br Introduction Expansion of hematopoietic stem cells HSCs i

Introduction Expansion of hematopoietic stem cells (HSCs) in vitro using growth factors has been thwarted by findings that there is loss of HSCs during in vitro expansion. In steady-state hematopoiesis, a majority of HSCs remain quiescent; however, in response to 5-fluorouracil (5-FU) treatment, majority of HSCs undergo rapid proliferation and lead to hematopoietic recovery (Harrison and Lerner, 1991). Therefore, a better understanding of molecular pathways that regulate HSC expansion and recovery of hematopoiesis following 5-FU treatment may allow us to develop protocols that will eventually lead to successful ex vivo expansion of HSCs. Mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two complexes, mammalian target of rapamycin complex (mTORC) 1 and 2; mTORC1 and 2 have been respectively characterized as the rapamycin-sensitive and rapamycin-insensitive complexes (Laplante and Sabatini, 2009). Growth factors stimulate the mammalian target of rapamycin complex1 (mTORC1) pathway that controls cell growth and division (Fingar and Blenis, 2004). Activation of mTOR is regulated by nitric oxide synthase inhibitors levels in the cell (Dennis et al., 2001). Oxidative phosphorylation (OXPHOS), an oxygen dependent process that occurs in the mitochondria, is the major source of cellular ATP. Taken together, these findings raise the possibility that the interaction between the molecular pathways, mTOR activation and mitochondrial biogenesis, plays a deterministic role in ability of hematopoietic stem and progenitor cells (HSPCs) to successfully expand following 5-FU treatment. Mitochondria have their own DNA that encodes for 13 essential proteins of inner membrane respiratory apparatus but nuclear genes encode for a majority of respiratory proteins and all other gene products necessary for a variety of mitochondrial functions. The nucleo-mitochondrial interactions depend on regulation of transcription factors and peroxisome proliferator-activated receptor gamma co-activator (PGC-1) family of transcriptional co-activators that includes PGC-1α, PGC-1β and PGC-1 related coactivators (PRC) (Scarpulla, 2008). Although various PGC-1 family members can regulate mitochondrial biogenesis (Scarpulla, 2002; Uldry et al., 2006), PGC-1α is considered the master regulator of mitochondrial biogenesis (Puigserver and Spiegelman, 2003). PGC-1α is expressed in BM hematopoietic stem progenitor cells (HSPCs) (Basu et al., 2013). While glycolysis is important for the maintenance of HSCs (Simsek et al., 2010; Takubo et al., 2010), the role of mitochondrial metabolism in hematopoiesis (Nakada et al., 2010; Gurumurthy et al., 2010), particularly in response to stress (Mortensen et al., 2011), is increasingly being realized. Indeed, recovery of peripheral blood cells (PBCs) following 5-FU treatment is significantly impaired in PGC-1α knockout (PGC-1α−/−) mice compared to wild type (WT) mice; however, steady-state hematopoiesis is not overtly affected in PGC-1α−/− mice (Basu et al., 2013). Moreover, the impairment of PBC recovery is more profound in older PGC-1α−/− mice (SB unpublished observation) consistent with age related degeneration of mitochondrial function (Pieri et al., 1993). These findings suggest a role of mitochondria in the recovery of hematopoiesis following 5-FU treatment. To get an insight into mechanisms regulating HSC proliferation in vivo, recovery of hematopoiesis following 5-FU treatment in WT and PGC-1α−/− mice was investigated in this study. The current study demonstrates that mitochondrial biogenesis is critical for HSPCs to optimally respond to mTORC1 activation and proliferate in response to mitogenic signals following 5-FU challenge. Moreover, the level of mTORC1 activation is regulated by the mitochondrial biogenesis potential of HSPCs. Interestingly, different members of the PGC-1 family play stage-specific roles in hematopoietic recovery following 5-FU treatment: while PRC is required for hematopoietic stem cell (HSC) proliferation during early recovery phase, PGC-1α is important for rapid proliferation of progenitors during late phase of hematopoietic recovery following 5-FU treatment.