Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases

The possibility of specifying functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (hPSCs) would overcome current limitations related to HSPC transplantation. However, generating hPSC-derived HSPCs has been elusive, necessitating a better understanding of the native developmental mechanisms that trigger HSPC specification. Here, we revealed in vivo an intrinsic inflammatory mechanism triggered by Nod1 that drives early hemogenic endothelium (HE) patterning to specify HSPCs. Our genetic and chemical experiments showed that HSPCs failed to specify in the absence of Nod1 and its downstream kinase Ripk2. Rescue experiments demonstrated that Nod1 and Ripk2 acted through NF-kB, and that small Rho GTPases are at the apex of this mechanism. Manipulation of NOD1 in a human system of hPSCs differentiation towards the definitive hematopoietic lineage indicated functional conservation. This work establishes the RAC1-NOD1-RIPK2-NFkB axis as the earliest inflammatory inductor that intrinsically primes the HE for proper HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs for the treatment of blood disorders. Keywords: NOD1, RIPK2, NF-kB, RAC1, Rho GTPases, Hematopoietic Stem and Progenitor Cell Specification, Hemogenic Endothelium. Overall design: AB wildtype x kdrl:mCherry zebrafish embryos were injected with Standard-MO, Nod1-MO2, Nod2-MO and Ripk2-MO at one-cell stage. Approximately 150 per morpholino injection 24hpf kdrl + zebrafish embryos were screened with Leica M165FC stereomicroscope, dechorionated with pronase, anesthetized in 1% tricane, gently shaken at 28C for 5 mins with 0.05mg/ml liberase TM(Roche) in PBS solution with Ca2+ and Mg2+. The resulting cell suspension was filtered and stained with Sytox Red to exclude dead cells. kdrl+ cells were sorted and collected with BD FACS Aria III. RNA was then isolated and purified with RNeasy Micro Kit (Qiagen) in triplicate per condition.