The embryonic origins of human haematopoiesis (2001)

Mammalian haematopoiesis can be subdivided into two main systems, the embryonic primitive system and the definitive system. During primitive haematopoiesis, nucleated erythroid cells arise in the extraembryonic yolk sac and circulate through the embryo supplying oxygen and nutrients to the developing tissues. As the embryo develops, this early circulation is superseded by the definitive multilineage blood system that seeds the liver, bone marrow and other haematopoietic tissues. Definitive haematopoiesis is sustained by pluripotent haematopoietic stem cells (HSCs) which have the capacity to self-renew and are capable of long-term repopulation in myeloablated recipients. However, HSCs exist only in small numbers in human bone marrow and peripheral blood. Consequently, their usefulness for clinical application is dependent upon recreating the appropriate microenvironment which will allow expansion of HSC in vitro while retaining their pluripotent phenotype and repopulating capacity. An understanding of the embryonic origin of long-term repopulating (LTR)-HSCs and the factors that regulate their development in vivo will provide invaluable information to facilitate their expansion in culture. As a consequence, the study of human embryonic haematopoiesis has stimulated a considerable amount of interest and research in recent years. In this article, we will describe what is currently known about the origin of definitive HSCs and the questions these findings have raised concerning their development. We will examine evidence from animal models which has provided many clues as to the regulatory factors which may be involved and relate these to recent findings in the human embryo. Finally, we will discuss possible mechanisms for HSC ontogenesis suggested by these data. Previously, it was thought that the embryonic liver became colonized by stem cells derived from the extraembryonic yolk sac blood islands and that these cells seeded the definitive haematopoietic system. However, experiments in which quail embryos were grafted onto chick yolk sac revealed that the major source of definitive HSCs was contained within the embryo itself (Dieterlen-Lievre, 1975). The subsequent detection of a territory with haematopoietic potential in the wall of the chick dorsal aorta suggested that definitive HSCs might therefore arise in this region (Cormier et al, 1986). In the mouse, an analogous intraembryonic region, termed the aorta–gonad–mesonephros (AGM), is derived from the para-aortic splanchnopleural (P-Sp) mesoderm and contains the dorsal aorta, putative gonadal ridge and mesonephros. At 7·5 d post-coitum (DPC), before circulation is established between the embryo and yolk sac, multipotent stem cells capable of lymphoid and myeloid differentiation are found in the P-Sp region surrounding the dorsal aorta but not in the yolk sac. Furthermore, at 10 DPC this region contains spleen colony-forming unit (CFU-S) activity at a frequency greater than yolk sac or liver (Medvinsky et al, 1993, 1996; Dzierzak & Medvinsky, 1995). Together, these findings suggest that HSC within the P-Sp-derived AGM at 10 DPC are not derived from circulating yolk sac progenitors, but are generated in situ (Cumano et al, 1996). Isolated AGM cultured in vitro also reveals this region to be a rich source of HSCs which arise autonomously and independently from the yolk sac and liver and which are capable of reconstituting irradiated recipient mice before the onset of liver haematopoiesis (Muller et al, 1994; Medvinsky & Dzierzak, 1996; Sanchez et al, 1996). It is thought that it is these AGM-derived HSCs which migrate to the embryonic liver and seed the definitive blood system. The contribution of yolk sac cells to the mammalian definitive system is still under discussion but, from the studies described above, appears to be minimal. Long-term multilineage repopulation by cells isolated from murine yolk sac at 9 DPC has been reported in newborn mice (Yoder et al, 1997); however, by this time circulation is established and it is possible that these LTR-HSCs are in fact AGM derived. In the human embryo, pluripotent haematopoietic progenitors with high-proliferative capacity also arise almost exclusively from non-hepatic intraembryonic tissue between 25 and 35 d gestation (Huyhn et al, 1995). Cells with haematopoietic potential within the AGM were first identified as clusters within the mesoderm underlying and adhering to the ventral floor of the chick dorsal aorta (Cormier et al, 1986). Subsequently, examination of tissue sections through human (Tavian et al, 1996; Marshall et al, 1999) and murine (Wood et al, 1997) embryos has also revealed clusters of round cells adhering to the ventral floor of the dorsal aorta within the AGM, although no clusters have thus far been detected in the underlying mesoderm. Similar adherent clusters of cells are also found in the proximal umbilical and vitteline arteries, which interconnect with the dorsal aorta (Wood et al, 1997; Tavian et al, 1999). The clear similarities of distribution between species suggest that intraembryonic haematopoiesis is a common developmental process. In common with endothelial cells lining the dorsal aorta, cells within the clusters express the membrane glycoprotein CD34 (Fig 1) (Tavian et al, 1996; Wood et al, 1997; Garcia-Porrero et al, 1998; Marshall et al, 1999). They also express the haematopoietic-specific marker CD45, which distinguishes them from adjacent CD34+/CD45− endothelial cells. Their appearance is highly restricted, both spatially and temporally, during embryogenesis. In the human, an accumulation of around 800 cells develops in the preumbilical dorsal aorta, often around the level of the upper limb bud between 28 and 38 d gestation (comparable with 9·5–11·5 DPC in the mouse). It is now widely accepted that the first definitive multipotent HSCs that seed the adult blood system are generated within the embryonic AGM region and, most likely, these stem cells are contained within these intravascular clusters from where they migrate to and colonize the fetal liver. (A) A 34-d human embryo showing heart (h), liver (l) and anterior limb bud (al). The aorta–gonad–mesonephros (AGM) region extends from the umbilicus to the anterior limb bud as indicated (agm) and contains the dorsal aorta (da). (B) Transverse section through the AGM region outlined in (A) showing the dorsal aorta (da) and surrounding mesenchyme, stained with a monoclonal antibody to the membrane glycoprotein CD34 (brown cells). A cluster of CD34+ haematopoietic cells (h) are associated with the ventral floor of the aorta within this region, in contact with endothelial cells (e) lining the wall of the aorta which also express CD34. (For immunohistochemistry method, see Marshall et al, 1999). Original magnification 39×. Analysis of the human and murine embryonic AGM has revealed that cells within the intra-aortic clusters express many markers known to be associated with haematopoiesis, including the stem cell factor receptor c-Kit and the transcriptional regulators GATA-2, acute myeloid leukaemia (AML)-1 and stem cell leukaemia (SCL) (Table I). Gene disruption studies have shown that GATA-2 and AML-1/Cbfa2 are essential for definitive haematopoiesis (Table II). GATA-2-null embryos survive up to E11·5, the onset of liver haematopoiesis, complicit with a critical role in definitive but not primitive haematopoiesis (Tsai et al, 1994). Disruption of AML-1/Cbfa2 expression also results in a block in all definitive lineages with failure of fetal liver haematopoiesis and embryonic death by E12·5 (Castilla et al, 1996; Okuda et al, 1996; Wang et al, 1996). In contrast, the T-cell leukaemia oncoprotein tal-1/SCL is essential for both primitive (yolk sac) and definitive (intraembryonic) blood formation. Disruption of the SCL gene in transgenic mice results in a complete lack of blood formation in SCL–/– embryos and is lethal between E8·5 and E10·5 at the commencement of circulation (Shivdasani et al, 1995). Moreover, the failure of SCL–/– murine embryonic stem cells to to or haematopoietic cells of that SCL may be involved in blood at a cell et al, 1996). the expression of early haematopoietic and has been in AGM cells. The of this isolated from AGM the and early factors GATA-2, and et al, 1999). In the to human embryo, cells within the CD34+ intra-aortic clusters express GATA-2, and c-Kit et al, 1996; et al, 1998; Marshall et al, 1999). They also express a of involved in and including et al, and et al, An from this is the of both by haematopoietic clusters within the clusters and by endothelial cells lining the aorta wall with which they are associated (Table I). it has been that both haematopoietic and endothelial cells may from a common the derived from are capable of to both lineages in vitro et al, however, the of the putative in vivo has marker for the is the receptor for endothelial factor also known as and is by endothelial cells and is essential for formation. In blood islands to in the yolk sac, not and haematopoietic progenitors only et al, 1997) (Table II). However, haematopoiesis in the of be a from the lack of a than to the of a cell In murine cells both haematopoietic and endothelial potential et al, and cells from early chick before are to both haematopoietic and endothelial cells et al, In this endothelial development the of is although it In with within the chick embryonic AGM, cells in the floor of the aorta to a from to as they haematopoietic et al, the human of is in of CD34+ blood cells experiments in mice have that all pluripotent LTR-HSCs are contained within the blood et al, 1999). The that expression is an early in haematopoietic human peripheral blood cells can also an adherent endothelial phenotype in et al, and the embryonic may cells with In the human embryonic AGM, is both endothelial cells lining the wall of the dorsal aorta and haematopoietic cells within the associated intra-aortic clusters et al, 1998; Marshall et al, 1999). the that haematopoietic clusters within the AGM are pluripotent stem cells that have from an The by which haematopoietic might arise has been a of considerable interest and It has been suggested that migrate into the wall of the dorsal aorta from the underlying mesenchyme, only to or other cells within the floor of the aorta to a haematopoietic in to a and A is that endothelial under are capable of and can to a haematopoietic The dorsal aorta is by the of from the of cells into an endothelial that the is established before intra-aortic definitive It therefore that this cell is within the endothelial wall at the time of However, it be that cells with haematopoietic potential from the mesoderm and into the at between formation and the appearance of It has also been suggested that in the ventral endothelial underlying intra-aortic through which haematopoietic cells migrate from the mesoderm of endothelial cell (Tavian et al, 1999). However, examination of tissue sections through the DPC murine AGM that the of the where it is associated with the is (Fig the adjacent endothelial cells are to other and to haematopoietic cells within the cluster by (Fig cells in the region, the are also by However, to be no between these two The and dorsal of the aorta are by a of endothelial cells (Fig In contrast, within the ventral floor of the aorta adjacent to the clusters and with the cells are into the of these cells to where they to the or to adjacent cells and to be from the wall (Fig the itself has the appearance of these cells in the floor of the aorta may therefore be to this activity than to the of the endothelial of the dorsal aorta in the murine AGM region at DPC showing the endothelial and underlying (A) floor of aorta adjacent to the haematopoietic cells within the endothelial are to other but not to cells in the underlying the and mesenchyme, a of is of at magnification showing (B) haematopoietic cells (h) within the cluster into the of the dorsal aorta by Cells are of the ventral endothelial an cells and to be from the the endothelial is of a of cells from a by nucleated primitive erythroid cells are found circulating in the of the cells underlying the ventral floor of the aorta are (A) than around the and dorsal Original is evidence that the factor may a role in the of haematopoietic cells of the and it has been suggested that it may also as a the from an endothelial to a haematopoietic et al, 1999). of into of the murine gene reveals that is by a of endothelial cells to of haematopoietic in the yolk sac, and umbilical and in the ventral floor of the dorsal aorta et al, 1999). In the aorta, expression is and at DPC before the of haematopoietic cells. Moreover, in embryos intra-aortic clusters to and, although extraembryonic primitive erythroid cells of haematopoietic cells from the of the yolk sac is expression may therefore an between the and to the endothelial or haematopoietic for the of intra-aortic from or within the is provided by the expression of the receptor and of the is endothelial cells at the level of the putative The of cells within the AGM within the and can to both endothelial and multipotent HSCs in et al, 1998; et al, 1999). role in haematopoiesis is thought to be the and of HSCs to endothelial thus their embryos have definitive haematopoiesis, to a failure of stem cell and expansion within the dorsal cells but death around DPC as a of in and (Table II). is also endothelial cells and is for cell and but for early endothelial differentiation or formation. expression a but before or from and to of blood cells through the et al, et al, (Table II). Analysis of both human and bone marrow that is also early multipotent CD34+ haematopoietic stem cells including long-term cells and LTR-HSCs et al, 1996; et al, expression is as the cells and are essential for the in haematopoiesis in embryos be to the of a than of a the other is not for formation it to a role in The of AGM haematopoietic development is therefore in and expression in CD34+ haematopoietic cells therefore a common A murine in which expression is to activity has the between endothelial and haematopoietic cells within the AGM to be At expression is between cells in the floor of the aorta and the associated haematopoietic cells. no cells are detected in the underlying mesoderm (Fig of the receptor in the murine embryonic AGM region at of the gene to results in of cells. (A) expression is to endothelial cells lining including the dorsal aorta (da) and (B) magnification of of is between the (e) and cells within the adherent haematopoietic cluster associated with the ventral floor of the aorta (h) a common Original (A) (B) 39×. In the chick embryo, of cells within the ventral mesoderm underlying the aorta and associated with the appearance of have been as putative of the to the et al, a of the expression of and in murine embryonic tissue has to the that these two factors may within the murine AGM and be involved in the of intraembryonic haematopoietic et al, is also found in cells the floor in the human AGM at et al, However, the between expression and haematopoietic is and, in the human, expression of known early haematopoietic as and not to with in this At it most that intra-aortic haematopoietic clusters are of cells from the floor of the aorta than cells through the wall from the underlying mesoderm. mechanisms this may the of endothelial cells the of within the wall of the The clusters of haematopoietic cells in the dorsal aorta are associated with the ventral an the A examination of the surrounding tissue in tissue sections A region of of between and in the human and of or in the mouse, the ventral wall et al, 1999). In contrast, cells surrounding the and dorsal of the aorta are is to the reported of cells around the quail embryonic aorta et al, 1996). of around is a critical in the wall and from ventral to in a of the ventral The of this region in AGM haematopoiesis is revealed by the expression of factors and other associated with the appearance of is by cells in human bone marrow et al, In the human embryonic AGM at d the of a cells are associated with the ventral wall of the dorsal aorta (Tavian et al, 1999). At a of cells the ventral wall associated with intra-aortic haematopoietic clusters with only a at the dorsal The that this ventral region a haematopoietic activity in the AGM is by the expression of and a of the murine cell is CD34+ HSCs and cells in haematopoietic and is to In the to human AGM, a small of cells the wall and are to the ventral region et al, 1999). An of expression is by the the human embryonic dorsal aorta at expression of is in the ventral region the endothelial wall and associated with the of intra-aortic haematopoietic clusters et al, 1999). At expression in this appears than in the surrounding During the cell by cell and at the in and et al, et al, is in murine bone cells et al, and although mice to have haematopoiesis the capacity of bone marrow cells derived from these mice to haematopoiesis is et al, The expression of in the ventral wall of the dorsal aorta that it may be involved in haematopoietic within the in the of factor an embryonic cell is by in the of factors or can cells to or are by a of and, by factors which gene expression in the models have shown that a of factors are for haematopoiesis, but their is As human embryonic tissue is and be in of the information we currently have haematopoietic development and from studies in other exist between and many and at the level are The and the numbers of embryos which the and have invaluable models in which to study the factors that blood cell formation. In primitive blood cells are in ventral blood to the mammalian yolk sac, and multilineage definitive blood cells are generated in dorsal analogous to the AGM The factor SCL is in these early in embryogenesis. SCL expression in animal which not to that SCL is to a haematopoietic & 1996; et al, In expression of SCL results in a in the of haematopoietic and endothelial et al, cells SCL expression endothelial including GATA-2 and but to express et al, 1997; et al, suggest that SCL is involved in the of to a haematopoietic and also in the of from cells. In SCL expression is by bone et al, a of the factor of factors which have a formation and cell 1996). In the mouse, between and is in the and and is essential at a of and in a of during development. In early it is also for mesoderm and mice expression are at the with or no mesoderm et al, 1995). embryos developing this have and an essential role for in and haematopoietic However, of the major from gene the of in the of intraembryonic haematopoiesis has been to study in these formation which are detected by cells. The of these appears to be to or of by as and than by & The of are and can or gene In with this of has been shown to expression in cultured murine cells in a & 1995). in this will also expression of but at a than that to blood cell formation. A is found in established At CD34+ stem cells to and at the capacity and stem cell phenotype is et al, 1999). of the in in vivo in the embryo, information about the of has from studies in The transcriptional regulators GATA-2 and SCL have been shown to be essential for definitive cells and but not to the blood of into cells results in GATA-2 expression and of expression of a receptor in ventral mesoderm GATA-2 et al, 1996). In cells to to the blood mesoderm has of SCL & 1996). in receptor is for haematopoietic from cells. It has been suggested that the role of in haematopoietic may be to the by gene in with other as factor and et al, ventral blood formation and GATA-2 of embryonic into ventral tissue appears to have the these et al, 1999). Moreover, expression of a receptor in which not blood results in a expansion of the expression of a which blood development. of these is that of may regulate of GATA-2 to allow expansion of the role for is by studies in murine cells. As cultured cells can to colony-forming which are thought to the and express cells in which the receptor gene has been and haematopoietic with et al, generated from express of but have c-Kit and of during differentiation the of and cells an which is by the of the other cells but not or haematopoietic cells. As cell differentiation is analogous to early embryonic it that is for the and the of and that SCL expression is for the from to haematopoietic It be that and not to haematopoietic activity in in the of in the haematopoietic & 1995). of the is a of blood cell in In the cell of LTR-HSCs et al, 1996). However, the of in vivo are to in the murine or receptor in embryonic death at DPC from as a of yolk sac and haematopoiesis, not endothelial and haematopoietic cell as might be et al, et al, 1996). During appears to regulate extraembryonic endothelial differentiation and primitive blood formation. role in intraembryonic haematopoiesis has not been in the it has been shown that the and of the dorsal aorta are derived from the mesoderm and endothelial cells which lack haematopoietic the ventral floor of cells with et al, 1996). experiments the of quail mesoderm onto chick have shown that potential can be mesoderm contact with or with for or & 1999). contact with potential in that a and a the and endothelial potential of cells. In the murine AGM, expression of in cells within the ventral floor of the aorta but not in the and also to between two of endothelial cells with et al, 1999). In with a role in ventral of the 34-d human embryo reveals a of expression the within the AGM is the ventral which contains the dorsal aorta, but is within the dorsal mesoderm et al, the aorta, expression is to the ventral floor within the underlying the intra-aortic haematopoietic At 28 clusters are to this to the floor is as the has not been 38 haematopoietic clusters have expression is around the expression is highly of a role for in the of haematopoiesis within the embryonic haematopoietic development in a as is suggested by cell a of in the ventral floor of the aorta haematopoietic activity is to this region and not detected around the and is also in cells surrounding blood islands in the human yolk sac and may also be involved in the of primitive blood cells. In to expression is around the dorsal aorta in the 34-d human AGM with no to the ventral However, haematopoietic cells within the intra-aortic cluster at than the adjacent et al, It has to be is involved in the of generated haematopoietic cells within the embryo, a role which it appears to in the extraembryonic or it of these cells within the aorta, a primitive before their to the fetal liver. During or are involved in the of a of of the and in a of including bud and In the developing murine expression in of bud formation and and of the et al, a of expression in the which to the of the and In this and regulate bud in a and to the In a and to regulate the of in the developing expression of the factor which the of formation and may a role in primitive et al, of is by and the of expression and therefore development. these has an tissue in the developing and to and to regulate cell in and can be into main which of of the and of and their to a and cell During cells express the receptor which the with and, in the of cells from to differentiation & 1995). of the including are thought to be involved in the differentiation of is during this and it may by as and receptor et al, 1995). and are also during expression in with is associated with of and expression during the differentiation & 1995). from studies in that of the haematopoiesis regulators SCL and of the of factors have also been to the is for differentiation during bone formation et al, In vitro of a expression of in both and cell et al, of a receptor expression and differentiation in cultured murine cells et al, can also and expression of in which to and expression of early that expression may a to an et al, 1999). In the of these the expression of in the floor of the dorsal aorta in with the of to this region is highly of a between and in haematopoietic and in the embryonic AGM (Fig A reported expression and gene disruption studies described in the for the of haematopoietic cells from embryonic mesoderm within the mammalian AGM (A) and the of transcriptional regulators have been in other systems, including bone in the of cells to a and are in before and during the formation of intra-aortic haematopoietic clusters and may a critical role in embryonic haematopoietic (B) and may to mesoderm (P-Sp) to and to the before may P-Sp-derived or endothelial in the floor of the dorsal aorta a of haematopoietic gene expression including The expression of and or may or an endothelial or haematopoietic expression in the AGM and the described above, we have a for and in the from through to haematopoietic in the human intraembryonic AGM (Fig is involved early in the of mesoderm and may with to cells to and to the before A of or with other a of haematopoietic gene including in or endothelial in the floor of the dorsal The or of and transcriptional regulators from to cell (Fig In the of and the expression of may or an endothelial or haematopoietic cells in the and of the dorsal aorta may be derived from than splanchnopleural mesoderm and may lack potential from the or developmental The role of intraembryonic haematopoiesis in the of a definitive blood system has and now appears to be a common developmental many However, the of haematopoietic from the and of a of gene expression within a In the may be and may be dependent of as and or of a may of and or of an The of intraembryonic HSCs is therefore to be a the of a of factors cells. The of that regulate haematopoietic cell and the of will to the involved in intraembryonic blood formation. An understanding of these will the development of for vivo expansion of human HSCs and, their usefulness in clinical The to the for and to and the for in of this in the for of human embryonic tissue through the and at the of for of for of the