ponder BA, Wilkinson MM, Wood M, Westwood JH. Immunohistochemical demonstration of H2 antigens in mouse tissue sections. J Histochem Cytochem. 1983 Jul;31(7):911–919. [PubMed]
  • Price J, Turner D, Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):156–160. [PMC free article] [PubMed]
  • Rastinejad F, Conboy MJ, Rando TA, Blau HM. Tumor suppression by RNA from the 3' untranslated region of alpha-tropomyosin. Cell. 1993 Dec 17;75(6):1107–1117. [PubMed]
  • Richler C, Yaffe D. The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev Biol. 1970 Sep;23(1):1–22. [PubMed]
  • Rudnicki MA, Braun T, Hinuma S, Jaenisch R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell. 1992 Oct 30;71(3):383–390. [PubMed]
  • Sanes JR, Rubenstein JL, Nicolas JF. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. [PMC free article] [PubMed]
  • Scharfmann R, Axelrod JH, Verma IM. Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4626–4630. [PMC free article] [PubMed]
  • Schultz E, Jaryszak DL, Valliere CR. Response of satellite cells to focal skeletal muscle injury. Muscle Nerve. 1985 Mar-Apr;8(3):217–222. [PubMed]
  • Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989 Jun 30;244(4912):1578–1580. [PubMed]
  • Tanabe Y, Esaki K, Nomura T. Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse. Acta Neuropathol. 1986;69(1-2):91–95. [PubMed]
  • Tremblay JP, Malouin F, Roy R, Huard J, Bouchard JP, Satoh A, Richards CL. Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant. 1993 Mar-Apr;2(2):99–112. [PubMed]
  • Watt DJ. Factors which affect the fusion of allogeneic muscle precursor cells in vivo. Neuropathol Appl Neurobiol. 1982 Mar-Apr;8(2):135–147. [PubMed]
  • Watt DJ. A comparison of long-term survival of muscle precursor cell suspensions and minced muscle allografts in the non-tolerant mouse. Adv Exp Med Biol. 1990;280:35–39. [PubMed]
  • Watt DJ, Lambert K, Morgan JE, Partridge TA, Sloper JC. Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse. J Neurol Sci. 1982 Dec;57(2-3):319–331. [PubMed]
  • Watt DJ, Morgan JE, Clifford MA, Partridge TA. The movement of muscle precursor cells between adjacent regenerating muscles in the mouse. Anat Embryol (Berl) 1987;175(4):527–536. [PubMed]
  • Watt DJ, Morgan JE, Partridge TA. Use of mononuclear precursor cells to insert allogeneic genes into growing mouse muscles. Muscle Nerve. 1984 Nov-Dec;7(9):741–750. [PubMed]
  • Watt DJ, Morgan JE, Partridge TA. Allografts of muscle precursor cells persist in the non-tolerized host. Neuromuscul Disord. 1991;1(5):345–355. [PubMed]
  • Wernig A, Irintchev A, Härtling A, Stephan G, Zimmermann K, Starzinski-Powitz A. Formation of new muscle fibres and tumours after injection of cultured myogenic cells. J Neurocytol. 1991 Dec;20(12):982–997. [PubMed]
  • Yao SN, Kurachi K. Expression of human factor IX in mice after injection of genetically modified myoblasts. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3357–3361. [PMC free article] [PubMed]

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