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. 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.

造血干细胞（hematopoietic stem cells, HSCs）通过在骨髓微环境（bone marrow niche）中维持静止状态以实现终身造血功能。目前体外培养体系尚无法重现其静止态，细胞会快速发生增殖与分化。通过调控模拟生理条件的环境因子，我们得以在低细胞因子浓度、低氧环境及高脂肪酸浓度的培养条件下，将可植入的静止态HSCs体外维持培养1个月；其中高脂肪酸浓度是必需的，因为此时细胞的内源性脂肪酸合成通路受到抑制。相比之下，高细胞因子浓度或常氧环境会诱导HSCs发生增殖与分化。本研究建立的新型培养体系，可为评估稳态造血干细胞的特性，以及在近似生理条件下体外检测特定因子的作用提供可行手段。本研究将10只小鼠骨髓混合后的CD150+CD41-CD48-CD34-Flt3-LSK细胞分选至SF-O3培养基中，随后分为新鲜样本组（直接裂解）及分别在4%牛血清白蛋白（nBSA）、4%重构牛血清白蛋白（reconstituted BSA）条件下培养7天的两组。培养后的细胞经4℃、340×g离心5分钟，使用75μL RLT缓冲液+0.75μL β-巯基乙醇（2-ME）进行裂解。RNA提取、cDNA合成、微阵列分析及数据标准化工作均委托至DNA芯片研究公司（DNA Chip Research Inc.）完成。