Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells.

The transplantation of autologous or HLA-compatible allogeneic hematopoietic multipotent progenitor (MPP) cells allows for the cure of patients with bone marrow failure and the restoration of hematopoiesis in cancer patients treated with high-dose chemoradiotherapy. Because of a shortage in donors for bone marrow transplantation, derivation of MPP cells from human pluripotent stem cells (hPSCs) provides alternative sources and should have a direct benefit on future stem cell therapy (Kaufman, 2009). Investigation of hematopoietic differentiation of hPSCs has led to remarkable advances in understanding of the mechanisms that underline hematopoietic specification. However, generation of functional hPSC-derived hematopoietic MPP cells, which are capable of multilineage hematopoietic differentiation and long-term engraftment in vivo, remain a significant challenge. Further discovery of critical factors and development of technology for de novo MPP generation from hiPSCs should greatly facilitate a realization of therapeutic applications of personalized hiPSCs. During embryogenesis, hemogenic endothelial cells (ECs), a specified subset of endothelial cells in the vascular endothelium, give rise to multipotent and self-renewable hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition (EHT) (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010). The bona fide HSCs emerge primarily from endothelium in the aortic-gonad-mesonephros (AGM) region (Zovein et al., 2008; Tavian et al., 2010; Rafii et al., 2013; Ivanovs et al., 2014), and are the origin of a full spectrum of blood cells sustained through the lifespan of an organism. Given the pivotal role of the hemogenic ECs in de novo generation of definitive HSCs, it is important to understand how definitive hematopoietic MPP cells generated from hemogenic ECs in the hPSC differentiation system. Several recent reports have focused on defining and characterization of hemogenic progenitors and definitive hematopoietic progenitors from various hPSC differentiation systems (Choi et al., 2012; Kennedy et al., 2012; Rafii et al., 2013), revealing the phenotypes and functionality of putative hemogenic progenitors in a specified context. Most recently, the first human HSCs are shown to emerge from the ventral domain of the dorsal aorta in the AGM region with an extensive defined phenotype including the expression of CD34, CD45, CD144 (VE-Cadherin), and CD117 (c-kit). Definitive hematopoietic MPP cells derived from hemogenic ECs of hPSCs have been reported (Lancrin et al., 2009; Choi et al., 2012; Kennedy et al., 2012; Rafii et al., 2013; Sturgeon et al., 2014; Uenishi et al., 2014; Ayllon et al., 2015), however, engraftment activity from these hematopoietic cells have not been demonstrated. A recent study demonstrated that vascular niche promotes engraftable human MPP production from hPSCs (Gori et al., 2015). The identity of hPSC-derived hematopoietic cells that possess long-term engraftment potential remains elusive. One of the possible factors contributing to the difficulty in de novo generation of engraftable hematopoietic cells from hPSCs is that definitive hemogenic ECs exist only briefly, thus definitive MPP generation via EHT must occur in a restricted developmental time window. We and others have identified hematopoietic and endothelial progenitors in differentiated hPSCs, based on markers expressed in endothelial and hematopoietic progenitor cells, including CD34, KDR (VEGFR2 or FLK1), CD31 (PECAM1), and CD144 (Kennedy et al., 2007; Choi et al., 2012; Kennedy et al., 2012; Wang et al., 2012; Bai et al., 2013; Rafii et al., 2013; Xie et al., 2015). We previously demonstrated that CD34+CD31+CD144+ population from hPSCs contains hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells (Bai et al., 2010; Bai et al., 2013; Xie et al., 2015). The key transcription factors required for definitive hematopoietic cell generation from hemogenic ECs, including SCL and RUNX1 (Lacaud et al., 2002; Patterson et al., 2005; Nottingham et al., 2007; Dzierzak and Speck, 2008; Chen et al., 2009; Lam et al., 2010; Ren and Gomez, 2010; Van et al., 2012; Ran et al., 2013; Zhen et al., 2013), are expressed in CD34+CD31+CD144+ cells (Bai et al., 2013). In the present study, we established a stepwise differentiation system to interrogate hematopoietic cells from the endothelial monolayer derived from HEPs. Our data indicated that the CD34+CD31+CD144+ population is endowed with hemogenic activity to generate definitive MPP cells that gave rise to multilineage hematopoietic cells, including definitive erythroid cells and T lymphocytes. Our study provides insight into the feature of hemogenic endothelial cells, and provides a unique system for future investigation of the mechanisms underlying the development and specification of human MPP cells through hemogenic ECs.