Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages

Abstract

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34+CD73−CD184− fraction of day 8 embryoid bodies (EBs) and it undergoes a NOTCH-dependent EHT to generate RUNX1C+ cells with multilineage potential. Arterial and venous VE progenitors, by contrast, segregate to the CD34+CD73medCD184+ and CD34+CD73hiCD184− fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells

“…A recent study, for example, demonstrated that AECs were confined to a CD184 + subfraction of CD34 + cells isolated from differentiating human embryonic stem cell-derived embryoid bodies (9). The human embryonic stem cellderived AECs described here also expressed high levels of CXCR4 (CD184), consistent with those previous results.…”

“…S3 A and B). EFNB2 and EPHB4 were the first identified and most widely used markers for AECs and VECs, respectively (6)(7)(8)(9)(10)(11)(12)(13)17). Specific targeting of the EFNB2 and EPHB4 locus was confirmed by junction PCR and Southern blot analysis (SI Appendix, Evaluation of Candidate Factors for Arteriovenous Specification.…”

“…1 C and D). We also tested the function of BMP4 as it is widely used for AEC differentiation (6,9,12,13). The result showed that BMP4 suppressed the arterial marker (EFNB2) expression ( Fig.…”

“…Previous studies have shown progress in AEC differentiation (6)(7)(8)(9)(10)(11)(12)(13)(14), but the arterial-specific functions in vitro and the protection of ischemic tissue in vivo have not been well demonstrated in the resulting cells. Here, we use single-cell RNA sequencing (RNA-seq) of early mouse AECs and a CRISPR/Cas9-generated EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to develop a protocol for differentiating human pluripotent stem cells into AECs using fully defined culture conditions.…”

An in vitro model of hemogenic endothelium commitment and hematopoietic production

“…Comparing in vitro cultures with the in vivo situation in the early mouse embryo, the blast colonyforming cell was shown to be the in vitro equivalent of the nascent mesoderm ingressing through the primitive streak (Huber et al, 2004) and was characterized by the expression of the receptor tyrosine kinase Flk1 (also known as Kdr and Vegfr2) and the T-box transcription factor brachyury (Robertson et al, 2000). Using wellestablished culture systems, it was possible to drive this early mesodermal progenitor to differentiate into hemogenic ECs able to give rise to blood cells through EHT and to document several aspects of blood formation from hemogenic ECs (Eilken et al, 2009;Lancrin et al, 2009), including some aspects of EHT (Ditadi et al, 2015). However, the number of hemogenic ECs generated in ESC cultures was reported to be low (1/3000) (Eilken et al, 2009), hampering the establishment of a versatile culture system that could be employed to address questions concerning hemogenic endothelium commitment from non-hemogenic ECs and the cellular and molecular changes that occur during EHT.…”