Retinoic acid as a stage-specific modulator of hematopoietic lineage fate from human pluripotent stem cells.

Retinoic acid (RA), a bioactive metabolite of vitamin A, plays roles in early embryogenesis and hematopoietic development. However, its precise function in directing the hematopoietic lineage outcomes of human pluripotent stem cells (hPSCs) remains unclear. Here, we uncovered a distinct, stage-specific role for RA as a lineage-specifying modulator during late-stage hematopoietic differentiation, rather than as a promoter of hematopoietic progenitor generation. Using a stepwise hPSC differentiation system, we demonstrated that RA exerted minimal or inhibitory effects when applied during early mesoderm or hemogenic endothelial stages. In contrast, RA treatment during days 13-15 significantly enhanced progenitor maturation, proliferation, and functional output. Notably, RA acted cooperatively with external cytokines to modulate lineage fate. In the presence of erythropoietin (EPO), RA strongly promoted erythroid differentiation by activating EPOR signaling and upregulating erythroid transcriptional programs, including GATA1, KLF1, and globin gene expression. Conversely, under GM-CSF/M-CSF stimulation, RA biased progenitor differentiation toward macrophages, consistent with its role as an amplifier of the prevailing cytokine-directed lineage fate rather than an independent suppressor of erythropoiesis. These effects were highly dose- and context-dependent, with low-dose RA optimally enhancing lineage bias without cytotoxicity. Importantly, RA modulated the transcriptional and proliferative dynamics of committed progenitors. Taken together, our findings reveal a previously unrecognized role of RA as a versatile and tunable modulator of hematopoietic lineage fate that offers a novel strategy for in vitro blood cell engineering. This study advances approaches for lineage-specific blood production relevant to disease modeling, drug screening, and regenerative medicine.