Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1^ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

通过用工程化的 Delta 样配体 (DELTA1 ^ext-IgG [DXI])处理激活人类造血干细胞/祖细胞 (HSPC) 中的 NOTCH 信号，使短期 HSPC 能够离体扩增，但对长期重新填充造血干细胞（LTR-HSCs）仍然不确定。在这里，我们证明了离体与常氧培养相比，在低氧张力下培养具有 DXI 的人类成人 HSPC 限制了 LTR-HSC 和谱系承诺祖细胞中的 ER 应激。在缺氧条件下用 DXI 培养的细胞中，明显的 HSC 基因特征上调，培养 21 天后，LTR-HSC 的频率相对于未培养的细胞增加 4.9 倍，与常氧 + DXI 组相比增加 4.2 倍。NOTCH 和缺氧途径相交以维持培养的 HSPCs 中未分化的表型。我们的工作强调了减轻 ER 应激扰动以在扩展培养物中保留功能性 LTR-HSC 的重要性，并为人类 HSPC 的扩展提供了一个临床上可行的平台。