Myotube driven myogenic recruitment of cells during in vitro myogenesis

Abstract

Muscular dysgenesis (mdg) is a recessive lethal mutation in the mouse which drastically affects skeletal muscle development during embryonic life. Physiologically, the disease is characterized by a complete paralysis resulting from a lack of excitation-contraction coupling. Existing electrophysiological, biochemical, and genetic evidence shows that mdg/mdg mice express a basic alteration of L-type voltage-sensitive Ca2+ channels in skeletal muscle. Studies on mdg/mdg myotubes in primary culture have shown that +/+ fibroblasts or +/+ Schwann cells may fuse with them and correct their functional deficiency by genetic complementation. As the spontaneous formation of heterocaryons is thought to be an exclusive property of myoblasts, we asked whether fibroblasts may have changed their properties before fusion occurred. We used primary cells issued from sciatic nerves dissected from newborn transgenic mice carrying the pHuDes1-nls-LacZ transgene (Des-LacZ cells) as non-muscle cells. These cells were mainly fibroblasts (80%) positive for Thy1.1 and Schwann cells positive for S100. The cultures were negative for myogenic markers (desmin, troponin T), did not form myotubes long-term, and did not display significant activation of the muscle reporter gene (pHuDes1-nls-LacZ). After a few days in coculture with dysgenic or normal myotubes, the muscle reporter gene (beta-galactosidase) was detected both within dysgenic myotubes, correlating with the restoration of normal contractile activity, and normal myotubes. As well as confirming that fusion takes place, this shows that Des-LacZ cells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleated Des-LacZ cells expressing beta-galactosidase were observed, indicating that myogenic genes were being expressed before fusion. This suggests a mechanism of myotube driven myogenic recruitment of cells during the in vitro myogenesis. Analysis of the distribution of the induced Des-LacZ cells (positive for beta-galactosidase) in compartmentalized muscle cocultures showed that in the presence of dysgenic myotubes, these cells were equally distributed in both myotube free and enriched areas, whereas in the presence of normal myotubes, the positive cells remained in close vicinity of the myotubes. This difference could be explained by the fact that the dysgenic phenotype might include release of the induction process from its normal controls. Our results are consistent with the idea of a transcellular mechanism triggering myogenic differentiation in non-muscle cells, and that myotubes themselves are able to drive myogenic recruitment of cells during the in vitro myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

“…The efficiency of this process is directly related to the lineage relationship with myoblasts, being highest in fibroblasts, lower in chondroblasts, and virtually null in hepatocytes or amnioblasts (9). In keeping with these observations, we and others have reported recently that fibroblasts undergo myogenic differentiation when cocultured with myogenic lines (C2C12) or primary cells, but not with other cell types (10)(11)(12). It is thus possible that a proportion of adult cells of mesodermal origin conserve a bi-or multipotential state of determination throughout life, thereby enhancing the regenerative capacity of the tissue in which they reside (13).…”

Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells

“…These include primary fibroblasts from different locations (Breton et al , 1995; Salvatori et al , 1995), BM-MSCs (Shi et al , 2004; Lee et al , 2005; Schulze et al , 2005), AT-MSCs (Di Rocco et al , 2006), pericytes (Dellavalle et al , 2007; Kirillova et al , 2007), and other BM-derived cells (Sacco et al , 2005). The fusion efficiency varies with cell types.…”

Myogenic fusion of human bone marrow stromal cells, but not hematopoietic cells

“…13 Previous studies have shown that coculturing C2C12 cells with dermal fibroblasts resulted in cell fusion and the subsequent correction of the dysgenic membrane in regenerating muscle. [14][15][16] The myogenic potential of neural stem cells was also demonstrated by coculture with C2C12 cells. 17,18 In this report we cocultured human hematopoietic and nonhematopoietic bone marrow cells with C2C12 and found that stromal cells have a significantly greater potential to contribute to myotube formation than enriched populations of hematopoietic progenitor cells, and that this contribution is due to cell fusion.…”