sigma 1 receptor Several studies have reported the existence

Several studies have reported the existence of rare CD34− HSCs upstream of the major population of CD34+ HSCs in the human hematopoietic hierarchy (Anjos-Afonso et al., 2013; Bhatia et al., 1998). Interestingly, the first HSCs found in the human embryo are CD34+ and give rise exclusively to a CD34+CD38−/lo daughter HSC population upon xenotransplantation (Ivanovs et al., 2011). Since the subsequent downregulation of CD34 during further development cannot be excluded, precise changes in the phenotype of human HSCs require further study. In summary, we have demonstrated that the first HSCs in the human embryo emerge in the ventral niche of the Ao and are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. By identifying the niche and phenotype of the first human definitive HSCs, this study lays a foundation for identifying and characterizing the molecular mechanisms that underlie the specification and high regenerative potential of the earliest human HSCs. Such knowledge may also be essential for developing protocols to derive human HSCs from embryonic and induced pluripotent stem cells for use in regenerative medicine.
Experimental Procedures
Acknowledgments
Introduction In the mouse embryo, the first blood-forming cells appear approximately 7.5 days into gestation (embryonic day [E] 7.5) within the blood islands that line the extraembryonic yolk sac (YS) (Moore and Metcalf, 1970). These “primitive” blood-forming cells appear to be lineage-restricted, form primarily large nucleated erythrocytes, and express embryonic globins (Palis et al., 1999). They also lack the ability to engraft when transplanted intravenously into lethally irradiated adult mice, a hallmark property of fully functional adult bone marrow HSCs (Müller et al., 1994). After the establishment of a circulatory system at e8.5, “definitive” erythromyeloid progenitors appear within the YS (Palis et al., 1999), the placenta (PL) (Alvarez-Silva et al., 2003), and the embryo proper (EP). The earliest intraembryonic hematopoietic progenitors are found within the para-aortic splanchnopleura (p-Sp), which develops into the aorta-gonad-mesonephros (AGM) that contains the dorsal sigma 1 receptor (Cumano et al., 1996; Godin et al., 1993, 1995; Medvinsky et al., 1993). Hematopoietic progenitors with the ability to self-renew appear within the YS and AGM at e9.0 and appear within the fetal liver (FL) a day or two later (Yoder and Hiatt, 1997). e9.5 YS cells lack the ability to home to the bone marrow when transplanted into adult mice, but their long-term self-renewal activity can be revealed in vivo by transplantation into the liver or facial vein of sublethally irradiated newborn mice (Yoder and Hiatt, 1997; Yoder et al., 1997a, 1997b) or alternatively by first coculturing with reaggregated AGM tissue (Taoudi et al., 2008) or on the OP9 bone marrow stromal line (Rybtsov et al., 2011), indicating that progenitors residing within the YS can mature into functional HSC. These embryonic progenitors were thought to be precursors to HSCs, or “pre-HSCs,” and whereas not precisely defined, pre-HSCs expressed markers associated with endothelial (VE-cadherin) and hematopoietic (CD41 then CD45) cells (Rybtsov et al., 2011). At e10.5, fully functional HSCs have been isolated from the dorsal aorta of the AGM region (Müller et al., 1994), the extraembryonic YS, PL (Gekas et al., 2005), and from the vitelline and umbilical vessels (de Bruijn et al., 2000). At e11.5, HSCs are also found within the FL, which then becomes the predominant site of hematopoiesis until the formation of a bone-marrow cavity several days later (Gekas et al., 2005; Müller et al., 1994). Thus, the maturation of blood-forming cells takes place in discrete steps and likely at several different sites. A fundamental unresolved question is whether definitive hematopoietic cells derive directly from the primitive precursors that first appear in the YS blood islands (Moore and Metcalf, 1970) or instead emerge separately from a hematoendothelial precursor in the dorsal aorta called hemogenic endothelium (Dzierzak and Medvinsky, 1995; Nishikawa et al., 1998). A large body of evidence supports the de novo generation of HSCs within the dorsal aorta, including ex vivo tissue explants of the dorsal aorta prior to circulation (Cumano et al., 1996, 2001; Medvinsky and Dzierzak, 1996). Also, time-lapse imaging of AGM sections in culture reveals the emergence of hematopoietic clusters from within the luminal wall of the dorsal aorta in mice, which express several HSC markers, such as KIT, SCA-1, and CD41 (Boisset et al., 2010). Definitive hematopoietic progenitors also exist within the YS (Huang and Auerbach, 1993; Kumaravelu et al., 2002). However, early studies could not exclude the possibility that such progenitors originated elsewhere and then migrated to the YS. Evidence supporting a distinct YS origin of definitive hematopoiesis comes from lineage-tracing experiments that used a Runx1 Cre-estrogen receptor (ER) reporter to exclusively label YS-derived hematopoietic cells; subsequent analysis of these mice revealed labeling of adult HSCs (Samokhvalov et al., 2007). Similarly, inducible rescue of Runx1 expression in Runx1 knockout embryos demonstrated that definitive hematopoiesis could only be rescued at the developmental stages when Runx1 expression was restricted to the YS (Tanaka et al., 2012). In Ncx1 embryos, which lack a heartbeat and thus circulation, all hematopoietic cells are found within the YS and PL prior to embryonic lethality at e10.5 (Lux et al., 2008; Rhodes et al., 2008). Additionally, transplantation of YS cells from e8 to e9 allogeneic donors into the YS cavities of e8 to e9 hosts in utero led to YS blood-island engraftment and, when analyzed several months after birth, gave rise to donor-derived spleen colony-forming myeloerythroid cells and thymic and peripheral T cells (Weissman et al., 1977, 1978). Therefore, maturation of early YS stem/progenitors to adult HSC was demonstrated, but the cellular identity of HSC precursors, their sites of maturation, and the molecular mechanisms involved remain a mystery.