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

Abstract

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 vessels from which RUNX1C blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Human haematopoietic stem cell development: from the embryo to the dish

“…Following an initial period of BMP4-based mesoderm induction, brief inhibition of activin (Kennedy et al, 2012), or provision of a WNT agonist (Gertow et al, 2013) were sufficient to inhibit the erythroid-biased primitive programme. Importantly, activin inhibition (Kennedy et al, 2012), WNT stimulation (Sturgeon et al, 2014) or a combination of the two (Ng et al, 2016) from days 2-4 of in vitro differentiation also resulted in a bias towards definitive haematopoietic lineages, as defined by the capacity to generate T lymphocytes (Kennedy et al, 2012;Sturgeon et al, 2014).…”

“…The selective expression of HOXA genes in human foetal liver and UCB-derived haematopoietic progenitor and stem cells clearly distinguished them from PSC-derived haematopoietic progenitors generated using conventional mesoderm induction protocols (Dou et al, 2016;Ng et al, 2016). HOX genes mark axial position during development and are first expressed at the primitive streak stage (Deschamps and van Nes, 2005).…”

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

“…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.…”

“…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.…”

“…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.…”