Identification of the Hemogenic Endothelial Progenitor and Its Direct Precursor in Human Pluripotent Stem Cell Differentiation Cultures

Abstract

Summary Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential to enable the investigation of the molecular determinants of HSC specification. Here we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by VE-cadherin+CD73−CD235a/CD43− phenotype. This phenotype discriminates true HEPs from VE-cadherin+CD73+ non-HEPs, and VE-cadherin+CD235a+CD41a− early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post primitive streak stage of differentiation directly from VE-cadherin-negative KDRbrightAPLNR+PDGFRαlow/− hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR+PDGFRα+ mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal to facilitate generation of HSCs from hPSCs.

HOXA9 promotes hematopoietic commitment of human embryonic stem cells

“…It has been described the existence of CD45 2 CD34 1 CD41a 1 CD235a 1 primitive hematopoietic progenitors within the HEP population. 33 As shown in supplemental Figure 2B, HOXA9 overexpression also increased (up to 12-fold) the proportion of primitive CD45 2 CD34 1 CD41a 1 CD235 1 cells. Functionally, we tested the clonogenic potential of hematopoietic progenitors derived from either day 8 of OP9-hESC cocultures ( Figure 3C) or day 15 EB development ( Figure 4C), measured by the ability to form CFUs in semisolid cultures.…”

A short history of hemogenic endothelium

“…This is reflected in the difficulty to generate bona fide HSCs de novo from ES/iPS cells [50,85,[91][92][93], or through direct reprogramming [94,95]. This indicates that critical components of EHT, responsible for regulating the hematopoietic nature of these cells are missing in these culture systems and indicate the importance of understanding the cellular, molecular and niche requirements for EHT in vivo.…”

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

“…The CD34 + CD43 + haematopoietic progenitors give rise to a broad range of erythroid and myeloid lineages (Choi et al, 2012;Kennedy et al, 2012;Ng et al, 2016), reminiscent of the broad potential of the yolk sac-derived erythro-myeloid progenitor (EMP) described in the mouse that transiently seeds the foetal liver (McGrath et al, 2015). Although EMP-type haematopoiesis has not been formally characterised in human embryos, kinetic studies report a rapid transition of clonogenic cells from the yolk sac to the foetal liver at around 5 weeks of gestation (Migliaccio et al, 1986).…”

“…In contrast to non-haematogenic vascular cells, hPSC-derived haematogenic endothelium lacks expression of CD73 (NT5E) (Choi et al, 2012;Ditadi et al, 2015), CXCR4 and DLL4 (Ditadi et al, 2015). Also, both mouse (North et al, 1999;Thambyrajah et al, 2016) and human (Choi et al, 2012;Ng et al, 2016) haematogenic endothelium are distinguished by their expression of the key transcription factors RUNX1 and GFI1, which are required for the endothelial to haematopoietic transition. Interestingly, hPSCs differentiated with a protocol that induces HOXA gene expression produce SOX17-expressing endothelial cells that bear similarity at the transcriptional level to developing dorsal aorta endothelium, and a SOX17…”

“…As well as the surface markers KDR and PDGFRα, blast colony-forming cells (BL-CFCs) also express the apelin receptor (Vodyanik et al, 2010;Choi et al, 2012;Yu et al, 2012), and CD235a (glycophorin A) (Sturgeon et al, 2014). As in the mouse (Huber et al, 2004), human blast colonies give rise to haematopoietic, endothelial and smooth muscle lineages (Yu et al, 2012).…”