Defining the heterogeneity of skeletal muscle-derived side and main population cells isolated immediately ex vivo.
Myoblast transfer therapy for Duchenne muscular dystrophy (DMD) largely fails due to cell death and inability of transplanted cells to engraft in diseased muscles. One method attempting to enrich for cell subpopulations is the Hoechst 33342 dye exclusion assay, yielding a side population (SP) thought to be progenitor enriched and a main population (MP). However, in vitro and transplant studies yielded inconsistent results relative to downstream progeny. Cell surface markers expressed by skeletal muscle-derived MP and SP cells have not been fully characterized directly ex vivo. Using flow cytometry, MP and SP cells were characterized based on their expression of several well-accepted progenitor cell antigens. Both the MP and SP populations are heterogeneous and overlapping in the cells they contain. The percentages of cells in each population vary with species and specific muscle examined. MP and SP populations contain both satellite and multipotent progenitor cells, based on expression of CD34, Sca-1, Pax7, and M-cadherin. Thus, isolation using this procedure cannot be used to predict downstream differentiation outcomes, and explains the conflicting literature on these cells. Hoechst dye also results in significant mortality of sorted cells. As defined subpopulations are easily obtained using flow cytometry, sorting immediately ex vivo based on accepted myogenic precursor cell markers will yield superior results in terms of cell homogeneity for transplantation therapy.
Kallestad
Kristen M
KM
Department of Ophthalmology and Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455, USA.
McLoon
Linda K
LK
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D000950:Antigens, Ly / Q000032:analysis; D001562:Benzimidazoles / Q000633:toxicity; D015415:Biomarkers / Q000032:analysis; D015820:Cadherins / Q000032:analysis; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000633:toxicity; D017493:Leukocyte Common Antigens / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D039902:Multipotent Stem Cells / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000166:cytology; D051763:PAX7 Transcription Factor / Q000032:analysis; D010641:Phenotype; D011817:Rabbits; D032496:Satellite Cells, Skeletal Muscle / Q000737:chemistry*
D018952:Antigens, CD34; D000950:Antigens, Ly; D001562:Benzimidazoles; D015415:Biomarkers; D015820:Cadherins; D005456:Fluorescent Dyes; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; C070896:M-cadherin; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse; C017807:bisbenzimide ethoxide trihydrochloride