First Printed in the R&D Systems 2003 Catalog
Within all adult animal systems, tissues and organs are comprised of specialized cells originally derived from pluripotent stem cells during embryonic development.
||Figure 1. The first definitive multipotent hematopoietic stem cells (HSCs) are generated within the embryonic aorta-gonad-mesonephros (AGM) region. The AGM extends from the umbilicus to the anterior limb bud of the human embryo and contains the dorsal aorta. Within the dorsal aorta, a cluster of CD34+ hematopoietic cells is associated with the ventral floor of the aorta.
Stem cells are defined by their potential to give rise to all differentiated cell types within a tissue and also by the ability to self-renew. For this reason, the use of stem cells in the treatment of genetic and degenerative diseases, either by ex vivo modification of defective genes or the regeneration of damaged tissues by transplantation, has enormous therapeutic potential. This type of therapy is particularly applicable to the hematopoietic system in which a number of immune diseases arising from single gene defects have been identified.
The utilization of stem cells as therapeutic tools, however, has thus far proved problematic. Although stem cells have been shown to exist in many adult tissues, they are normally quiescent. In adult tissues, truly pluripotent stem cells are extremely rare and in culture, in the absence of regulatory signals such as those that occur during embryogenesis, tend to lose their capacity to self-renew and become lineage-restricted. Consequently, their effectiveness in the treatment of disease would be greatly increased if we could recreate in vitro the appropriate microenvironment to allow expansion of isolated stem cells whilst retaining their pluripotency and ability to repopulate. Unfortunately, our current understanding of the growth factors and cytokines that regulate stem cell generation and maintenance is very limited. It is likely to involve the complex interplay of a number of different molecules. The study of embryonic stem cells has therefore stimulated a considerable amount of interest and research in recent years.
The following mini-review will focus on the mammalian embryonic hematopoietic system: describing how the hematopoietic hierarchy is created from stem cells, where adult-type stem cells are initially generated during embryogenesis and how analysis of these embryonic sites has provided key information as to the factors involved in stem cell regulation. What has emerged from these studies is the existence of a highly regulated microenvironment, or niche, which ensures that pluripotent stem cells are generated in sufficient numbers and at an appropriate developmental stage to seed subsequent hematopoietic tissues.
The Hematopoietic Hierarchy
Hematopoiesis is the process by which all the different cell lineages that form the blood and immune system are generated from a common pluripotent stem cell. During the life of an individual, two separate hematopoietic systems exist, both arising during embryonic development but only one persisting in the adult. The primitive system is derived from the extraembryonic yolk sac and consists mainly of nucleated erythroid cells, which carry oxygen to the developing embryonic tissues. As the embryo increases in size, this early circulatory system is superseded by the more complex definitive system, which originates within the embryo itself and continues throughout adult life. This definitive hematopoietic system is made up of all adult blood cell types including erythrocytes and cells of the myeloid and lymphoid lineages. All these cells are derived from pluripotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential under the control of specific cytokines such as interleukins and granulocyte/monocyte-stimulating factors. In most cases, the cytokines that determine differentiation to a particular lineage are well defined. The factors that regulate HSC generation and maintenance of pluripotency, however, remain largely unknown.
The Primary Site of Definitive HSC Emergence in the Embryo: the AGM Region
The adult hematopoietic tissues (i.e. bone marrow, thymus and spleen) are seeded by multilineage blood cells derived from the fetal liver. During embryogenesis, however, fetal liver hematopoiesis is preceded by the emergence of pluripotent HSCs in a region of the para-aortic splanchnopleural mesoderm containing the dorsal aorta, gonadal ridge and mesonephros, named the aorta-gonad-mesonephros or AGM region (Figure 1). In vitro studies and repopulation analyses in myeloablated recipient mice have established this region as a major source of long-term repopulating (LTR)-HSCs between 8.5-11.5 days post coitum (dpc) in the mouse and 4-6 weeks gestation in the human embryo, prior to the onset of liver hematopoiesis.1, 2, 3, 4, 5, 6 Coincident with this period of LTR-HSC activity, clusters of rounded cells adhering to the ventral wall of the dorsal aorta and the umbilical and vitelline arteries (where they connect with the dorsal aorta) have been identified in both human and murine AGM regions in vivo.5, 7 Expression analysis of these cell clusters reveals that they express the hematopoietic-specific marker CD45 as well as a number of markers in common with adjacent endothelial cells including the membrane glycoprotein CD34, which is commonly used to identify HSCs in bone marrow and peripheral blood.8 CD34-positive cells isolated from murine AGM at 10.5 dpc can give rise to cells of all hematopoietic lineages in vitro.9 It is now widely accepted that these intra-aortic cell clusters constitute the first site of definitive HSC generation during development and represent the origins of adult hematopoiesis.
The source of these first HSCs and the mechanisms by which their tightly regulated appearance and subsequent disappearance are controlled are currently under investigation. The shared expression patterns of a number of molecules by both intra-aortic cluster cells and underlying endothelial cells supports the existence of a hemangioblast or endothelial-like cell with hemogenic potential that resides within the ventral floor of the dorsal aorta.8 Within the embryonic AGM region, the eventual fate of these hemangioblasts would be determined by factors that bind at the cell surface, triggering downstream signaling pathways that culminate in the activation of hematopoietic or other lineage-specific genes. The identification of a morphologically distinct region of cells, resembling a stromal layer, underlying the ventral floor of the dorsal aorta within the AGM has suggested that this region could represent a microenvironment, or niche, supporting HSC development.10 This highly defined ventral region, coupled with the absence of contaminating committed hematopoietic progenitors within the AGM at this stage of development, provides an ideal environment in which to investigate the factors involved in the generation and regulation of HSCs in vivo.
Identification of Candidate Factors Regulating Embryonic HSC Development
Studies using transgenic mouse models, in which specific genes have been deleted, have shown that a number of transcription factors are essential for specification of the hematopoietic program and for normal hematopoietic development during embryogenesis. For example, stem cell leukemia (SCL) deficiency results in a complete failure of both primitive (yolk sac) and definitive (intra-embryonic) hematopoiesis leading to embryonic death as early as 8.5 dpc, at the onset of circulation.11 In contrast, disruption of the GATA-2 gene appears mainly to affect the development of definitive blood cells resulting in failure of fetal liver hematopoiesis and embryonic death at around 11.5 dpc.12
Within the human embryonic AGM region, SCL and GATA-2 are expressed in both endothelial cells lining the dorsal aorta and in the associated hematopoietic clusters.10 Studies in mouse embryos show that expression of transcription factor AML-1 (Runx1/Cbfa2) is restricted to cells within the intra-aortic clusters and, immediately preceding cluster emergence, to a subset of cells located within the ventral floor of the dorsal aorta.13 Moreover, adherent intra-aortic HSC clusters fail to appear in AML-1-deficient embryos suggesting that AML-1 may play a role in cluster formation, or possibly in the specification of HSCs from a pre-hematopoietic cell. A number of other hematopoietic- associated transcription factors, including c-myb, GATA-3 and Lmo-2, are also expressed within the human AGM region and further studies may reveal roles for these factors in embryonic HSC emergence.8
Since transcription factors are under the control of signaling pathways that begin with the binding of extracellular ligands to cell surface receptors, identification of the upstream growth factors involved in their induction may provide clues as to how and from which precursor cells HSCs are generated. One possible candidate is vascular endothelial growth factor (VEGF). VEGF receptor 2 (VEGF R2, also known as Flk-1 in the mouse and KDR in the human) is required for endothelial development and the number of hematopoietic progenitors is dramatically reduced in Flk-1-deficient embryos.14 In human postnatal hematopoietic tissues, VEGF R2 is expressed on all postnatal pluripotent LTR-HSCs within the CD34+ blood fraction.15 VEGF R2 is also expressed on the surface of both aortic endothelial cells and hematopoietic cells within the intra-aortic clusters in the human embryonic AGM region.10 VEGF R2 expression appears to be down-regulated at the RNA level within the AGM clusters, however, as cells move away from the ventral aortic floor VEGF R2 expression disappears as postnatal bone marrow-derived cells become lineage-committed.15, 16 Studies in the chick embryo suggest that VEGF signaling is required for endothelial cell development but that HSC specification is VEGF-independent.17 It therefore seems likely that VEGF is involved at a pre-hematopoietic stage, but not directly in HSC formation.
Receptors for the cytokines stem cell factor (SCF) and flt-3 ligand (flt-3L), c-kit/SCF R and flt-3/stem cell tyrosine kinase-1 (STK-1) respectively, are also expressed on the surface of intra-aortic cluster cells in the human embryonic AGM region.10, 18 In established hematopoiesis, SCF and flt-3L are thought to synergize in HSC regulation and proliferation and are routinely used in cultures of CD34+ cells isolated from human blood and bone marrow.19 In embryonic hematopoiesis, however, SCF alone is insufficient to maintain HSC pluripotency in extended AGM-derived cultures and would therefore appear to play a role in stem cell development rather than generation or maintenance (Marshall, unpublished).
Macrophage colony stimulating factor (M-CSF) may also play a role in the development of HSC precursors within the embryonic AGM region. In cultures of cells derived from the AGM of murine embryos in which the gene encoding M-CSF has been deleted, immature hematopoietic progenitors appear to accumulate coincident with a reduction in the number of cells expressing endothelial markers including Flk-1.20 This data suggests that M-CSF may negatively regulate hematopoiesis by promoting endothelial differentiation from hemangioblast precursors. By implication, cultures of hemangioblasts could potentially be pushed towards the hematopoietic lineage via the suppression of M-CSF. Aside from its established role in macrophage development, however, the involvement of M-CSF in embryonic hematopoiesis and HSC regulation in vivo has yet to be fully investigated.
Two very important families of morphogens involved in the formation of a variety of tissues at various stages of embryogenesis are the fibroblast growth factors (FGFs) and transforming growth factors (TGFs). These molecules exert their influence on cell fate specification and maintenance through the establishment of concentration gradients in conjunction with antagonistic factors.21 Considerable information on the possible role of these growth factors in hematopoietic development has come from studies on amphibian Xenopus embryos and murine embryonic stem (ES) cells, which differentiate to form embryoid bodies. In Xenopus, definitive blood cells are generated in a region of embryonic mesoderm analogous to the mammalian AGM region, the dorsal lateral plate. The transcription factor SCL is normally expressed in this region prior to blood cell development. Ectopic SCL expression and hematopoietic specification can be induced in regions that do not normally give rise to blood cells by a member of the TGF family, bone morphogenetic protein-4 (BMP-4).22, 23 This potential role for BMP-4 in hematopoietic specification is supported by studies using murine ES cell cultures in which globin expression is induced by BMP-4 but not by other TGF or FGF family members.24 BMP-4 can also influence the development of pre-existing LTR-HSCs derived from human cord blood. The addition of BMP-4 at low concentrations to cultures of these cells appears to have little effect. The stem cells proliferate but become lineage-restricted over time. Higher concentrations of BMP-4, however, appear to prolong the long-term repopulating capacity of HSCs in culture.25 Interestingly, during human embryogenesis, BMP-4 is expressed at high concentrations within the stromal layer underlying the ventral floor of the dorsal aorta coincident with the appearance of intra-aortic hematopoietic clusters.26 In contrast, expression of a related family member, TGF-beta 1, within the human AGM is restricted to the hematopoietic cluster.26 TGF-beta 1 is a potent inhibitor of murine definitive LTR-HSC proliferation in vitro.27 Its role in intra-embryonic hematopoiesis, however, remains unclear.
The story for FGF is rather more complicated. Embryoid bodies generated from murine ES cells that lack a functional FGF receptor-1 gene have reduced expression of SCL, VEGF R2/Flk-1, c-kit/SCF R and globin genes and produce fewer hematopoietic colonies compared to wild-type cells.28 Addition of FGF basic to cultures of differentiating ES cells increases the number of hemangioblast-like cells and VEGF R2/Flk-1+ cells suggestive of a role in pre-hematopoietic cell formation rather than HSC induction.
Within the context of a supportive microenvironment, cell adhesion and extracellular matrix molecules also play an important role in HSC development. Within the adult bone marrow, these molecules mediate interactions between stromal and hematopoietic cell components that in turn regulate cell proliferation, differentiation and migration. A number of cell adhesion and extracellular matrix molecules are expressed in distinct spatial and temporal patterns within the embryonic AGM, thus suggesting that they are involved at distinct stages during HSC ontogenesis. For example, the vascular adhesion molecules VCAM-1 and VE-cadherin are expressed both on endothelial cells lining the dorsal aorta and on cells within the associated hematopoietic clusters within the human embryonic AGM (Marshall unpublished). Expression of the hematopoietic cell adhesion molecule HCAM/CD44 is restricted to cells within the intra-aortic clusters.29 In contrast, hematopoietic cell antigen (HCA/ALCAM/CD166), which is expressed on the primitive CD34+ hematopoietic cell population in fetal liver and bone marrow, is mainly concentrated within the ventral stromal region underlying the hematopoietic clusters.30 The extracellular matrix molecule tenascin-C, which is associated with cell proliferation and transformation in a number of embryonic and adult systems, is also expressed in a distinctive pattern in this region, specifically associated with the presence of adherent hematopoietic cell clusters.8, 10 These diverse expression patterns suggest that a variety of extracellular ancillary molecules may be involved in facilitating cell-cell and cell-cytokine communication in the generation and maintenance of HSCs.
Mechanisms of HSC Ontogenesis: How Do the Regulators Work?
One feature that has become clear through all the various studies into in vivo expression patterns and in vitro effects is that the determination of cell fate is controlled by a great many intrinsic and extrinsic factors. Moreover, cell lineage commitment is likely to involve not only the activation of specific permissive pathways but also the suppression of inhibitory signals. While a number of factors can influence hematopoietic development in vitro, deciphering which are actually involved and their precise roles in HSC regulation requires an understanding of how they connect with appropriate transcriptional regulators, such as SCL, GATA-2 and AML-1. Fortunately, parallels in other developmental systems and studies in lower animal models have provided some insight as to how the influence of key factors may result in activation of the hematopoietic program.
For example, during embryogenesis, members of the FGF and BMP families interact to influence cell fate determination in a number of systems including lung bud and tooth formation. During murine lung development, FGF-10 induces a gradient of BMP-4 expression. At high concentrations, BMP-4 inhibits FGF-induced cell proliferation, thus regulating distal branching of alveoli.31 Similarly, members of the BMP family initially inhibit the FGF-induced expression of transcription factors to restrict domains of tooth formation in the developing mandibular arch.32 Subsequently, BMP-4 has the opposite (inductive) effect on odontogenesis after positional demarcation has been established.
Studies in Xenopus suggest that FGF inhibits blood cell formation via the suppression of GATA-2 and the induction of another transcription factor PU.1.33 Paradoxically, in the murine ES cell system, in which hematopoietic development can be induced in an analogous sequence to that which occurs during embryogenesis in vivo, disruption of FGF signaling, instead of promoting blood formation as might be expected, appears to have the contrary effect of reducing hematopoietic potential.28 In the same system, addition of FGF results in an increase in the number of VEGF R2/Flk-1+ cells and hemangioblasts.
In contrast to FGF, addition of BMP-4 induces GATA-2 and SCL expression, globin synthesis and hematopoietic specification in Xenopus ectoderm-derived animal cap (AC) cells, which do not normally give rise to blood.22, 34 Members of the BMP family have also been linked with expression of AML transcription factors. AML-3/Cbfa1 is required for osteoblast differentiation during bone formation.35 In cultured murine calvariae cells, disruption of BMP receptor signaling blocks AML-3 expression and subsequent osteoblast differentiation.36 Muscle cell precursors can be induced to transiently express AML-3 and to divert to an osteogenic fate following treatment with exogenous BMP-2.37
||Figure 2. A potential model for the generation and maintenance of HSCs, under the regulation of specific factors in the AGM region, is depicted (note: this illustration is adapted from the model presented in Marshall, C.J. & A.J. Thrasher, 2001, British Journal of Haematology, 112:838). Members of the Hedgehog family of signaling molecules may influence HSC proliferation via BMP regulation. BMP-4 may play a role in patterning the mesoderm by influencing gene expression. FGF and activin may interact with BMP-4 to convert splanchnopleuric mesoderm to hemangioblasts, increasing the hemangioblast pool while preventing further lineage commitment. BMP-4 may subsequently act on hemangioblasts or endothelial intermediates, inducing transcription of hematopoietic-specific genes. Expression levels of VEGF R2 and SCL may influence an endothelial or hematopoietic cell fate. (For more details, please refer to the main text.)
Reviewing all these observations in the context of embryonic hematopoiesis, a possible model emerges for the generation and maintenance of HSC under the regulation of specific factors that concurs well with their observed expression patterns in the AGM region (see Figure 2). Prior to the onset of definitive hematopoiesis, FGF may act on naïve mesodermal cells to increase the hemangioblast pool whilst preventing further lineage commitment. Subsequently, a localized concentration of BMP expression within the stromal region ventral to the dorsal aorta induces expression of AML-1 in a small population of cells at the luminal surface. Under the influence of BMP, transcription of hematopoietic-specific genes by SCL and GATA-2 and possibly other transcription factors is induced, stabilized, or possibly de-repressed in this subpopulation. At the same time, acquisition of a hematopoietic phenotype is accompanied by a down-regulation of endothelial-specifying genes such as VEGF R2/Flk-1. As a consequence, an expanding number of nascent HSCs start to bud out from the aortic wall and form a cluster maintained by signals emanating from the underlying stroma or niche. During this process, stromal cell-stem cell interactions and ligand-receptor binding are facilitated by a variety of cell adhesion and extracellular matrix molecules. As cells within the developing cluster move away from this niche, further progression from stem cell to progenitor is inhibited by TGF-beta 1. Finally, as inductive stromal signals (e.g. BMP-4) decline, HSC generation ceases and the cluster is released into circulation to migrate to the fetal liver.
To date, the treatment of many diseases has been limited by difficulties in replacing damaged or defective tissues. The ability to maintain and expand stem cells in culture will potentially revolutionize current therapies. The identification of cytokines involved in these processes is central to the development of culture systems that will allow stem cells to be used safely and effectively in clinical applications. In the hematopoietic system, clues as to the factors that regulate HSC generation and development during embryogenesis are beginning to emerge and the use of new technologies, such as gene chip expression analysis, will increase our understanding of how pluripotency and self-renewal are maintained in these fascinating cells.
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