title: Derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells.
abstract: Human pluripotent stem cells (hPSCs) constitute a promising resource for use in cell-based therapies and a valuable in vitro model for studying early human development and disease. Despite significant advancements in the derivation of specific fates from hPSCs, the generation of skeletal muscle remains challenging and is mostly dependent on transgene expression. Here, we describe a method based on the use of a small-molecule GSK3β inhibitor to derive skeletal muscle from several hPSC lines. We show that early GSK3β inhibition is sufficient to create the conditions necessary for highly effective derivation of muscle cells. Moreover, we developed a strategy for stringent fluorescence-activated cell sorting-based purification of emerging PAX3+/PAX7+ muscle precursors that are able to differentiate in postsort cultures into mature myocytes. This transgene-free, efficient protocol provides an essential tool for producing myogenic cells for in vivo preclinical studies, in vitro screenings, and disease modeling.
authors.affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Bianca
authors.initials: B
authors.lastname: Borchin
authors.affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Joseph
authors.initials: J
authors.lastname: Chen
authors.affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Tiziano
authors.initials: T
authors.lastname: Barberi
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor / Q000378:metabolism*; D051761:Paired Box Transcription Factors / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051763:PAX7 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3