Partial Laminin ct2 Chain Restoration in ct2 Chain-deficient dy/dy Mouse by Primary Muscle Cell Culture Transplantation

Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin or2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the 13-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologicaUy-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the 13-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo. AMININ-2 belongs to the laminin family (Burgeson et al., 1994) and is present in basal laminae of cardiac and skeletal muscle cells (Ehrig et al., 1990). Some cells of mesenchymal origin, Schwann cells, and thyroid cells would participate in its secretion (Leivo and Engvall, 1988; Engvall, 1993; Andre et al., 1994). Laminin-2 is constituted by the assembly of three subunits called et2 chain (400 kD), 131, and ~1 chains (200 kD each). Laminins have cross-like three-dimensional structures which interact with cellular receptors such as integrins (Engvall, 1993). Laminin-2 interacts with the 156-kD dystrophin-associated glycoprotein (o~-dystroglycan; Campbell, 1995). The laminins promote cell spreading, migration, proliferation, and/or differentiation, and laminin-2 also promotes neurite outgrowth and nerve-muscle interactions (Engvall et al., Address all correspondence to J.P. Tremblay, Centre de Recherche en Neurobiologie, H6pital de L'Enfant-J6sus, 1401, 18 e Rue, Qu6bec (Qu6) Canada G1J1Z4. Ph.: (418) 649-5593. Fax: (418) 649-5910. E-mail: [email protected]. 1992). Laminin or2 chain (LAMA2) 1 also promotes Schwann cell migration in vitro and nerve regeneration in vivo (Anton et al., 1994). The gene coding for human ct2 chain has been cloned and located on chromosome 6 in position q22/ 23. The cDNA is about 9.5 kb in length and encodes for a more than 3,100-amino acid protein (Vuolteenaho et al., 1994). Hayashi et al. (1993) showed that the LAMA2 expression was reduced in muscle cell basal membranes of some Fukuyama-type congenital muscular dystrophy patients, and Tom6 et al. (1994) showed that LAMA2 expression was deficient in 13 of 20 congenital muscular dystrophy (CMD) patients (i.e., 65%). Out of 25 CMD patients, Sewry et al. (1995) also showed that LAMA2 expression was undetectable in seven and reduced to trace amounts in five. Thus, 48% of CMD patients in this series were deficient for LAMA2 expression. The LAMA2 deficiency has been linked to the Lama2 gene on chromosome 6 (Hillaire 1. Abbreviations used in this paper: 13-gal, 13-galactosidase; CMD, congenital muscular dystrophy; IFN-~,, interferon ~/; LAMA2, laminin-2 et2 chain; TA, Tibialis anterior. © The Rockefeller University Press, 0021-9525/96/04/185/13 $2.00 The Journal of Cell Biology, Volume 133, Number 1, April 1996 185-197 185 on July 8, 2017 jcb.rress.org D ow nladed fom et al., 1994), and a direct implication of the Lama2 gene in the development of the LAMA2-negative CMD has been reported in man (Helbling-Leclerc et al., 1995). One LAMA2-negative CMD patient also presented LAMA2 mRNA deficiency (Hayashi et al., 1995). The LAMA2deficient CMD patients usually have a more severe clinical phenotype than LAMA2-positive CMD patients. They regularly present white matter changes on brain imaging (Philpot et al., 1995). The identification of mutations on the Lama2 gene could provide a useful tool in categorizing various CMD which show clinical heterogeneity (for review see Dubowitz and Fardeau, 1995; Helbling-Leclerc et al., 1995). The Fukuyama-type CMD would not be directly linked to the Lama2 gene. Three groups showed that the dystrophia muscularis (dy/dy) mouse model was deficient for LAMA2 expression (Arahata et al., 1993; Sunada et al., 1994; Xu et al., 1994a). In these studies, LAMA2 was undetectable in immunohistofluorescence in skeletal and cardiac muscle cell basement membranes and in peripheral nerve and some very low levels of LAMA2 mRNA could be detected only after RT-PCR amplification (Arahata et al., 1993; Xu et al., 1994a). The LAMA2 deficiency has been linked to the Lama2 gene on chromosome 10 in dy/dy mouse (Sunada et al., 1994) and a mutation has been identified in the dye~ dy 2s mouse, an allelic variant of the LAMA2-negative dy/ dy model (Xu et al., 1994b; Sunada et al., 1995). First described in 1955, the dy/dy mouse develops early signs of muscular dystrophy (Michelson et al., 1955). The progressive and lethal dystrophy is characterized first by ataxia, convulsive head movements, spasmodic hind leg flexion, and flaccid extension, followed by progressive muscle atrophy and paralysis extending to axial musculature and anterior legs. At the late stages, the posterior leg locomotion is lost and the mouse develops marked kyphosis. The size, number and diameter of muscle fibers, are greatly variable in dy/dy mice, but always less than in normal mice. The muscles are invaded by connective tissues. The neural system of the dy/dy mice also show many abnormalities. The Schwann cell basal lamina are patchy, and the myelination of the peripheral nervous system is variably defective (Bradley and Jenkison, 1973; Madrid et al., 1975; Montgomery and Swenarchuk, 1977). The life span of the dyldy mouse is rarely more than six months, and the disease is of autosomal recessive transmission. The milder allelic variant of the dy/dy mouse, the dy2S/ dy 2s mouse model, express a truncated protein (Xu et al., 1994b; Sunada et al., 1995). This model would not truly reflect at least the majority of the human CMD because of the expression of a truncated but partially functional LAMA2. Muscle regeneration was investigated in the dy2S/dy 2s mouse following muscle tissue, G8 clonal muscle cells, and primary fetal or newborn muscle cell transplantations (Law, 1982; Law et al., 1988a,b). The aetiology and molecular basis of this neuromuscular pathology were unknown at the time of these experiments and the use of this animal model seems now partially inadequate. Actually, G8 myoblasts did not form hybrid muscle fibers (Law et al., 1988a), and some models were not compatible for minor antigens (Law et al., 1988b). Some, but few animals showed behavioral improvements 2 to 7 mo after transplantation (Law et al., 1988a). Myoblast transplantations have been performed in the mdx/mdx mouse, which is a biochemical model of Duchenne muscular dystrophy. Transplantations of cultured muscle cells promoted myoblast fusion, formation of new and/or hybrid muscle fibers in vivo, and intracellular dystrophin restoration in mdxlmdx mice. One of the major drawbacks of myoblast transplantation experiments is the host immune response against donor cells (Kinoshita et al., 1994b; Gu6rette et al., 1995). Immunosuppressive treatments, immunodeficient or immunocompatible animals, were used to overcome this problem (Partridge et al., 1978, 1989; Grounds et al., 1980; Watt et al., 1984; Morgan et al., 1990, 1992, 1993, 1994; Huard et al., 1994; Kinoshita et al., 1994a,b; Pavlath et al., 1994; Asselin et al., 1995; Vilquin et al., 1995a,c). Our group has obtained the best success either using FK506 immunosuppression to control nonhistocompatible myoblast transplantation, or using fully compatible host/donor combinations (Kinoshita et al., 1994b; Vilquin et al., 1995c). In the present study, LAMA2 restoration in the dy/dy mouse after transplantation of cultured primary muscle ceils was evaluated with respect to age at transplantation, type of transplantation, and muscle pretreatment. In addition, pure myoblast cultures were used to study the role of myoblasts in LAMA2 deposition in vivo. Materials and Methods