Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation.
ABSTRACT: Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.
Project description:Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence.
Project description:The data presented in this article are related to the research articles entitled "APOBEC2 negatively regulates myoblast differentiation in muscle regeneration" and "Data supporting possible implication of APOBEC2 in self-renewal functions of myogenic stem satellite cells: toward understanding the negative regulation of myoblast differentiation" (Ohtsubo et al., 2017a, 2017b) [1,2]. This article provides in vivo phenotypical data to show that Paired Box Transcription Factor 7 (Pax7)-positive cell number (per myofiber) is significantly lower in APOBEC2 (a member of apoB mRNA editing enzyme, catalytic polypeptide-like family)-knockout muscle than the control wild-type tissue at the same age of 8-wk-old in mice. The emerging results support an essential role for APOBEC2 in the self-renewal functions of myogenic stem satellite cells, namely the re-establishment of quiescent status after activation and proliferation of myoblasts.
Project description:Muscle satellite cells are myogenic stem cells whose quiescence, activation, self-renewal, and differentiation are influenced by oxygen supply, an environmental regulator of stem cell activity. Accordingly, stem cell-specific oxygen signaling pathways precisely control the balance between muscle growth and regeneration in response to oxygen fluctuations, and hypoxia-inducible factors (HIFs) are central mediators of these cellular responses. However, the in vivo roles of HIFs in quiescent satellite cells and activated satellite cells (myoblasts) are poorly understood. Using transgenic mouse models for cell-specific HIF expression, we show here that HIF1? and HIF2? are preferentially expressed in pre- and post-differentiation myoblasts, respectively. Interestingly, double knockouts of HIF1? and HIF2? (HIF1?/2? dKO) generated with the MyoDCre system in embryonic myoblasts resulted in apparently normal muscle development and growth. However, HIF1?/2? dKO produced with the tamoxifen-inducible, satellite cell-specific Pax7CreER system in postnatal satellite cells delayed injury-induced muscle repair due to a reduced number of myoblasts during regeneration. Analysis of satellite cell dynamics on myofibers confirmed that HIF1?/2? dKO myoblasts exhibit reduced self-renewal but more pronounced differentiation under hypoxic conditions. Mechanistically, the HIF1?/2? dKO blunted hypoxia-induced activation of Notch signaling, a key determinant of satellite cell self-renewal. We conclude that HIF1? and HIF2? are dispensable for muscle stem cell function under normoxia but are required for maintaining satellite cell self-renewal in hypoxic environments. Our insights into a critical mechanism in satellite cell homeostasis during muscle regeneration could help inform research efforts to treat muscle diseases or improve muscle function.
Project description:Studies using mouse models have established a critical role for resident satellite stem cells in skeletal muscle development and regeneration, but little is known about this paradigm in human muscle. Here, using human muscle stem cells, we address their lineage progression, differentiation, migration, and self-renewal. Isolated human satellite cells expressed alpha7-integrin and other definitive muscle markers, were highly motile on laminin substrates and could undergo efficient myotube differentiation and myofibrillogenesis. However, only a subpopulation of the myoblasts expressed Pax7 and displayed a variable lineage progression as measured by desmin and MyoD expression. Analysis identified a differentiation-resistant progenitor cell population that was Pax7+/desmin- and capable of self-renewal. This study extends our understanding of the role of Pax7 in regulating human satellite stem cell differentiation and self-renewal.
Project description:Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-J?, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.
Project description:Satellite cells are skeletal muscle stem cells capable of self-renewal and differentiation after transplantation, but whether they contribute to endogenous muscle fiber repair has been unclear. The transcription factor Pax7 marks satellite cells and is critical for establishing the adult satellite cell pool. By using a lineage tracing approach, we show that after injury, quiescent adult Pax7(+) cells enter the cell cycle; a subpopulation returns to quiescence to replenish the satellite cell compartment, while others contribute to muscle fiber formation. We demonstrate that Sprouty1 (Spry1), a receptor tyrosine kinase signaling inhibitor, is expressed in quiescent Pax7(+) satellite cells in uninjured muscle, downregulated in proliferating myogenic cells after injury, and reinduced as Pax7(+) cells re-enter quiescence. We show that Spry1 is required for the return to quiescence and homeostasis of the satellite cell pool during repair. Our results therefore define a role for Spry1 in adult muscle stem cell biology and tissue repair.
Project description:PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.
Project description:Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7+ and Myf5-) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.
Project description:Satellite cells play a central role in mediating the growth and regeneration of skeletal muscle. However, whether satellite cells are stem cells, committed progenitors, or dedifferentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre-reporter alleles, we observed that in vivo 10% of sublaminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells gave rise to Pax7(+)/Myf5(+) satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7(+)/Myf5(-) and an apical Pax7(+)/Myf5(+) cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7(+)/Myf5(+) cells exhibited precocious differentiation, Pax7(+)/Myf5(-) cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of neuromuscular diseases.
Project description:The remarkable regeneration capability of skeletal muscle depends on the coordinated proliferation and differentiation of satellite cells (SCs). The self-renewal of SCs is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in SCs in vivo remains largely unknown. Here, we demonstrate that SCs are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of SCs by maintaining their quiescence, increasing their self-renewal, and blocking their myogenic differentiation. HIF2A stabilization in SCs cultured under normoxia augments their engraftment potential in regenerative muscle. Conversely, HIF2A ablation leads to the depletion of SCs and their consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerates muscle regeneration by increasing SC proliferation and differentiation. Mechanistically, HIF2A induces the quiescence and self-renewal of SCs by binding the promoter of the Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in SCs and may be therapeutically targeted to improve muscle regeneration.