Prospective Isolation of Skeletal Muscle Stem Cells with a Pax7 Reporter

Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA.
Stem Cells (Impact Factor: 6.52). 10/2008; 26(12):3194-204. DOI: 10.1634/stemcells.2007-1017
Source: PubMed


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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Available from: Zhaohui Xu, Oct 06, 2015
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    • "A major barrier to the development of stem cell-based therapies is the inability to generate large numbers of transplantable stem cells with the potential to both self-renew and differentiate. In general, the contribution of donor SCs to muscle regeneration has been shown to correlate with the number of cells transplanted (Bosnakovski et al., 2008; Sacco et al., 2008). Although transplantation of SCs in association with donor muscle fibers has been shown to enhance engraftment efficiency (Collins et al., 2005; Hall et al., 2010), biopsies, surgical specimens, and post-mortem tissue donations are expected to yield few cells relative to the number that will be required for therapeutic huSC engraftment , and techniques for the growth and manipulation of progenitor cells ex vivo are, therefore, expected to be an important element of cell-based therapies. "
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    ABSTRACT: Adult skeletal muscle stem cells, or satellite cells (SCs), regenerate functional muscle following transplantation into injured or diseased tissue. To gain insight into human SC (huSC) biology, we analyzed transcriptome dynamics by RNA sequencing of prospectively isolated quiescent and activated huSCs. This analysis indicated that huSCs differentiate and lose proliferative potential when maintained in high-mitogen conditions ex vivo. Further analysis of gene expression revealed that p38 MAPK acts in a transcriptional network underlying huSC self-renewal. Activation of p38 signaling correlated with huSC differentiation, while inhibition of p38 reversibly prevented differentiation, enabling expansion of huSCs. When transplanted, expanded huSCs differentiated to generate chimeric muscle and engrafted as SCs in the sublaminar niche with a greater frequency than freshly isolated cells or cells cultured without p38 inhibition. These studies indicate characteristics of the huSC transcriptome that promote expansion ex vivo to allow enhanced functional engraftment of a defined population of self-renewing huSCs.
    Full-text · Article · Sep 2015 · Stem Cell Reports
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    • "Furthermore, FOXO3 loss decreases the ability of neural stem cells to self-renew in vitro (Renault et al., 2009; Paik et al., 2007). Although the FOXO factors were shown to be expressed in different cell types in muscle and FOXO3 germline knockout mice were reported to display a delay in muscle regeneration (Bosnakovski et al., 2008; Hu et al., 2008), none of the members of the FOXO family have been shown to regulate SC function. "
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    ABSTRACT: Skeletal muscle stem cells, or "satellite cells" (SCs), are required for the regeneration of damaged muscle tissue. Although SCs self-renew during regeneration, the mechanisms that govern SC re-entry into quiescence remain elusive. We show that FOXO3, a member of the forkhead family of transcription factors, is expressed in quiescent SCs (QSCs). Conditional deletion of Foxo3 in QSCs impairs self-renewal and increases the propensity of SCs to adopt a differentiated fate. Transcriptional analysis of SCs lacking FOXO3 revealed a downregulation of Notch signaling, a key regulator of SC quiescence. Conversely, overexpression of Notch intracellular domain (NICD) rescued the self-renewal deficit of FOXO3-deficient SCs. We show that FOXO3 regulates NOTCH1 and NOTCH3 receptor expression and that decreasing expression of NOTCH1 and NOTCH3 receptors phenocopies the effect of FOXO3 deficiency in SCs. We demonstrate that FOXO3, perhaps by activating Notch signaling, promotes the quiescent state during SC self-renewal in adult muscle regeneration.
    Full-text · Article · Apr 2014 · Stem Cell Reports
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    • "Existing literature was surveyed to identify candidate antigenic markers that might discriminate live myogenic from nonmyogenic cells (Fukada et al., 2004; Kuang et al., 2007; Lecourt et al., 2010; Pisani et al., 2010a, 2010b; Sacco et al., 2008; Sherwood et al., 2004). PAX7, the canonical marker of muscle satellite cells in mouse and human postnatal muscle (Bosnakovski et al., 2008; Seale et al., 2000), is inappropriate for such an approach because as a nuclear protein, antibody staining requires cell fixation/permeabilization . Flow cytometric analysis of hMFA cells revealed differential expression of 11 candidate cell surface markers (CD45, CD11b, glycophorin A [GlyA] "
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    ABSTRACT: Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells. These CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(+) fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7(+) CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.
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