Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo

Abstract

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

ATP turnover and glucose dependency in hematopoietic stem/progenitor cells are increased by proliferation and differentiation

“…To determine the metabolic characteristics of HSCs and HPCs at steady state and during proliferation, we utilized ex vivo culture conditions that maintained quiescence or induced proliferation. We previously established these culture conditions for HSCs [12]; therefore, we first determined the minimal cytokine combinations and concentrations that maintained quiescence (quiescent conditions) of HPCs. Culture in the presence of increased concentrations of the minimal cytokine combinations was used to induce proliferation (proliferative conditions) of HPCs.…”