Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cell Ex Vivo. Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cell Ex Vivo

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. By modulating environmental factors mimicking physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under low cytokine concentrations, hypoxia, and high fatty acid concentration, the latter required due to suppression of intrinsic fatty acid synthesis. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors in vitro under near-physiological conditions. Overall design: CD150+CD41-CD48-CD34-Flt3-LSK cells of pooled bone marrow from 10 mice were sorted into SF-O3 medium and then and either lysed for the fresh sample or cultured for 7 days either in the 4% nBSA or 4% reconstituted BSA conditions. Cultured cells were centrifuged at 340 x g for 5 min at 4°C and lysed with 75µL RLT buffer + 0.75µL 2-ME. RNA extraction, cDNA synthesis, microarray analysis, and data normalization were outsourced to DNA Chip Research Inc.