title: Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.
abstract: Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Shelton
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada; The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.
authors.forename: Avetik
authors.initials: A
authors.lastname: Kocharyan
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada. Electronic address: iskerjan@uottawa.ca.
authors.forename: Ilona S
authors.initials: IS
authors.lastname: Skerjanc
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada; The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada; Ottawa Institute of Systems Biology, Ottawa, ON, Canada. Electronic address: wstanford@ohri.ca.
authors.forename: William L
authors.initials: WL
authors.lastname: Stanford
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D000066449:Human Embryonic Stem Cells / Q000502:physiology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D008856:Microscopy, Fluorescence; D032342:Muscle Cells / Q000502:physiology; D032446:Myoblasts / Q000502:physiology
keywords: CHIR99021; Embryonic stem cells; Induced pluripotent stem cells; Myogenesis; PAX7; Skeletal muscle