Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

中文翻译：

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适应离体培养会降低人类造血干细胞的活性，与细胞周期无关


离体造血干细胞 (LT-HSC) 长期功能的丧失阻碍了依赖培养的临床方案的成功。然而，发生这种情况的动力学和机制仍未完全表征。在这项研究中，通过时间分辨单细胞 RNA 测序、匹配的体内功能分析以及使用早期 G 阻滞的可逆体外系统，我们定义了第一次离体过程中发生的转录和功能事件的序列。人类 LT-HSC 的划分。我们证明，LT-HSC 重新增殖能力的最严重损失发生在培养的 6 至 24 小时之间，即 LT-HSC 进入细胞周期进程之前。在此时间窗口内，LT-HSC 适应培养环境，限制基因表达的全局变异性，并短暂上调参与信号传导和应激反应的基因网络。从 24 小时开始，LT-HSC 进展超过 G 早期，有助于在培养物中建立分化程序。然而，与目前的假设相反，我们证明离体 HSC 功能的丧失与细胞周期进程无关。最后，我们发现通过抑制 JAK/STAT 信号的早期激活来靶向 LT-HSC 对培养物的适应可改善 HSC 的长期体外增殖功能。总的来说，我们的研究表明，控制早期 LT-HSC 对离体培养的适应（例如通过 JAK 抑制）对于改善 HSC 基因治疗和扩增方案至关重要。