Para-aortic Splanchnopleura Research Articles

The changing cellular environments of hematopoiesis in human development in utero

The hematopoietic system is indispensable from the earliest stages of development and adapts to the rapidly changing anatomy of the embryo and fetus; this takes place in such different anatomic locations as the yolk sac blood island, hepatic parenchyme, aorta-gonads-mesonephros paravascular mesenchyme, and bone marrow primary logette. We herein summarize our investigation of these serial blood-forming events in the human embryo and fetus. The access to early stages of human development, availability of a large panoply of molecular markers for human blood cell lineages, and recent development of robust assays for the earliest human hematopoietic stem cells have allowed us to gain relatively clear insight into the developmental sequence that underlies the ontogeny of human blood cells. Conversely, the control exerted by these diverse cellular environments on the emergence of human hematopoietic cells remains elusive, as is the case in animal models. We nonetheless present preliminary attempts to decipher the structure and molecular characteristics of the distinct cellular "niches" in which blood cells are produced during human gestation.

GATA Motifs Regulate Early Hematopoietic Lineage-Specific Expression of the Gata2 Gene

Transcription factor GATA-2 is essential for definitive hematopoiesis, which developmentally emerges from the para-aortic splanchnopleura (P-Sp). The expression of a green fluorescent protein (GFP) reporter placed under the control of a 3.1-kbp Gata2 gene regulatory domain 5' to the distal first exon (IS) mirrored that of the endogenous Gata2 gene within the P-Sp and yolk sac (YS) blood islands of embryonic day (E) 9.5 murine embryos. The P-Sp- and YS-derived GFP(+) fraction of flow-sorted cells dissociated from E9.5 transgenic embryos contained far more CD34(+)/c-Kit(+) cells than the GFP(-) fraction did. When cultured in vitro, the P-Sp GFP(+) cells generated both immature hematopoietic and endothelial cell clusters. Detailed transgenic mouse reporter expression analyses demonstrate that five GATA motifs within the 3.1-kbp Gata2 early hematopoietic regulatory domain (G2-EHRD) were essential for GFP expression within the dorsal aortic wall, where hemangioblasts, the earliest precursors possessing both hematopoietic and vascular developmental potential, are thought to reside. These results thus show that the Gata2 gene IS promoter is regulated by a GATA factor(s) and selectively marks putative hematopoietic/endothelial precursor cells within the P-Sp.

The Vascular Wall as a Source of Stem Cells

We have characterized the emerging hematopoietic system in the human embryo and fetus. Two embryonic organs, the yolk sac and aorta, support the primary emergence of hematopoietic stem cells (HSCs), but only the latter contributes lymphomyeloid stem cells for definitive, adult-type hematopoiesis. A common feature of intra- and extraembryonic hematopoiesis is that in both locations hematopoietic cells emerge in close vicinity to vascular endothelial cells. We have provided evidence that a population of angiohematopoietic mesodermal stem cells, marked by the expression of flk-1 and the novel BB9/ACE antigen, migrate from the paraaortic splanchnopleura into the ventral part of the aorta, where they give rise to hemogenic endothelial cells and, in turn, hematopoietic cells. HSCs also appear to develop from endothelium in the embryonic liver and fetal bone marrow, albeit at a much lower frequency. This would imply that the organism does not function during its whole life on a stock of hematopoietic stem cells established in the early embryo, as is usually accepted. We next examined whether the vessel wall can contribute stem cells for other cell lineages, primarily in the model of adult skeletal muscle regeneration. By immunohistochemistry and flow cytometry, we documented the existence in skeletal muscle, besides genuine endothelial and myogenic cells, of a subset of satellite cells that coexpress endothelial cell markers. This suggested the existence of a continuum of differentiation from vascular cells to endothelial cells that was confirmed in long-term culture. The regenerating capacity of these cells expressing both myogenic and endothelial markers is being investigated in skeletal and cardiac muscle, and the results are being compared with those generated by satellite cells. Altogether, these results point to a generalized progenitor potential of a subset of endothelial, or endothelium-like, cells in blood vessel walls, in pre- and postnatal life.

L’ontogenèse du système hématopoïétique revisitée

The ontogenesis of hematopoiesis is classically described as a series of successive steps: the first takes in the yolk sac where blood islands differentiate. Then, cells deriving from these structures migrate and populate the transient hematopoietic organs such as the liver and the spleen. At last, the eventual migration allows the establishment of bone marrow hematopoiesis. This theory described in almost all the textbooks of Human Embryology does not fit with recent experimental data. Indeed, the construction of quail-chick chimeras shows that the yolk sac does not contribute to the adult hematopoiesis in birds. Adult hematopoietic cells arise from a population located on the ventral side of the aorta both in birds and mammals. The aortic population derives from the para-aortic splanchnopleura and its derivative, the so-called AGM (Aorta-Gonad-Mesonephros). These new data provide new concepts to understand the process of ontogenesis of the hematopoietic system in vertebrates.

Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro

AbstractThe embryonic origin and development of hematopoietic and endothelial cells is highly interdependent. We hypothesized that primary endothelial cells from murine yolk sac and para-aortic splanchnopleura (P-Sp) may possess the capacity to expand hematopoietic stem cells (HSCs) and progenitor cells ex vivo. Using Tie2-GFP transgenic mice in combination with fluorochrome-conjugated monoclonal antibodies to vascular endothelial growth factor receptor-2 (Flk1) and CD41, we have successfully isolated pure populations of primary endothelial cells from 9.5-days after coitus (dpc) yolk sac and P-Sp. Adult murine bone marrow Sca-1+c-Kit+lin-cells were cocultured with yolk sac or P-Sp Tie2-GFP+Flk-1+CD41-endothelial cell monolayers for 7 days and the total number of nonadherent cells increased 47- and 295-fold, respectively, and hematopoietic progenitor counts increased 9.4- and 11.4-fold, respectively. Both the yolk sac and P-Sp endothelial cell cocultures facilitated long-term (> 6 months) HSC competitive repopulating ability (2.8- to 9.8-fold increases, respectively). These data suggest that 9.5-dpc yolk sac- and P-Sp-derived primary Tie2-GFP+Flk-1+CD41-endothelial cells possess the capacity to expand adult bone marrow hematopoietic progenitor cell and HSC repopulating ability ex vivo. (Blood. 2003;102:4345-4353)

Notch1 but Not Notch2 Is Essential for Generating Hematopoietic Stem Cells from Endothelial Cells

Hematopoietic stem cells (HSCs) are thought to arise in the aorta-gonad-mesonephros (AGM) region of embryo proper, although HSC activity can be detected in yolk sac (YS) and paraaortic splanchnopleura (P-Sp) when transplanted in newborn mice. We examined the role of Notch signaling in embryonic hematopoiesis. The activity of colony-forming cells in the YS from Notch1 −/− embryos was comparable to that of wild-type embryos. However, in vitro and in vivo definitive hematopoietic activities from YS and P-Sp were severely impaired in Notch1 −/− embryos. The population representing hemogenic endothelial cells, however, did not decrease. In contrast, Notch2 −/− embryos showed no hematopoietic deficiency. These data indicate that Notch1, but not Notch2, is essential for generating hematopoietic stem cells from endothelial cells.

Ontogenic emergence of definitive hematopoietic stem cells.

Research in the past 10 years has dramatically increased our knowledge of the development of the mammalian hematopoietic system and has provided insight into the embryonic sites of hematopoietic cell generation, the variety of hematopoietic cell types produced, and some of the microenvironmental influences on the rapidly growing blood system. Indeed, within mammalian embryos, it is now widely accepted that the embryo proper produces the first adult repopulating hematopoietic stem cells. This mesodermally derived intraembryonic region, known as the aorta-gonad-mesonephros region or, at a slightly earlier developmental stage, the paraaortic splanchnopleura, produces, respectively, potent hematopoietic stem cells and multipotent progenitor cells before their appearance in the yolk sac. This review focuses on the most recent findings concerning qualitative and quantitative aspects of hematopoietic stem-cell development, the endothelium as a possible direct precursor population of hematopoietic stem cells, and the microenvironment leading to the onset and maintenance of hematopoietic stem cells in the mammalian embryo.

Hematopoietic stem cell emergence in the human embryo and fetus.

Two waves of hematopoietic stem cell generation take place in the first month of human gestation. The first one has long been known to occur in the yolk sac; only recently was another one identified that results in the development, from the 27th day, of clusters of hematopoietic cells on the ventral endothelium of the aorta and vitelline artery. This latter, intra-embryonic phase of blood cell progenitor production is undoubtedly local since its presumptive territory of occurrence, the para-aortic splanchnopleura, is endowed with blood-forming potential in vitro from, at least, day 19 of development. The first multipotent, lympho-myeloid stem cells emerge within that intra-embryonic territory, whereas the yolk sac only produces myeloid precursor cells devoid of lymphoid potential. The forerunners of the hematopoietic stem cells born to intra-embryonic arteries appear to migrate from the splanchnopleura as KDR(+) angio-hematopoietic cells that colonize the ventral wall of the aorta to give rise to hematogenous endothelium. All these results indicate that stem cells for human definitive hematopoiesis emerge within the embryo, in the walls of truncal arteries, from splanchnopleural ancestors and through a vascular endothelial intermediate stage. Less expectedly, blood-forming endothelial cells were also encoutered in the embryonic and fetal liver and in the fetal and, even, adult bone marrow, suggesting that a filiation between vascular and hematopoietic cells may persist during the whole life.

Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta.

Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5-9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells.

Stromal cells expressing ephrin-B2 promote the growth and sprouting of ephrin-B2+ endothelial cells

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arterial endothelial cells (ECs) and surrounding cells and interacts with multiple EphB class receptors. Conversely, EphB4, a specific receptor for ephrin-B2, is expressed on venous ECs, and both ephrin-B2 and EphB4 play essential roles in vascular development. The bidirectional signals between EphB4 and ephrin-B2 are thought to be specific for the interaction between arteries and veins and to regulate cell mixing and the making of particular boundaries. However, the molecular mechanism during vasculogenesis and angiogenesis remains unclear. Manipulative functional studies were performed on these proteins in an endothelial cell system. Using in vitro stromal cells (OP9 cells) and a paraaortic splanchnopleura (P-Sp) coculture system, these studies found that the stromal cells expressing ephrin-B2 promoted vascular network formation and ephrin-B2(+) EC proliferation and that they also induced the recruitment and proliferation of alpha-smooth muscle actin (alpha-SMA)-positive cells. Stromal cells expressing EphB4 inhibited vascular network formation, ephrin-B2(+) EC proliferation, and alpha-SMA(+) cell recruitment and proliferation. Thus, these data suggest that ephrin-B2 and EphB4 mediate reciprocal interactions between arterial and venous ECs and surrounding cells to form each characteristic vessel. (Blood. 2001;98:1028-1037)

Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region

In the mammal, definitive hematopoietic stem cells (HSCs) are first derived from mesodermal cells within a region of the embryonic para-aortic splanchnopleura known as the aorta-gonad-mesonephros (AGM). Within this region, HSCs are thought to arise from hemangioblast precursors located in the ventral wall of the dorsal aorta. However, the factors that regulate HSC development in vivo are still largely unknown. Bone morphogenetic protein (BMP)-4, a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors, is a potent ventralizing factor and has been implicated in the commitment of embryonic mesodermal cells to a hematopoietic fate in a number of systems. In the human AGM, we find that BMP-4 is expressed at high levels, and with striking polarity, in a region of densely packed cells underlying intra-aortic hematopoietic clusters. In contrast, TGF-beta1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-beta1 in the initiation and regulation of hematopoiesis in the human AGM. Furthermore, the distribution of BMP-4 expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in vivo. (Blood. 2000;96:1591-1593)

Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region

AbstractIn the mammal, definitive hematopoietic stem cells (HSCs) are first derived from mesodermal cells within a region of the embryonic para-aortic splanchnopleura known as the aorta-gonad-mesonephros (AGM). Within this region, HSCs are thought to arise from hemangioblast precursors located in the ventral wall of the dorsal aorta. However, the factors that regulate HSC development in vivo are still largely unknown. Bone morphogenetic protein (BMP)-4, a member of the transforming growth factor beta (TGF-β) superfamily of growth factors, is a potent ventralizing factor and has been implicated in the commitment of embryonic mesodermal cells to a hematopoietic fate in a number of systems. In the human AGM, we find that BMP-4 is expressed at high levels, and with striking polarity, in a region of densely packed cells underlying intra-aortic hematopoietic clusters. In contrast, TGF-β1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-β1 in the initiation and regulation of hematopoiesis in the human AGM. Furthermore, the distribution of BMP-4 expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in vivo.

Fetal and Adult Hematopoietic Stem Cells Require β1 Integrin Function for Colonizing Fetal Liver, Spleen, and Bone Marrow

Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that β1 integrin–deficient HSCs from the para-aortic splanchnopleura and the fetal blood had hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult β1 integrin null HSCs isolated from mice carrying loxP-tagged β1 integrin alleles and ablated for β1 integrin expression by retroviral cre transduction failed to engraft irradiated recipient mice. Moreover, absence of β1 integrin resulted in sequestration of HSCs in the circulation and their reduced adhesion to endothelioma cells. These findings define β1 integrin as an essential adhesion receptor for the homing of HSCs.

The Ikaros family and the development of early intraembryonic hematopoietic stem cells.

It is currently well established that across evolutionarily distant species the first blood-forming stem cells appear in the blood islands of both the extraembryonic yolk sac and the embryo proper. Following this initial and only transient generation of primitive erythroid-lineage cells, the major hematopoietic regions are thereafter located in the intraembryonic mesenchyme of the early embryos. Taking advantage of avian yolk sac-embryo chimeras it was originally shown that lymphoid cells are of intraembryonic origin [1, 2]. Two decades after the avian grafting experiments, the murine para-aortic splanchnopleura (P-Sp) and the aorta-gonad-mesonephrosregion (AGM) were functionally characterized to have lymphoid potential [3, 4]. These results confirmed the intraembryonic sources for murine lymphopoiesis. Thus, mesodermal foci, i.e. the splanchnopleura and the ventrally derived intraaortic clusters as well as the later appearing dorsal mesentery-derived para-aortic loci, initiate development of hematopoietic stem cells (HSCs). Although the AGM region is already known to harbor committed progenitors, and lymphoid potential has been shown to reside even in the para-aortic splanchnopleura, the exact time schedule of fate decisions is still rather controversial and unresolved. Contradictory evidence exists concerning the role of the P-Sp/AGM area on one hand as a de novo generator of intraembryonic hematopoietic stem cells and on the other hand as a site of hematopoietic differentiation. It has been postulated that these early hematopoietic environments only give rise to hematopoietic stem cells and further differentiation occurs in the lymphoid organs after colonization [5]. By contrast however, there are data showing the presence of committed lymphocyte progenitors not only in the fetal liver, but also in the murine AGM [6]. These results that are based on a clonal assay system support the idea of early lineage-determination in the lymphoid system. Taken together, these findings confirm not only the intraembryonic origin of the lymphoid cells, but also support the idea of early fate determination occurring before seeding of the primordial lymphoid organs.KeywordsHematopoietic Stem CellLymphocyte DevelopmentStern CellIkaros FamilyIkaros ProteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

An SCL 3' enhancer targets developing endothelium together with embryonic and adult haematopoietic progenitors.

The SCL gene encodes a basic helix-loop-helix transcription factor which is expressed in early haematopoietic progenitors throughout ontogeny and is essential for the normal development of blood and blood vessels. Transgenic studies have characterised spatially distinct 5' enhancers which direct lacZ expression to subdomains of the normal SCL expression pattern, but the same elements failed to produce appropriate haematopoietic expression. We now describe an SCL 3' enhancer with unique properties. It directed lacZ expression in transgenic mice to extra-embryonic mesoderm and subsequently to both endothelial cells and to a subset of blood cells at multiple sites of embryonic haematopoiesis including the yolk sac, para-aortic splanchnopleura and AGM region. The 3' enhancer also targeted expression to haematopoietic progenitors in both foetal liver and adult bone marrow. Purified lacZ(+ )cells were highly enriched for clonogenic myeloid and erythroid progenitors as well as day-12 spleen colony forming units (CFU-S). Within the total gated population from bone marrow, 95% of the myeloid and 90% of the erythroid colony-forming cells were contained in the lacZ(+) fraction, as were 98% of the CFU-S. Activation of the enhancer did not require SCL protein. On the contrary, transgene expression in yolk sacs was markedly increased in an SCL-/- background, suggesting that SCL is subject to negative autoregulation. Alternatively the SCL-/- environment may alter differentiation of extra-embryonic mesoderm and result in an increased number of cells capable of expressing high levels of the transgene. Our data represents the first description of an enhancer that integrates information necessary for expression in developing endothelium and early haematopoietic progenitors at distinct times and sites throughout ontogeny. This enhancer provides a potent tool for the manipulation of haematopoiesis and vasculogenesis in vivo.

Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites.

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/ aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1+ cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization.

The Mouse GATA-2 Gene is Expressed in the Para-Aortic Splanchnopleura and Aorta-Gonads and Mesonephros Region

AbstractWe previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2–expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5′ end of the IS first exon direct expression of β-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5′ end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5′ to the IS exon.

The Mouse GATA-2 Gene is Expressed in the Para-Aortic Splanchnopleura and Aorta-Gonads and Mesonephros Region

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2–expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5′ end of the IS first exon direct expression of β-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5′ end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5′ to the IS exon.

Differential expression of KDR/VEGFR-2 and CD34 during mesoderm development of the early human embryo

Recent findings on vertebrate embryos have provided compelling evidence for the existence of hemangioblasts, i.e. common precursors for endothelial and hematopoietic cells, characterized by expression of the VEGFR2/Flk1 receptor. We describe here a population of KDR + CD34 − mesoderm cells that emerges in early-somitic human embryos, by the beginning of the 4th week of gestation. In the developing blood vessels, KDR-expressing CD34 − cells gradually coexpress increasing levels of CD34 antigen. Remarkably, as development proceeds, a KDR + CD34 − contingent persists in the paraaortic splanchnopleura until just prior to the emergence of aorta-associated hematopoietic cell clusters. These observations suggest that KDR + CD34 − mesodermal cells might represent the putative hemangioblastic precursor of human hematopoietic and endothelial lineages.

Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo.

Hepatic hematopoiesis in the mouse embryo is preceded by two hematopoietic waves, one in the yolk sac, and the other in the paraaortic splanchnopleura, the presumptive aorta-gonad-mesonephros region that gives rise to prenatal and postnatal blood stem cells. An homologous intraembryonic site of stem cell emergence was previously identified at 5 weeks of human gestation, when hundreds of CD34(++ )Lin- high-proliferative potential hematopoietic cells border the aortic endothelium in the preumbilical region. In the present study, we have combined immunohistochemistry, semithin section histology, fluorescence-activated cell sorting and blood cell culture in an integrated study of incipient hematopoiesis in the human yolk sac, truncal arteries and embryonic liver from 21 to 58 days of development. The chronology of blood precursor cell emergence in these distinct tissues suggests a pivotal role in the settlement of liver hematopoiesis of endothelium-associated stem cell clusters, which emerge not only in the dorsal aorta but also in the vitelline artery. Anatomic features and in vitro functionality indicate that stem cells develop intrinsically to embryonic artery walls from a presumptive territory whose blood-forming potential exists from at least 24 days of gestation.