Myogenic progenitors and imaging single-cell flow analysis: a model to study commitment of adult muscle stem cells.
Research on skeletal muscles suffers from a lack of appropriate human models to study muscle formation and regeneration on the regulatory level of single cells. This hampers both basic understanding and the development of new therapeutic approaches. The use of imaging multicolour flow cytometry and myogenic stem cells can help fill this void by allowing researchers to visualize and quantify the reaction of individual cultured cells to bioactives or other physiological impulses. As proof of concept, we subjected human CD56+ satellite cells to reference bioactives follistatin and Malva sylvestris extracts and then used imaging multicolor flow cytometry to visualize the stepwise activation of myogenic factors MyoD and myogenin in individual cells. This approach enabled us to evaluate the potency of these bioactives to stimulate muscle commitment. To validate this method, we used multi-photon confocal microscopy to confirm the potential of bioactives to stimulate muscle differentiation and expression of desmin. Imaging multicolor flow cytometry revealed statistically significant differences between treated and untreated groups of myogenic progenitors and we propose the utilization of this concept as an integral part of future muscle research strategies.
Faculty of Medicine, Institute of Physiology, University of Maribor, Taborska ulica 8, 2000, Maribor, Slovenia, martin.trapecar@gmail.com.
Martin
M
Trapecar
Robi
R
Kelc
Lidija
L
Gradisnik
Matjaz
M
Vogrin
Marjan Slak
MS
Rupnik
D019002:CD56 Antigen / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D003893:Desmin / Q000096:biosynthesis; D005434:Flow Cytometry / Q000379:methods; D038681:Follistatin / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D028501:Malva / Q000737:chemistry; D018613:Microscopy, Confocal; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D017570:MyoD Protein / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D010936:Plant Extracts / Q000494:pharmacology; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
D019002:CD56 Antigen; D003893:Desmin; D038681:Follistatin; D009124:Muscle Proteins; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D018008:Myogenin; C522918:NCAM1 protein, human; D010936:Plant Extracts