Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.

揭示造血规范的调节机制不仅可以克服当前与造血干细胞和祖细胞（HSPC）移植相关的局限性，而且还可以推进细胞免疫疗法。然而，生成功能性人类诱导多能干细胞 (hiPSC) 衍生的 HSPC 及其衍生物一直难以实现，因此需要更好地了解触发 HSPC 规范的发育机制。在这里，我们揭示了内皮细胞 (EC) 中 Nod1-Ripk2-NF-kB 炎症通路的早期激活促使它们将命运转变为最终的造血内皮细胞，这是指定 HSPC 的先决条件。我们的遗传和化学胚胎模型表明，由于造血内皮 (HE) 编程失败，HSPC 在缺乏 Nod1 及其下游激酶 Ripk2 的情况下无法指定，并且小型 Rho GTPases 协调该途径的激活。在人类确定性造血分化系统中对 NOD1 的操作表明其功能保守。这项工作将 RAC1-NOD1-RIPK2-NF-kB 轴确立为关键的固有电感器，可在 HE 命运切换和 HSPC 规范之前启动 EC。对该途径的操纵可以帮助获得有能力的 HE，能够指定功能性患者特异性 HSPC 及其衍生物，用于治疗血液疾病。