Hematopoietic stem cell (HSC) self-renewal and lineage commitment depend on complex interactions with the microenvironment, and the ability to maintain or expand HSCs for clinical applications or for basic research has been significantly limited because these interactions are not well defined. Recent evidence suggests that HSCs reside in a low perfusion, reduced nutrient niche and that nutrient sensing pathways contribute to HSC homeostasis. Here we report that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/β-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/β-catenin signaling and inhibit mTOR increases the number (but not the proportion) of long-term HSCs in vivo.

Skeletal muscle stem/progenitor cells, which give rise to terminally differentiated muscle, represent potential therapies for skeletal muscle diseases. Delineating the factors regulating these precursors will facilitate their reliable application in human muscle repair. During embryonic development and adult regeneration, skeletal muscle progenitors reside in low-O 2 environments before local blood vessels and differentiated muscle form. Prior studies established that low O 2 levels (hypoxia) maintained muscle progenitors in an undifferentiated state in vitro, although it remained unclear if progenitor differentiation was coordinated with O 2 availability in vivo. In addition, the molecular signals linking O 2 to progenitor differentiation are incompletely understood. Here we show that the muscle differentiation program is repressed by hypoxia in vitro and ischemia in vivo. Surprisingly, hypoxia can significantly impair differentiation in the absence of hypoxia-inducible factors (HIFs), the primary developmental effectors of O 2 . In order to maintain the undifferentiated state, low O 2 levels block the phosphatidylinositol 3-kinase/AKT pathway in a predominantly HIF1␣-independent fashion. O 2 deprivation affects AKT activity by reducing insulin-like growth factor I receptor sensitivity to growth factors. We conclude that AKT represents a key molecular link between O 2 and skeletal muscle differentiation.

IGF-I is a key regulator of muscle development and growth. The pre-pro-peptide produced by the Igf1gene undergoes several posttranslational processing steps to result in a secreted mature protein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). The goals of this study were to determine what forms of IGF-I exist in skeletal muscle, and whether the mature IGF-I protein was the only form able to activate the IGF-IR. We measured the proportion of IGF-I species in murine skeletal muscle and found that the predominant forms were nonglycosylated pro-IGF-I and glycosylated pro-IGF-I, which retained the C-terminal E peptide extension, instead of mature IGF-I. These forms were validated using samples subjected to viral expression of IGF-I combined with furin and glycosidase digestion. To determine whether the larger molecular weight IGF-I forms were also ligands for the IGF-IR, we generated each specific form through transient transfection of 3T3 cells and used the enriched media to perform kinase receptor activation assays. Compared with mature IGF-I, nonglycosylated pro-IGF-I had similar ability to activate the IGF-IR, whereas glycosylation of pro-IGF-I significantly reduced receptor activation. Thus, it is important to understand not only the quantity, but also the proportion of IGF-I forms produced, to evaluate the true biological activity of this growth factor.

Deeper insight into the molecular pathways that orchestrate skeletal myogenesis should enhance our understanding of, and ability to treat, human skeletal muscle disease. It is now widely appreciated that nutrients, such as molecular oxygen (O 2 ), modulate skeletal muscle formation. During early stages of development and regeneration, skeletal muscle progenitors reside in low O 2 environments before local blood vessels and differentiated muscle form. Moreover, low O 2 availability (hypoxia) impedes progenitor-dependent myogenesis in vitro through multiple mechanisms, including activation of hypoxia inducible factor 1α (HIF1α). However, whether HIF1α regulates skeletal myogenesis in vivo is not known. Here, we explored the role of HIF1α during murine skeletal muscle development and regeneration. Our results demonstrate that HIF1α is dispensable during embryonic and fetal myogenesis. However, HIF1α negatively regulates adult muscle regeneration after ischemic injury, implying that it coordinates adult myogenesis with nutrient availability in vivo. Analyses of Hif1a mutant muscle and Hif1a-depleted muscle progenitors further suggest that HIF1α represses myogenesis through inhibition of canonical Wnt signaling. Our data provide the first evidence that HIF1α regulates skeletal myogenesis in vivo and establish a novel link between HIF and Wnt signaling in this context.