High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells

Abstract

SummaryInvestigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC) pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs.

Coding Cell Identity of Human Skeletal Muscle Progenitor Cells Using Cell Surface Markers: Current Status and Remaining Challenges for Characterization and Isolation

“…CD29, or integrin beta-1, is a well-established marker widely utilized to isolate or identify human myogenic cells. CD29 expression has been confirmed in PSC-derived SMPCs (Awaya et al, 2012; Darabi et al, 2012; Abujarour et al, 2014; Magli et al, 2017; Sakai-Takemura et al, 2018), fetal muscle (Castiglioni et al, 2014), postnatal muscle (Garcia et al, 2018), and adult muscle (Lecourt et al, 2010; Woodard et al, 2014; Charville et al, 2015; Xu et al, 2015; Lorant et al, 2018). An isoform of CD29, integrin beta-1D, has been shown to be severely reduced in patients with limb girdle muscular dystrophy type 2C (Anastasi et al, 2004) or sensitive-motor polyneuropathy (Anastasi et al, 2008).…”

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

“…The major benefit of this approach is that multiple assays and experiments can be performed on hMPCs from the same donor, allowing for maintenance of donor phenotype across experiments while introducing little inter-experiment variability. Our method differs from recently published methods that focus on extracting the purest population of Pax7 expressing cells directly out of biopsy tissue where the focus is xenotransplantation 8 . The challenge with these previous methods however, is that they require a starting tissue weight of greater than one gram.…”

Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine

“… 18 , 83 To overcome these, microenvironmental alterations to favor human hematopoiesis have been described (Figure 2 ). Methods include (1) mutation of critical murine growth factor receptors, such as the c-kit receptor (NSGW41, NSGWv/+, NSGWv, NBSGW, SRG-W41, and BRgWv mice), 23 , 30 (2) inhibition of growth factor receptor function (eg, anti-c-kit receptor antibody), 84 (3) exogenous human cytokine administration (eg, B lymphocyte stimulator for mature human B cell reconstitution 63 and IL-7 analogues for T cell reconstitution 75 ), and (4) knock-in of human cytokine genes, such as in SGM3, 27 MISTRG, 42 SRG-15, 85 and hIL-6 Tg NSG 28 strains, which include knock-ins of IL-3, IL-15, GM-CSF, M-CSF, or IL-6 to support engraftment of the wider human hematopoietic repertoire, including innate cells and regulatory T cells (Treg). 27 …”