Molecular dissection of Hematopoietic Stem Cell specification triggered by inflammatory mediators - Project Summary Due to the unique property of hematopoietic stem cells (HSCs) to reconstitute the entire blood system of the organism, these stem cells are utilized clinically to treat blood disorders. The possibility of culturing and expanding HSCs in vitro would make hematopoietic stem cell transplantation (HSCT)-based therapies more feasible. However, this has eluded the field for more than three decades, necessitating a closer examination of the native developmental mechanisms that govern the emergence of HSCs. Many years of investigation have revealed that HSCs require multiple molecular inputs for proper specification, including activity of the Notch, nitric oxide (NO), Wnt, FGF, and BMP signaling pathways. In addition, inflammatory signaling (Tnfa, NF-kB, Tlr4, interferons, Il1b and inflammasome) have been recently reported as a novel group of HSC fate modulators, yet the underlying molecular mechanisms are unclear. Addressing this knowledge gap will be critical to help develop in vitro protocols for the generation of patient-specific HSCs. The goal of this proposal is to reveal in vivo the inflammatory network that unlocks HSC specification from the hemogenic endothelium (HE), and its relationship with the Notch and nitric oxide pathways. To attain this goal, the following three specific aims will be pursued: (1) Identify the role of Nod1 signaling during HSC development; (2) determine the mechanism of NF-kB-directed HSC specification; and (3) analyze the impact of the NOD1/RIPK2/NF-kB inflammatory axis on human pluripotent stem cell-derived definitive hematopoietic progenitor cells. Since hematopoietic development is highly conserved between vertebrate species, the zebrafish model provides a unique opportunity to circumvent the challenges of in utero experimentation, permitting non-invasive experiments that avoid the artifactual inflammation caused by cellular stress. To achieve this application's goals, a combination of novel zebrafish reporter and mutant lines, new methods to perform epigenomic and transcriptomic profiling of the HE by CUT&RUN-sequencing and RNA-sequencing, live imaging of HSC development by confocal and light-sheet microscopy, qPCR, FACS-sorting, and lineage tracing using Cre- mediated reporter systems will be utilized. In addition, to translate these in vivo findings to human health, this proposal will be complemented with a model of hematopoietic differentiation from human pluripotent stem cells (hPSCs). Upon successful completion of the proposed research, a previously undescribed inflammatory pathway affecting HSC specification will be identified, in addition to the central molecular mechanism by which inflammatory signaling drives HSC fate and crosstalk to other main HSC inductors. These new findings could provide key insights needed to instruct HSC fate, informing in vitro approaches to generate HSCs from pluripotent precursors for the treatment of blood disorders.
