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

Abstract

Abstract. 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 fl-galactosidase (fl-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 fl-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 cellmediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

“…Mononucleated myoblasts spontaneously fuse with large multinucleated myotubes, 'sharing' their gene products. 29 Myoblasts are reportedly more efficiently transduced than myotubes by adenovirus recombinants; 30 the same comparison has not been done using rAAV. The efficient transduction of myotubes that we observed could result from primary AAV infection of differentiated myotubes or infection of myoblasts with subsequent formation of fusion myofibers.…”

Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse

“…17 In vitro transfection and transduction Primary myoblasts derived from 3-day-old S4/129 (wildtype) and W1/129 (ANT1-deficient) mouse gastrocnemius muscle were purified to 499%. 34 Myoblasts grown in GM medium (Ham's F10 (Gibco), 20% FBS (Hyclone), 5 ng/ml bFGF (Promega)/5% CO 2 /371C) on dishes coated with E-C-L matrix (Upstate Biotech) were plated in 24-well tissue culture plates and plating efficiency was controlled to avoid significant variation among groups. At 48 h after plating, cells were transduced with the viral vectors, either with AAV-ANT1 plus pTR-UF1 at a 1:1 ratio or with AAV-ANT1 alone.…”

Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction

“…Previous experiments have shown that early preplates (pp1-2) contain a majority of fibroblasts (5-15% desmin-positive) and the late preplates (pp5-6) are enriched with desmin-positive cells (>80%). [20][21][22]41 The cells used in this experiment were taken from the late preplate (pp6) and were shown to express myogenic and stem cell markers. 9 The proliferation medium used to grow the cells was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10% horse serum (HS) and 1% penicillin/streptomycin.…”

“…20 These cells were harvested from a muscle biopsy and purified by the preplate technique based on their adherence characteristics to collagen-coated flasks. [20][21][22] The purified MDC (pp6) used for these experiments took about 6 days to adhere to collagen-coated flasks and stained positive for myogenic (desmin) and well-known stem cell markers, including Sca-1 and FLK-1. 9 These results suggest that this population of highly purified MDC may contain stem cells.…”