3022 – GENERATION OF RETINOIC ACID-DEPENDENT HEMOGENIC ENDOTHELIAL PROGENITORS FROM HUMAN PLURIPOTENT STEM CELLS

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The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a- mesodermal population that gives rise to a clonally multipotent definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes, and is functionally unresponsive to RA treatment. Thus, the specification of a RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNAseq analyses, we identified that two distinct KDR+CD235a- populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, CYP26A1, a RA degrading enzyme, was expressed in KDR+CD235a-CXCR4- mesoderm, and this was the only mesodermal population harboring definitive hematopoietic potential. In sharp contrast, KDR+CD235a-CXCR4+ mesoderm expressed ALDH1A2, a key enzyme in the synthesis of RA and lacked any hematopoietic potential. However, the stage-specific application of retinoic acid signaling to this CXCR4+ mesodermal population resulted in the robust specification of CD34+ HE with multi-lineage definitive erythroid, myeloid and lymphoid hematopoietic potential. Further, in vivo correlates to these functionally-distinct mesodermal precursors could be found within a scRNA-seq dataset of gastrulating murine embryos. Additional comparison to human embryos demonstrated that CD34+ cells specified with retinol from CXCR4+ mesoderm have fetal-like HOXA expression. Collectively, this represents the first ever characterization of stage-specific RA-dependent hPSC-derived definitive hematopoiesis, and its mesodermal progenitor. This novel insight into human hematopoietic development will ultimately provide the basis for the de novo specification of HSCs.