Abstract
Decoding the gene regulatory mechanisms mediating self-renewal of hematopoietic stem cells (HSCs) during their amplification in the fetal liver (FL) is relevant for advancing therapeutic applications aiming to expand transplantable HSCs, a long-standing challenge. Here, to explore intrinsic and extrinsic regulation of self-renewal in FL-HSCs at the single cell level, we engineered a culture platform designed to recapitulate the FL endothelial niche, which supports the amplification of serially engraftable HSCs ex vivo. Leveraging this platform in combination with single cell index flow cytometry, serial transplantation assays, and single cell RNA-sequencing, we elucidated previously unrecognized heterogeneity in immunophenotypically defined FL-HSCs and demonstrated that differentiation latency and transcriptional signatures of biosynthetic dormancy are distinguishing properties of self-renewing FL-HSCs with capacity for serial, long-term multilineage hematopoietic reconstitution. Altogether, our findings provide key insights into HSC expansion and generate a novel resource for future exploration of the intrinsic and niche-derived signaling pathways that support FL-HSC self-renewal.