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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    • "An even more marked reduction in transplantation linked to cell culture was observed in comparisons of cultured and freshly isolated canine muscle progenitor cells (Parker et al., 2012). Although the transplantation efficiency of muscle progenitors from model organisms has been enhanced previously by manipulation of Notch signaling (Parker et al., 2012) or substrate elasticity (Gilbert et al., 2010), there are no techniques currently for the expansion of self-renewing huSCs ex vivo. "
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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.
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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.
    Full-text · Article · Jan 2014 · BMC Developmental Biology
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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.
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