STAT3 protects hematopoietic stem cells from intrinsic interferon signaling and loss of long-term blood-forming activity. STAT3 protects hematopoietic stem cells from intrinsic interferon signaling and loss of long-term blood-forming activity

The transcriptional regulator signal transducer and activator of transcription 3 (STAT3) has a well-established anti-inflammatory function in mature myeloid cells. This role, however, has precluded an understanding of STAT3 function in hematopoietic stem and progenitor cells (HSPCs), as Stat3 deletion in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. Thus, novel approaches to uncouple STAT3 function in HSPCs from the effects of systemic inflammation are needed. To address this, we established competitive mixed bone marrow (BM) chimeric mice with CreER-mediated conditional Stat3 deletion in 20% of the hematopoietic compartment. After confirming a lack of detectable inflammation in mice with Stat3-deficient BM, we found that, in contrast to Stat3-sufficient controls, Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM. Single-cell RNA sequencing of Lin-ckit+Sca1+ (LSKs) BM cells revealed an altered transcriptional landscape in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, as evidenced by the intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient LSKs accumulated γH2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs. Overall design: Lin-ckit+Sca1+ cells (LSKs) were purified by fluorescence-activated cell sorting (FACS) from mixed bone marrow chimeric mice and processed for single cell RNA sequencing. The mixed bone marrow chimeric mice contained Stat3-sufficient CreER+ cells and wild-type competitor cells (control group), or Stat3-deficient CreER+ Stat3f/f cells and wild-type competitor cells (test group) in a 1:1 ratio. Stat3 gene deletion was enforced by tamoxifen treatment of the bone marrow chimeric mice 8 weeks prior to LSK purification.

转录调节因子信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）在成熟髓系细胞中具有明确的抗炎功能。然而，这一功能却阻碍了学界对其在造血干细胞和祖细胞（hematopoietic stem and progenitor cells, HSPCs）中功能的理解——因为在造血系统中敲除Stat3会诱发全身性炎症，进而影响HSPC的活性。因此，亟需开发能够将STAT3在HSPC中的功能与全身性炎症的影响解耦的新方法。
为解决这一问题，我们构建了竞争性混合骨髓（bone marrow, BM）嵌合小鼠模型，使20%的造血室中发生CreER介导的条件性Stat3敲除。在确认Stat3缺陷型骨髓小鼠未出现可检测的炎症后，我们发现：与Stat3充足的对照组相比，Stat3缺陷型HSPC的造血活性受损，且无法在骨髓中完成扩增。
对Lin⁻cKit⁺Sca1⁺（LSKs）骨髓细胞进行单细胞RNA测序后显示，Stat3缺陷型造血干细胞（hematopoietic stem cells, HSCs）和多能祖细胞的转录谱发生改变，具体表现为细胞周期、应激反应及干扰素信号通路的内在激活。与上述调控异常一致的是，Stat3缺陷型LSKs会随时间积累γH2AX。
在次级骨髓移植后，Stat3缺陷型HSPC无法有效重建外周血，提示其造血干细胞室存在严重功能缺陷。本研究揭示了STAT3在造血干细胞中的核心作用，并提示可通过靶向合成致死策略结合STAT3抑制，以清除缺陷型或病变型HSPC。
整体实验设计：从混合骨髓嵌合小鼠中通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）纯化Lin⁻cKit⁺Sca1⁺（LSKs）细胞，用于单细胞RNA测序。混合骨髓嵌合小鼠的造血组分按1:1比例包含两类细胞：一组为Stat3充足的CreER⁺细胞与野生型竞争细胞（对照组），另一组为Stat3缺陷型CreER⁺ Stat3f/f细胞与野生型竞争细胞（实验组）。在纯化LSKs前8周，通过他莫昔芬处理诱导骨髓嵌合小鼠的Stat3基因敲除。