Activation of Notch Signaling During Ex Vivo Expansion Maintains Donor Muscle Cell Engraftment

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, District of Columbia, USA. .
Stem Cells (Impact Factor: 6.52). 10/2012; 30(10):2212-20. DOI: 10.1002/stem.1181
Source: PubMed


Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. However, a single donor muscle biopsy is unlikely to provide enough cells to effectively transplant the muscle mass of a patient affected by muscular dystrophy. Expansion of cells ex vivo using traditional culture techniques significantly reduces engraftment potential. We hypothesized that activation of Notch signaling during ex vivo expansion would maintain donor cell engraftment potential. In this study, we expanded freshly isolated canine muscle-derived cells on tissue culture plates coated with Delta-1(ext) -IgG to activate Notch signaling or with human IgG as a control. A model of canine-to-murine xenotransplantation was used to quantitatively compare canine muscle cell engraftment and determine whether engrafted donor cells could function as satellite cells in vivo. We show that Delta-1(ext) -IgG inhibited differentiation of canine muscle-derived cells and increased the level of genes normally expressed in myogenic precursors. Moreover, cells expanded on Delta-1(ext) -IgG resulted in a significant increase in the number of donor-derived fibers, as compared to cells expanded on human IgG, reaching engraftment levels similar to freshly isolated cells. Importantly, cells expanded on Delta-1(ext) -IgG engrafted to the recipient satellite cell niche and contributed to further regeneration. A similar strategy of expanding human muscle-derived cells on Notch ligand might facilitate engraftment and muscle regeneration for patients affected with muscular dystrophy. STEM Cells2012;30:2212-2220.

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Available from: William Duddy, May 26, 2015
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    • "A seemingly contradictory observation is that freshly isolated canine satellite cells plated on Dll1 ligand, expanded more than non-treated cells [65]. One major difference between this and the Pax7CreERT2: R26stop-NICD experiment cited above, is that cells were exposed to the ligand after their isolation, hence they were already activated. "
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    ABSTRACT: Notch signalling acts in virtually every tissue during the lifetime of metazoans. Recent studies have pointed to multiple roles for Notch in stem cells during quiescence, proliferation, temporal specification, and maintenance of the niche architecture. Skeletal muscle has served as an excellent paradigm to examine these diverse roles as embryonic, foetal, and adult skeletal muscle stem cells have different molecular signatures and functional properties, reflecting their developmental specification during ontology. Notably, Notch signalling has emerged as a major regulator of all muscle stem cells. This review will provide an overview of Notch signalling during myogenic development and postnatally, and underscore the seemingly opposing contextual activities of Notch that have lead to a reassessment of its role in myogenesis.
    BMC Developmental Biology 01/2014; 14(1):2. DOI:10.1186/1471-213X-14-2 · 2.67 Impact Factor
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    • "Inhibition of MMP-9 improves the activation of endogenous satellite cells and facilitates exogenous myoblast engraftment and consequent expression of dystrophin in myofibers of mdx mice. Clinical trials utilizing signaling pathways such as Notch as a mean to expand myoblast prior to transplantation have been successful in achieving more functional engraftment but are still limited by the ability of cells to migrate after entering a dystrophic environment [66]. Given that inhibition of MMP-9 was sufficient to facilitate muscle cell survival through cooperative activation of multiple signaling pathways including Notch and Wnt, pharmacological inhibition of MMP-9 can be an important approach to improve satellite cell proliferation, migration, and subsequent gene expression inside the diseased muscle after transplantation. "
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    ABSTRACT: Duchenne muscular dystrophy (DMD) caused by loss of cytoskeletal protein dystrophin is a devastating disorder of skeletal muscle. Primary deficiency of dystrophin leads to several secondary pathological changes including fiber degeneration and regeneration, extracellular matrix breakdown, inflammation, and fibrosis. Matrix metalloproteinases (MMPs) are a group of extracellular proteases that are involved in tissue remodeling, inflammation, and development of interstitial fibrosis in many disease states. We have recently reported that the inhibition of MMP-9 improves myopathy and augments myofiber regeneration in mdx mice (a mouse model of DMD). However, the mechanisms by which MMP-9 regulates disease progression in mdx mice remain less understood. In this report, we demonstrate that the inhibition of MMP-9 augments the proliferation of satellite cells in dystrophic muscle. MMP-9 inhibition also causes significant reduction in percentage of M1 macrophages with concomitant increase in the proportion of promyogenic M2 macrophages in mdx mice. Moreover, inhibition of MMP-9 increases the expression of Notch ligands and receptors, and Notch target genes in skeletal muscle of mdx mice. Furthermore, our results show that while MMP-9 inhibition augments the expression of components of canonical Wnt signaling, it reduces the expression of genes whose products are involved in activation of non-canonical Wnt signaling in mdx mice. Finally, the inhibition of MMP-9 was found to dramatically improve the engraftment of transplanted myoblasts in skeletal muscle of mdx mice. Collectively, our study suggests that the inhibition of MMP-9 is a promising approach to stimulate myofiber regeneration and improving engraftment of muscle progenitor cells in dystrophic muscle.
    PLoS ONE 08/2013; 8(8):e72121. DOI:10.1371/journal.pone.0072121 · 3.23 Impact Factor
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    • "Although the therapeutic potentials of SCs are attractive, their regenerative ability is greatly reduced after cell culture, rendering expansion in vitro unsatisfactory [14], [27]. Several studies demonstrated expansion of SCs maintaining engraftment potential [42], [43] but acquisition of enough amounts of SCs for transplantation remains to be a major challenge. Thus other novel cell sources of large quantities of cells are required. "
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    ABSTRACT: Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3(GFP/+) embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+) mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease.
    PLoS ONE 05/2013; 8(5):e63016. DOI:10.1371/journal.pone.0063016 · 3.23 Impact Factor
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