Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, in vitro.These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells.Most muscular dystrophies result from a defect within myogenic cells that leads to progressive muscle weakness and wasting, and in severe cases, to the replacement of muscle fibers by connective tissue and/or fat. At advanced stages, skeletal muscle is replaced by fibroblasts and adipocytes. Although these cell types probably promote disease in later stages, it is generally believed that the root cause leading to most muscular dystrophies is a defect originating in myogenic cells. We therefore developed a protocol to enrich, immortalize and isolate rare muscle progenitor cells from severely affected dystrophic muscles to obtain clonal myogenic cell lines. In this report, the protocol is described for cells isolated from skeletal muscle of people with facioscapulohumeral muscular dystrophy (FSHD), but the technique is also applicable to other muscular dystrophies.FSHD has been linked to deletions of D4Z4 tandem repeats at chromosome 4q [1], but it is still unclear how this deletion causes disease. Primary skeletal-muscle cultures have been used in attempts to model the disease, and several FSHD-specific phenotypes, such as increased vacuolization and sensitivity to oxidative stress [2] have been descr