The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF

The differentiation of embryonic stem (ES) cells offers a powerful approach to study mechanisms implicated in cell fate decision. A major hurdle, however, is to promote the directed and efficient differentiation of ES cells toward a specific lineage. Here, we define in serum-free media the minimal factor requirement controlling each step of the differentiation process, resulting in the production of highly enriched hematopoietic progenitors. Four factors - Bmp4, activin A, bFGF (Fgf2) and VEGF (VegfA) - are sufficient to drive the selective and efficient differentiation of mouse ES cells to hematopoiesis. Each of these factors appears to regulate a step of the process: Bmp4 promotes the very efficient formation of mesoderm; bFGF and activin A induce the differentiation of these mesodermal precursors to the hemangioblast fate; and VEGF is required for the production of fully committed hematopoietic progenitors. The stimulation of mesodermal precursors by bFGF and activin A switches on very rapidly the hematopoietic program, allowing us to dissect the molecular events leading to the formation of the hemangioblast. Runx1, Scl (Tal1) and Hhex expression is upregulated within 3 hours of stimulation, whereas upregulation of Lmo2 and Fli1 is observed later. Interestingly, increased expression levels of genes such as cMyb, Pu.1 (Sfpi1), Gata1 and Gata2 are not observed at the onset of hemangioblast commitment. This stepwise control of differentiation is extremely efficient, giving rise to a very high frequency of hematopoietic precursors, and provides an optimal system for understanding the molecular machineries involved in blood progenitor commitment.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

Hemangioblast development and regulation.

Hematopoietic and endothelial cell lineages are the first to mature from mesoderm in the developing embryo. However, little is known about the molecular and (or) cellular events leading to hematopoietic commitment. The recent applications of technology utilizing gene targeted mice and the employment of many available in vitro systems have facilitated our understanding of hematopoietic establishment in the developing embryo. It is becoming clear that embryonic hematopoiesis occurs both in the extra-embryonic yolk sac and within the embryo proper in the mouse. The existence of the long pursued hemangioblast, a common progenitor of hematopoietic and endothelial cells, is now formally demonstrated. Based on this new information, many studies are being conducted to understand hematopoietic commitment events from mesoderm. In this review, we will first discuss the establishment of the hematopoietic system with special emphasis on the most primitive hematopoietic committed cells, the hemangioblast. We will then discuss mesoderm-inducing factors and their possible role in hematopoietic lineage commitment.     

Endoglin is required for hemangioblast and early hematopoietic development

Endoglin (ENG), an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, has a well-studied role in endothelial function. Here, we report that endoglin also plays an important role early in development at the level of the hemangioblast, an embryonic progenitor of the hematopoietic and endothelial lineages. Eng(-/-), Eng(+/-) and Eng(+/+) mouse embryonic stem (ES) cells were differentiated as embryoid bodies (EBs) and assayed for blast colony-forming cells (BL-CFCs). Our results showed a profound reduction in hemangioblast frequency in the absence of endoglin. Furthermore, cell-sorting experiments revealed that endoglin marks the hemangioblast on day 3 of EB differentiation. When analyzed for hematopoietic and endothelial activity, replated Eng(-/-) BL-CFCs presented limited hematopoietic potential, whereas endothelial differentiation was unaltered. Analysis of hematopoietic colony formation of EBs, at different time points, further supports a function for endoglin in early hematopoiesis. Taken together, these findings point to a role for endoglin in both hemangioblast specification and hematopoietic commitment.     

Differentiation of hemangioblasts from embryonic mesothelial cells? A model on the origin of the vertebrate cardiovascular system

The existence of the hemangioblast, a common progenitor of the endothelial and hematopoietic cell lineages, was proposed at the beginning of the century. Although recent findings seem to confirm its existence, it is still unknown when and how the hemangioblasts differentiate. We propose a hypothesis about the origin of hemangioblasts from the embryonic splanchnic mesothelium. The model is based on observations collected from the literature and from our own studies. These observations include: (1) the extensive population of the splanchnic mesoderm by mesothelial-derived cells coinciding with the emergence of the endothelial and hematopoietic progenitors; (2) the transient localization of cytokeratin, the main mesothelial intermediate filament protein, in some embryonic vessels and endothelial progenitors; (3) the possible origin of cardiac vessels from epicardial-derived cells; (4) the origin of endocardial cells from the splanchnic mesoderm when this mesoderm is an epithelium; (5) the evidence that mesothelial cells migrate to the hemogenic areas of the dorsal aorta. (6) Biochemical and antigenic similarities between mesothelial and endothelial cells. We suggest that the endothelium-lined vascular system arose as a specialization of the phylogenetically older coelomic cavities. The origin of the hematopoietic cells might be related to the differentiation, reported in some invertebrates, of coelomocytes from the coelomic epithelium. Some types of coelomocytes react against microbial invasion and other types transport respiratory pigments. We propose that this phylogenetic origin is recapitulated in the vertebrate ontogeny and explains the differentiation of endothelial and blood cells from a common mesothelial-derived progenitor.     

The hemangioblast--between blood and vessels

The hemangioblast is a bipotential cell that gives rise to hematopoietic and endothelial cells. Although the existence of the hemangioblast was first postulated early last century, a cell with this activity has yet to be unequivocally identified in mammals. In the last decade, gene targeting experiments in the mouse have uncovered genes which are required for development of both the hematopoietic and endothelial lineages, and this, together with increasing recognition that the two cell types share gene expression patterns, has renewed interest in the hemangioblast. The murine embryonic stem cell differentiation system has been used to demonstrate the existence of a Fft-1 positive progenitor cell, called the BL-CFC, which has the properties of the hemangioblast and this system is now being used to dissect the molecular regulation of hemangioblast development and differentiation.     

Lysophosphatidic acid acts as a nutrient‐derived developmental cue to regulate early hematopoiesis

Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX‐LPA signaling was mediated by PI3K/Akt‐Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient‐derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation.     

The Hemangioblast: Between Blood and Vessels

The hemangioblast is a bipotential cell that gives rise to hematopoietic and endothelial cells. Although the existence of the hemangioblast was first postulated early last century, a cell with this activity has yet to be unequivocally identified in mammals. In the last decade, gene targeting experiments in the mouse have uncovered genes which are required for development of both the hematopoietic and endothelial lineages, and this, together with increasing recognition that the two cell types share gene expression patterns, has renewed interest in the hemangioblast. The murine embryonic stem cell differentiation system has been used to demonstrate the existence of a Flk-1 positive progenitor cell, called the BL-CFC, which has the properties of the hemangioblast and this system is now being used to dissect the molecular regulation of hemangioblast development and differentiation.     

The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic growth factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and is thought to play an important role in the mesoderm induction. Although hematopoietic cells derive from the mesoderm, relatively few studies have, until recently, addressed the role of FGF-2 in hematopoiesis. FGF-2 is expressed in cells of the bone marrow including stromal cells, and possibly cells from several hematopoietic cell lineages. It is stored in the bone marrow extra-cellular matrix and released by enzymes such as heparanase, plasmin, or phospholipase C and D. FGF-receptors (FGF-Rs) are expressed in leukemic cell lines and in hematopoietic cells. FGF-2 positively regulates hematopoiesis, by acting on stromal cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. The action of FGF-2 is most likely indirect since its action, on megakaryocytopoiesis for example, is abrogated by anti-IL6 antibodies. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of TGF-β Taken together, these results demonstrate that FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.     

Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation

The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP(-)Flk1(-), GFP(+)Flk1(-) and GFP(+)Flk1(+) cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.     

Characterization of GATA-1(+) hemangioblastic cells in the mouse embryo

Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1(+) cells isolated from E7.0-7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1(+) cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1(+) hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo.     

Cross-talk between hematopoiesis and angiogenesis signaling pathways

The relationship between hematopoietic cells and endothelial cells has been seen as an indication that a common progenitor, the hemangioblast, gives rise to both cell types in the yolk sac, the initial site of hematopoiesis and blood vessel formation during mammalian development. The existence of angioblast-like circulating endothelial precursor cells in adults humans has recently been suggested. In this review, we have summarized the principle mechanisms involved in the cross-talk signaling pathway between hematopoiesis and angiogenesis in order to further understand how the hematopoietic and vascular systems are established during the development.