Blood is one of the most highly regenerative tissues and is generated by a rare population of hematopoietic stem cells (HSCs). Generation of HSCs seems to be restricted during early embryonic development to particular fetal tissues, such as yolk sac, aorta gonad mesonephros (AGM) region and placenta. The competence of HSCs to self-renew and differentiate towards multiple lineages sustains the generation of blood cells for an entire lifespan. Human HSCs are defined by their self-renewal and multilineage differentiation in functional xenotransplantation assays. Differentiation of human embryonic stem cells (hESCs) into transplantable HSCs continues to be a major challenge in the field. We believe that generation of functional multipotent HSCs from hESC requires an approach that recapitulates early human development both temporally and spatially. Thus, we have developed an innovative in vitro system that mimics the early events of human embryonic development prior to hematopoietic generation; where 1) induction of a single ESC monolayers to mesodermal fate; 2) single cell mesoderm progenitors are placed into a fetal AGM stromal environment under conditions that favor endothelial differentiation; and 3) hematopoietic inductors are added generating single free-floating hematopoietic progenitors in a temporal fashion. We have identified both hemogenic endothelium (adherent) and hematopoietic (non-adherent) cells in the AGM/mesoderm coculture. Hemogenic endothelium defined by expression of VE-Cadherin cocultured on AFT024 fetal liver stromal cells is able to generate hematopoietic precursors. Non-adherent cells start budding up by day 18 in the AGM/mesoderm coculture. Flow cytometry analysis shows that CD31 is the prevalent marker in this floating population. Non-adherent VE-Caherin-CD31+CD34+ cells arising between day 24 till 33 have shown the highest hematopoietic activity as measure by colony forming units (CFU) cells, cobblestone area-forming cell assays (week 4–5 CAFC) and their potential to generate both B and T-cells. Molecular characterization of hemogenic endothelium the non-adherent VE-Caherin-CD31+CD34+ cells by qRT-PCR confirm that also a temporal gene expression is taken place as cells transit from endothelium to hematopoietic cells. VE-Caherin-CD31+CD34+ cells also express CRCX4, suggesting their potential for engrafting in mice models.
This novel approach provides a unique opportunity to study the early events and to identify molecular targets in the human hematopoietic commitment, lineage specification, and maturation; these hematopoietic cells may represent a promise for clinical therapies in a diverse range of blood diseases.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.