Prospective Isolation of Skeletal Muscle Stem Cells with a Pax7 Reporter
ABSTRACT Muscle regeneration occurs through activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to make new myofibers. We used a transgenic Pax7-ZsGreen reporter mouse to prospectively isolate stem cells of skeletal muscle by flow cytometry. We show that Pax7-expressing cells (satellite cells) in the limb, head, and diaphragm muscles are homogeneous in size and granularity and uniformly labeled by certain cell surface markers, including CD34 and CD29. The frequency of the satellite cells varies between muscle types and with age. Clonal analysis demonstrated that all colonies arising from single cells within the Pax7-sorted fraction have myogenic potential. In response to injury, Pax7(+) cells reduce CD34, CD29, and CXCR4 expression, increase in size, and acquire Sca-1. When directly isolated and cultured in vitro, Pax7(+) cells display the hallmarks of activation and proliferate, initially as suspension aggregates and later distributed between suspension and adherence. During in vitro expansion, Pax7 (ZsGreen) and CD34 expression decline, whereas expression of PSA-NCAM is acquired. The nonmyogenic, Pax7(neg) cells expand as Sca1(+) PDGRalpha(+) PSA-NCAM(neg) cells. Satellite cells expanded exclusively in suspension can engraft and produce dystrophin(+) fibers in mdx(-/-) mice. These results establish a novel animal model for the study of muscle stem cell physiology and a culture system for expansion of engraftable muscle progenitors.
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ABSTRACT: Diminished regenerative capacity of skeletal muscle occurs during adulthood. We identified a reduction in the intrinsic capacity of mouse adult satellite cells to contribute to muscle regeneration and repopulation of the niche. Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week-old relative to 18-month-old mice. Knockdown of Jak2 or Stat3 significantly stimulated symmetric satellite stem cell divisions on cultured myofibers. Genetic knockdown of Jak2 or Stat3 expression in prospectively isolated satellite cells markedly enhanced their ability to repopulate the satellite cell niche after transplantation into regenerating tibialis anterior muscle. Pharmacological inhibition of Jak2 and Stat3 activity similarly stimulated symmetric expansion of satellite cells in vitro and their engraftment in vivo. Intramuscular injection of these drugs resulted in a marked enhancement of muscle repair and force generation after cardiotoxin injury. Together these results reveal age-related intrinsic properties that functionally distinguish satellite cells and suggest a promising therapeutic avenue for the treatment of muscle-wasting diseases.Nature Medicine 09/2014; 20(10). DOI:10.1038/nm.3655 · 28.05 Impact Factor
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ABSTRACT: The skeletal muscle has the capacity to repair damage by the activation and differentiation of fiber sub-laminar satellite cells. Regeneration impairment due to reduced satellite cells number and/or functional capacity leads to fiber substitution with ectopic tissues including fat and fibrous tissue and to the loss of muscle functions. Muscle mesenchymal cells that in physiological conditions sustain or directly contribute to regeneration differentiate in adipocytes in patients with persistent damage and inflammation of the skeletal muscle. These cells comprise the fibro-adipogenic precursors, the PW1-expressing cells and some interstitial cells associated with vessels (pericytes, mesoangioblasts and myoendothelial cells). Resident fibroblasts that are responsible for collagen deposition and extracellular matrix remodeling during regeneration yield fibrotic tissue and can differentiate into adipose cells. Some authors have also proposed that satellite cells themselves could transdifferentiate into adipocytes, although recent results by lineage tracing techniques seem to put this theory to discussion. This review summarizes findings about muscle resident mesenchymal cell differentiation in adipocytes and recapitulates the molecular mediators involved in intramuscular adipose tissue deposition.Cellular and Molecular Life Sciences CMLS 02/2015; 72(11). DOI:10.1007/s00018-015-1857-7 · 5.86 Impact Factor
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ABSTRACT: Previously, in an attempt to isolate stem cells that would be capable of regenerating injured skeletal muscle, we cultured cells derived from muscle, non-adherently, in serum-free media. As a result of the culture conditions used, these cells formed spheres, and thus were referred to as myospheres. It was found that myosphere-derived cells expressed Sca-1, a marker that is not typically associated with myogenic cells, and as a result has generated some questions as to the origin of these cells. The goal of this study was to clearly determine the origin of myosphere-derived cells, and in particular to answer the question of whether myospheres contain myogenic cells. To determine if myospheres were composed of myogenic cells without altering the structure of myospheres or the culture conditions used to maintain myospheres, I isolated these cells from yellow fluorescent protein (YFP)-Myf5, YFP-MyoD, and ZsGreen-Pax7 lineage-tracing mice and monitored their growth over time. I found that myospheres do contain myogenic cells, but that these cells are gradually lost over time (within 2 months). Additionally, the use of the lineage-tracing mice gave an interesting perspective into the composition of myospheres. I found that myospheres were composed of two distinct cell types, one that is myogenic (α7 integrin+) and contains cells expressing Myf5, MyoD, and Pax7, and a second that is non-myogenic (α7 integrin-) expressing platelet-derived growth factor receptor alpha (PDGFRα) and Sca-1, both of which have been associated with fibro/adipocyte mesenchymal cells.PLoS ONE 02/2015; 10(2):e0116956. DOI:10.1371/journal.pone.0116956 · 3.53 Impact Factor