Isolation and characterization of human muscle cells.

Abstract

We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations ofmuscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, aactin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.

Increased apoptosis, huntingtin inclusions and altered differentiation in muscle cell cultures from Huntington's disease subjects

“…Myoblasts were derived in accordance with Blau and Webster 35 and grown in HAM's F10 medium (Sigma, St Louis, MO, USA) supplemented with 15% fetal bovine serum (Gibco/Invitrogen, San Diego, CA, USA), 0.5 mg/ ml bovine serum albumin, 10 ng/ml epidermal growth factor, 4 ng/ml insulin, 0.39 mg/ml dexamethasone, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cultures were stained by immunocytochemistry using antibodies to desmin (Chemicon, Temecula, CA; Calbiochem/Oncogene Research Products, Cambridge, MA, USA), a protein expressed only in myogenic cells.…”

Enhancement of skeletal muscle regeneration

“…After muscle injury, satellite cells become activated in response to both positive and negative growth signals and enter the cell cycle (Allen and Rankin, 1990;Bischoff, 1990a,c). The cells are capable of extensive proliferation when challenged by repeated cycles of injury and regeneration in vivo (Sadeh et al, 1985;Morlet et al, 19891, or by clonal growth in culture (Blau and Webster, 1981;Ham et al, 1990), while maintaining the ability to differentiate. Activated satellite cells are also capable of crossing the myofiber basal lamina in both directions (Lipton and Schultz, 1979;Blau and Hughes, 1990), and migrating considerable distances within the muscle (Klein-Ogus and Harris, 1983;Schultz et al, , 1988.…”

Methylguanine DNA Methyltransferase-Mediated Drug Resistance-Based Selective Enrichment and Engraftment of Transplanted Stem Cells in Skeletal Muscle

“…The contribution of bone marrow cells to regenerating muscle has been documented in a number of studies [37,49,60,61]. It was not clear until recently, however, whether the incorporation of bone marrow cells was the result of random fusion of inflammatory cells into the regenerating muscle or whether some cells in the bone marrow possessed the capacity to differentiate into the myogenic lineage.…”

“…With mounting evidence showing that circulating cells in the peripheral blood are able to contribute to muscle [37,49,60,61], it has been hypothesized that stem cells that contribute to a variety of cell and tissue types may reside in peripheral blood as a means to maintain pools of stem cells that are readily mobilized [57]. Recently, mesenchymal stem cells (or multipotent stromal cells) have been identified as contributors to the regeneration of a range of mesenchymal tissues including bone, muscle, cartilage, tendon, and adipose tissue, with a remarkable ability to migrate and home to sites of injury (reviewed in [65]).…”

“…Cell Culture and Retroviral Transduction C2C12 cells and human myoblasts were cultured as described previously [37]. MFG-MGMT-P140K retroviral vectors were constructed using the retroviral backbone MFG as described previously [38].…”