Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros

The production of definitive haematopoietic stem/progenitor cells from human pluripotent stem cells (hPSCs) remains a significant challenge. Using reporter lines to track the endothelial (SOX17) to haematopoietic (RUNX1C) transition, we found that hPSC differentiated in growth factor supplemented serum free medium generated RUNX1C+CD34+ clonogenic cells that homed to the bone marrow, but did not engraft. Compared to repopulation-competent cord blood CD34+ cells, RUNX1C+CD34+ progenitors lacked HOXA gene expression, indicating incorrect mesoderm patterning. This deficiency was ameliorated by a timed pulse of WNT activation combined with ACTIVIN antagonism. Significantly, these HOXA+ cultures now formed complex SOX17+ vessels that produced fetal liver-like haematopoietic cells, similar to the human aorta-gonad-mesonephros (AGM). Comparison of transcriptional profiles of these nascent haematopoietic stem/progenitors with cells isolated from human AGM confirmed significant similarities, consistent with the assignment of our in vitro generated cells to the definitive human haematopoietic lineage. Our findings argue that HOXA codes established early in differentiation predict cellular potential and provide correct cell patterning for the specification of definitive haematopoietic lineages from hPSCs. Overall design: mRNA profiles of 26 samples were obtained for 5 different cell populations and 2 different treatments.

26

Ng ES, Azzola L, Bruveris FF, Calvanese V, Phipson B, Vlahos K, Hirst C, Jokubaitis VJ, Yu QC, Maksimovic J, Liebscher S, Januar V, Zhang Z, Williams B, Conscience A, Durnall J, Jackson S, Costa M, Elliott D, Haylock DN, Nilsson SK, Saffery R, Schenke-Layland K, Oshlack A, Mikkola HK, Stanley EG, Elefanty AG