Rapamycin Enhances Long-Term Hematopoietic Reconstitution of Ex Vivo Expanded Mouse Hematopoietic Stem Cells by Inhibiting Senescence

Abstract

The mammalian target of rapamycin (mTOR) is an important regulator of hematopoietic stem cell (HSC) self-renewal and its overactivation contributes to HSC premature exhaustion in part via induction of HSC senescence. Inhibition of mTOR with rapamycin has the potential to promote long-term hematopoiesis of ex vivo expanded HSCs to facilitate the clinical application of HSC transplantation for various hematologic diseases. A well-established ex vivo expansion system for mouse bone marrow HSCs was used to investigate whether inhibition of overactivated mTOR with rapamycin can promote long-term hematopoiesis of ex vivo expanded HSCs and to elucidate the mechanisms of action of rapamycin. HSC-enriched mouse bone marrow LSK cells exhibited a time-dependent activation of mTOR after ex vivo expansion in a serum-free medium supplemented with stem cell factor, thrombopoietin, and Flt3 ligand. The overactivation of mTOR was associated with induction of senescence but not apoptosis in LSK cells and a significant reduction in the ability of HSCs to produce long-term hematopoietic reconstitution. Inhibition of overactivated mTOR with rapamycin promoted ex vivo expansion and long-term hematopoietic reconstitution of HSCs. The increase in long-term hematopoiesis of expanded HSCs is likely attributable in part to rapamycin-mediated up-regulation of Bmi1 and down-regulation of p16, which prevent HSCs from undergoing senescence during ex vivo expansion. These findings suggest that mTOR plays an important role in the regulation of HSC self-renewal in vitro and inhibition of mTOR hyperactivation with rapamycin may represent a novel approach to promote ex vivo expansion and their long-term hematopoietic reconstitution of HSCs.