Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions.

1Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone 2 marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly 3 proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations 4 and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable 5 quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty 6 acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis 7 by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced 8 HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of 9 steady state HSCs and test effects of defined factors in vitro under near-physiological conditions. 10 11

Metabolic process governs cell cycle and differentiation status of hematopoietic stem cells (HSCs) through regulating the balance between oxidative phosphorylation and glycolysis. Cell cycle and self-renewal capacity dynamically changes during developmental and aging process; in early stage of life, HSCs are highly cycling and self-renew, whereas adult HSCs exhibit quiescent in cell-cycle, and gradually decline in its function as they age. How functional alteration of HSCs is coupled with metabolic program is an important issue for understanding the lifecourse of HSCs. Phosphoglycerate mutase 1 (Pgam1) is an enzyme catalyzing conversion of 3-phosphoglycerate into 2-phosphoglycerate. By deleting Pgam1, ATP production through glycolysis was abrogated while pentose-phosphate pathway (PPP) was spared, allowing us to examine dependence on glycolytic energy production without affecting nucleotide and NADPH production through PPP. Following inducible Pgam1 deletion in mice at various ages, the number of HSCs was measured. At an early life stage (5-week old), Pgam1 deletion resulted in decreased number of HSCs, suggesting an essential role of glycolysis for HSC maintenance. Notably, the number of HSCs in Pgam1-deleted mice restored in an age-dependent manner, with 2-year old mice exhibiting almost comparable number of HSCs with wild type mice. By contrast, upon HSC transplantation, the repopulation capacity of Pgam1-deficient HSCs from any stages of life was uniformly abrogated, indicating that glycolytic energy production is essential for stressed condition in which HSCs are forced to proliferate. Reverse transplantation of wild type HSCs into Pgam1-floxed recipients followed by conditional deletion in non-hematopoietic cells showed no phenotypes in hematopoietic lineages, suggesting a cell-autonomous requirement of Pgam1 for blood cells. To further determine the relevance of glycolysis with maintenance/proliferation balance, we performed in vitro culture utilizing newly-developed quiescence-maintaining condition with or without glucose. The number of HSCs under the quiescence-maintaining condition was irrelevant to the presence or absence of glucose, while cell number under the proliferating condition was markedly decreased in glucose-deprived condition. Furthermore, lymphoid-primed multipotential progenitors (LMPP) with marker phenotype of Flt3+ linage markers- Sca-1+ c-Kit+ cells, which show high cycling status in vivo, were highly dependent on glucose for survival in vitro. Collectively, proliferating cells require glucose irrespective to its cell type, while quiescent HSCs are less dependent on glycolytic energy production in line with age-dependent decrease of requirement for glycolysis in vivo. ATP concentration directly reflects ATP production/consumption rate inside cells. We utilized ATP-biosensor (GO-ATeam2)-transgenic mice to monitor ATP concentration in live HSCs under various metabolic environments in vitro. HSCs cultured in the proliferating condition exhibited gradual decrease in ATP concentration during 7-day culture, whereas in the quiescence-maintaining condition ATP was kept high. In the steady state, cycling multipotent progenitors (MPPs) exhibited lower ATP concentration than quiescent HSCs. These results suggest that quiescent state is characterized by lower ATP production/consumption rate than the proliferating cells. We then compared ATP concentration in young and old HSCs using the same system. The decrease of ATP under the proliferating condition was less prominent in old HSCs, while ATP concentration was comparable in the quiescence-maintaining condition. The result indicates the lower ATP production/consumption rate in old HSCs, presumably explaining lower dependence on glycolytic energy production in the steady state hematopoiesis. Altogether, we concluded that the dependence on glycolysis-driven ATP production derived from glucose decreased with age, underlying mechanism of which is explained by lower ATP consumption rate in old HSCs. Disclosures Okamoto: Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.