Among the key properties that distinguish adult mammalian stem cells from their more differentiated progeny is the ability of stem cells to remain in a quiescent state for prolonged periods of time1,2. However, the molecular pathways for the maintenance of stem cell quiescence remain elusive. Using adult muscle stem cells (“satellite cells” (SCs)) as a model system, we show that the microRNA (miRNA) pathway is essential for the maintenance of the quiescent state. SCs lacking a functional miRNA pathway spontaneously exit quiescence and enter the cell cycle. We identified quiescence-specific miRNAs in the SC lineage by microarray analysis. Among these, microRNA-489 (miR-489) is highly expressed in quiescent SCs and quickly down-regulated during SC activation. Further analysis revealed that miR-489 functions as a regulator of SC quiescence by post-transcriptionally suppressing the oncogene DEK, a protein that localizes to the more differentiated daughter cell during asymmetric division of SCs and promotes the transient proliferative expansion of myogenic progenitors. Our results provide the first evidence of the miRNA pathway in general, and a specific miRNA, miR-489, in actively maintaining the quiescent state of an adult stem cell population.

SummaryAdult 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.

Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.

SUMMARY The decline of tissue regenerative potential with age correlates with impaired stem cell function. However, limited strategies are available for therapeutic modulation of stem cell function during aging. Using skeletal muscle stem cells (MuSCs) as a model system, we identify cell death by mitotic catastrophe as a cause of impaired stem cell proliferative expansion in aged animals. The mitotic cell death is caused by a deficiency in Notch activators in the microenvironment. We discover that ligand-dependent stimulation of Notch activates p53 in MuSCs via inhibition of Mdm2 expression through Hey transcription factors during normal muscle regeneration and this pathway is impaired in aged animals. Pharmacologic activation of p53 promotes the expansion of aged MuSCs in vivo. Taken together, these findings illuminate a Notch-p53 signaling axis that plays an important role in MuSC survival during activation and that is dysregulated during aging, contributing to the age-related decline in muscle regenerative potential.