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      Derivation and FACS-Mediated Purification of PAX3+/PAX7+ Skeletal Muscle Precursors from Human Pluripotent Stem Cells

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      1 , 1 , 1 ,
      Stem Cell Reports
      Elsevier

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          Summary

          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.

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          Highlights

          • GSK-3 inhibition efficiently promotes the derivation of skeletal muscle from hPSCs

          • C-MET and CXCR4 are used to isolate highly pure PAX3+/PAX7+/LBX1+ precursors

          • Isolated precursors undergo terminal muscle differentiation in culture

          • Nicotinic ACh receptor is used as single marker to isolate skeletal myocytes

          Abstract

          Borchin, Chen, and Barberi describe a method to derive skeletal muscle from hPSC. Early GSK3β inhibition is sufficient to create the conditions necessary for effective muscle derivation. Moreover, they present a strategy for FACS purification of PAX3+/PAX7+ muscle precursors. This protocol provides an essential tool for producing myogenic cells for in vivo studies, in vitro screenings, and disease modeling.

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          Most cited references35

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          A Pax3/Pax7-dependent population of skeletal muscle progenitor cells.

          During vertebrate development, successive phases of embryonic and fetal myogenesis lead to the formation and growth of skeletal muscles. Although the origin and molecular regulation of the earliest embryonic muscle cells is well understood, less is known about later stages of myogenesis. We have identified a new cell population that expresses the transcription factors Pax3 and Pax7 (paired box proteins 3 and 7) but no skeletal-muscle-specific markers. These cells are maintained as a proliferating population in embryonic and fetal muscles of the trunk and limbs throughout development. Using a stable green fluorescent protein (GFP) reporter targeted to Pax3, we demonstrate that they constitute resident muscle progenitor cells that subsequently become myogenic and form skeletal muscle. Late in fetal development, these cells adopt a satellite cell position characteristic of progenitor cells in postnatal muscle. In the absence of both Pax3 and Pax7, further muscle development is arrested and only the early embryonic muscle of the myotome forms. Cells failing to express Pax3 or Pax7 die or assume a non-myogenic fate. We conclude that this resident Pax3/Pax7-dependent progenitor cell population constitutes a source of myogenic cells of prime importance for skeletal muscle formation, a finding also of potential value in the context of cell therapy for muscle disease.
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            Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud.

            Limb muscles develop from cells that migrate from the somites. The signal that induces migration of myogenic precursor cells to the limb emanates from the mesenchyme of the limb bud. Here we report that the c-met-encoded receptor tyrosine kinase is essential for migration of myogenic precursor cells into the limb anlage and for migration into diaphragm and tip of tongue. In c-met homozygous mutant (-/-) mouse embryos, the limb bud and diaphragm are not colonized by myogenic precursor cells and, as a consequence, skeletal muscles of the limb and diaphragm do not form. In contrast, development of the axial skeletal muscles proceeds in the absence of c-met signalling. The specific ligand of the c-met protein, the motility and growth factor scatter factor/hepatocyte growth factor, is expressed in limb mesenchyme and can thus provide the signal for migration which is received by c-met. We have therefore identified a paracrine signalling system that regulates migration of myogenic precursor cells.
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              Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice.

              A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies. Copyright © 2012 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                27 November 2013
                27 November 2013
                17 December 2013
                : 1
                : 6
                : 620-631
                Affiliations
                [1 ]Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia
                Author notes
                []Corresponding author tb.stemcell.lab@ 123456gmail.com
                Article
                S2213-6711(13)00119-7
                10.1016/j.stemcr.2013.10.007
                3871395
                24371814
                6096cd0d-d98b-432a-93b3-d69260acad14
                © 2013 The Authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 August 2013
                : 15 October 2013
                : 16 October 2013
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