p65 signaling dynamics drive the developmental progression of hematopoietic stem and progenitor cells through cell cycle regulation
While hematopoietic stem and progenitor cells (HSPCs) and the signaling pathways that specify them have been well defined in vivo, deriving functional HSPCs from human pluripotent stem cells (hPSCs) is still an outstanding challenge in the stem cell field. This has impaired efforts to use patient-specific HSPCs and their derivatives to treat leukemia, lymphoma, anemia, and solid cancers. Identifying the missing element to derive functional human HSPCs will be critical to advance these therapies. Here, we found that in contrast to in vitro protocols where signaling pathways are typically manipulated statically, the natural developmental progression of HSPC fate in the embryo requires a precise and temporally controlled oscillatory signaling dynamics.  Particularly, our work reveals that NF-kB signaling works as an oscillatory clock that controls the developmental progression of HSPCs by driving phases of quiescence and proliferation precisely during embryonic development. This was revealed by live imaging of a custom NF-kB zebrafish reporter line, in conjunction with transcriptomic profiling and precise temporal manipulations of the NF-kB family member p65, the driver of these signaling events. We also show that in vitro protocols of human hematopoietic differentiation from iPSCs depend on initial pro-inflammatory signaling activation. In summary, our results uncover the functional role of pro-inflammatory signaling during HSPC specification, revealed their oscillatory dynamics, and define this signaling pathway as the main driver of HSPC fate progression. The integration of these dynamics in vitro will be fundamental to achieve the long-standing goal of generating and expanding patient derived functional HSPCs. Overall design: To gain insights into how NF-kB in endothelium was contributing to HSPC specification, NFKB+ or NFKB- endothelial cells (kdrl+) were sorted and collected from 22hpf zebrafish embryos in triplicate and subject to RNA-seq.