Modulation of reactive oxygen species (ROS) levels in hematopoietic stem cells (HSC) is crucial to control HSC quiescence and blood formation. High ROS levels are required for leukocyte formation while low ROS levels are essential to maintain HSC quiescence. However, regulation of ROS content in HSC is poorly understood. Adhesion interactions between HSC and their bone marrow (BM) stromal cells (BMSC) via CXCL12/CXCR4 maintain HSC in a quiescence nonmotile state, protecting them from 5FU chemotherapy insult (Sugiyama, Immunity, 2006). Surface CXCL12 expression by BMSC is dependent on connexin43 (Cx43) gap junctions mediated cell contact (Schajnovitz, Nat. Immunol., 2011) and BM hematopoietic stem and progenitor cells (HSPC) survive and eliminate excess ROS levels post 5FU chemotherapy treatment, by transferring ROS to BMSC in a Cx43 dependent manner (Taniguchi, PNAS 2012).
Here, we report that ROS content of BM HSPC inversely correlates with ROS levels in adjacent BMSC. Administration of the pro inflammatory cytokine GCSF results in decreased HSPC Cx43 expression, elevated ROS levels and increased glucose uptake. Conversely, in the BM stromal microenvironment, GCSF administration generated lower ROS level and reduced glucose uptake. Upregulation of BM Sphingosine 1Phosphate (S1P), a downstream target of GCSF required for ROS production in HSPC, reduced stromal ROS content and proliferation. Accordingly, mice with reduced BM S1P levels (S1P low) have lower BM content of HSPC, accompanied by reduced ROS, glucose uptake and lactate production in these cells. More importantly, BM from S1P low mice has a 3 fold increased frequency of primitive ROS low/ EPCR + longterm repopulating cells, as evident by immunophenotypic analysis and longterm competitive repopulation assays. Concomitantly, S1P low mice have increased content of BMSC with higher ROS levels and glucose uptake, leading to higher BM content of colonyforming unit fibroblasts. Our results reveal a dynamic and inverse metabolic relationship between BM HSC and the stroma microenvironment. We hypothesized that the opposite metabolic state of HSPC and BMSC is due to mitochondrial transfer between the two populations. Therefore, we created chimeric mice by transplanting mitochondria labeled GFP (mitoGFP) HSPC to wild type (WT) mice and detected 88% of the host BMSC to contain donorderived mitochondria, indicating the existence of mitochondria transfer from hematopoietic cells to BMSC in vivo. This transfer is bidirectional, albeit at a lesser degree, as determined in reverse chimeric mice where up to 26% of the donorderived HSPCs acquired recipient mitochondria. Mitochondrial transfer can be recapitulated also in vitro in an overnight coculture system of mitoGFP HSPC and primary BMSC, resulting in mitochondrial transfer and increased ROS content in a subpopulation of osteogenic BM PDGFRα + / Sca1 /CD48 dim stromal cells. Mitochondrial transfer is cell contact dependent and mediated by Cx43 gap junctions. In vitro coculture of mitoGFP HSPC from Cx43 deficient (KO) mice with WT or Cx43 KO BMSC reduced 50% mitochondrial transfer to PDGFRα + /Sca1 /CD48 dim stromal cells. Contrarily, the mitochondrial transfer from WT HSPC to Cx43 KO stromal cells was not affected, revealing that Cx43 expression on HSPC, but not on BM stromal cells, is specifically required for mitochondrial transfer. Interestingly, in vitro inhibition of AMPactivated protein kinase (AMPK), a crucial metabolic regulator, dramatically increased mitochondrial transfer from HSPC to BMSC. Administration of the AMPK inhibitor BML in vivo increased ROS content of PDGFRα + /Sca1 BMSC while decreasing it in HSPC, further suggesting that AMPK inhibition regulates mitochondrial transfer and ROS production. Our results imply that mitochondria are scavenged by the BM osteogenic microenvironment to prevent excessive ROS levels in the HSC pool and in parallel to activate bone formation. Altogether, we have discovered a dynamic, inverse metabolic state between BM HSPC and their supporting stromal microenvironment during quiescence, proliferation and differentiation of these two populations. Thus, blood cell production and bone generation take place at the expense of the other. This metabolic seesaw is mediated by mitochondrial transfer from HSPC to osteogenic BM stroma in a HSPC Cx43 gapjunction dependent manner and regulated through AMPK signaling.