Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

Tissue homeostasis requires the presence of multipotent adult stem cells that are capable of efficient self-renewal and differentiation; some of these have been shown to exist in a dormant, or quiescent, cell cycle state. Such quiescence has been proposed as a fundamental property of hematopoietic stem cells (HSCs) in the adult bone marrow, acting to protect HSCs from functional exhaustion and cellular insults to enable lifelong hematopoietic cell production. Recent studies have demonstrated that HSC quiescence is regulated by a complex network of cell-intrinsic and -extrinsic factors. In addition, detailed single-cell analyses and novel imaging techniques have identified functional heterogeneity within quiescent HSC populations and have begun to delineate the topological organization of quiescent HSCs. Here, we review the current methods available to measure quiescence in HSCs and discuss the roles of HSC quiescence and the various mechanisms by which HSC quiescence is maintained.

The BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steadystate conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C ؊/؊ ) mice showed normal steadystate hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wildtype hematopoietic cells to regenerate hematopoiesis was reduced in TN-C ؊/؊ recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin ␣9-dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57 Kip2 IntroductionThe BM is the main hematopoietic organ in the adult. It provides an efficient microenvironment for hematopoiesis, which contributes to the maintenance, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPCs). A wellaccepted concept regarding the hematopoietic microenvironment is that of the hematopoietic stem cell (HSC) niche. 1-3 The HSC niche is subdivided into the osteoblastic niche 4-7 and the vascular niche. 8,9 The BM vasculature is surrounded by perivascular niche cells such as macrophages 10,11 and stromal cells (ie, reticular cells) of mesenchymal lineage, 12,13 which cooperatively regulate HSC activity.In contrast to the well-investigated cellular niches, the functions of ECM proteins as a niche are poorly understood. The ECM of the BM comprises fibrous proteins such as types I and IV collagen and fibronectin (FN) 14 and nonfibrous proteins such as tenascin-C (TN-C). 15 We have shown previously that longterm bromodeoxyuridine (BrdU)-label-retaining cells reside in the hypoxic areas distant from the endothelial tubes closely attached to nonendothelial ECM structures. 16 In vitro culture systems also suggest the importance of the ECM in the maintenance of HSPCs. 17 Therefore, a role for the ECM as a BM niche has been suggested, yet little is known about how the ECM affects HSPCs in vivo.TN-C is a highly conserved ECM glycoprotein that is expressed mainly during embryogenesis. 18 TN-C-deficient mice show normal development with no defects in gross organization. 18 In adult tissues, TN-C expression is restricted to sites of active tissue remodeling (eg, inflammation 19,20 and wound healing 21 ) and plays a significant function in these pathologies. [19][20][21] Expression of TN-C in the BM is l...

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Most of the hematopoietic stem cells (HSCs) within the bone marrow (BM) show quiescent state with a low mitochondrial membrane potential (ΔΨ). In contrast, upon stress hematopoiesis, HSCs actively start to divide. However, the underlying mechanism for the initiation of HSC division still remains unclear. To elucidate the mechanism underlying the transition of cell cycle state in HSCs, we analyzed the change of mitochondria in HSCs after BM suppression induced by 5-fluoruracil (5-FU). We found that HSCs initiate cell division after exhibiting enhanced ΔΨ as a result of increased intracellular Ca level. Although further activation of Ca-mitochondria pathway led to loss of HSCs after cell division, the appropriate suppression of intracellular Ca level by exogenous adenosine or Nifedipine, a Ca channel blocker, prolonged cell division interval in HSCs, and simultaneously achieved both cell division and HSC maintenance. Collectively, our results indicate that the Ca-mitochondria pathway induces HSC division critically to determine HSC cell fate.