Direct Conversion from Mouse Fibroblasts Informs the Identification of Hemogenic Precursor Cells In Vivo

Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in the aorta-gonad-mesonephros (AGM) region and placenta. We have recently demonstrated the induction of hematopoietic stem/progenitors (HSPCs) from mouse fibroblasts with a combination of transcription factors progressing through endothelial-like precursors. Here, guided by our in vitro programming experiments we analyzed mouse placentas for the presence of the precursor phenotype. We identified a small population of CD34+ Sca1+Prom1+ (34PS) cells in mid-gestation placentas that do not express the pan-hematopoietic marker CD45. After isolation and culture 34PS cells acquire CD45 and generate large hematopoietic as well as cobblestone colonies. Prom1+ cells localize to the placental vascular labyrinth where HSPCs emerge. 34PS cells express markers associated with the hemogenic endothelium (CD31, Tie2, VE-Cadherin, Sox17, Runx1, Scl) and also markers identified by direct induction (Itga6/CD49f). This population is heterogeneous for the early hematopoietic marker CD41 and expresses the programming transcription factors. Remarkably, global gene expression profiles of placental 34PS cells correlate with AGM-derived hemogenic endothelium and fibroblast-derived precursors. Finally, when co-cultured with stroma placental 34PS cells give rise to B/T lymphoid cells as well as mixed colonies containing erythroid, myeloid and megakaryocytic cell lineages. In summary, we show that direct in vitro conversion provided a cell surface phenotype for the isolation of hemogenic precursors in vivo. Our findings provide insights into the specification of definitive hemogenesis in the placenta, in depth characterization of hemogenic precursor populations and the first evidence that direct in vitro conversion approaches can be used as a valuable tool to address basic developmental questions in vivo.