Expansion of hematopoietic stem cells (HSCs) is a ‘holy grail’ of regenerative medicine, as successful stem cell transplantations depend on the number and quality of infused HSCs. Although many attempts have been pursued to either chemically or genetically increase HSC numbers, neither clonal analysis of these expanded cells nor their ability to support mature blood lineages has been demonstrated. Here we show that miR-125a, at the single cell level, can expand murine long-term repopulating HSCs. In addition, miR-125a increases clone longevity, clone size and clonal contribution to hematopoiesis. Unexpectedly, we found that miR-125a expanded HSCs clones were highly homogenously distributed across multiple anatomical sites. Interestingly, these miR-125a overexpressing cells had enhanced mobility and were more frequently detected in the spleen. Our study reveals a novel, cell-intrinsically controlled mechanism by which HSC migration is regulated.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression posttranscriptionally by binding to the 3 0 untranslated regions of their target mRNAs. The evolutionarily conserved microRNA-125a (miR-125a) is highly expressed in both murine and human hematopoietic stem cells (HSCs), and previous studies have found that miR-125 strongly enhances selfrenewal of HSCs and progenitors. In this study we explored whether temporary overexpression of miR-125a would be sufficient to permanently increase HSC self-renewal or, rather, whether persistent overexpression of miR-125a is required. We used three complementary in vivo approaches to reversibly enforce expression of miR-125a in murine HSCs. Additionally, we interrogated the underlying molecular mechanisms responsible for the functional changes that occur in HSCs on overexpression of miR-125a. Our data indicate that continuous expression of miR-125a is required to enhance HSC activity. Our molecular analysis confirms changes in pathways that explain the characteristics of miR-125a overexpressing HSCs. Moreover, it provides several novel putative miR-125a targets, but also highlights the complex molecular changes that collectively lead to enhanced HSC function.

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