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.     

Ontogeny of the mouse hematopoietic system

In vertebrates the extraembryonic mesoderm of the yolk sac (YS) is the first site during embryogenesis where morphologically discernible hematopoiesis may be found. Later hematopoiesis shifts into the embryo proper, first to the liver, the major fetal hematopoietic site, then to definitive hematopoietic territories, the spleen and bone marrow. It is widely accepted that in the mouse this picture reflects the migration of pluripotent hematopoietic stem cells (HSC) from the YS accompanied by subsequent colonization of the hematopoietic tissues during embryogenesis. However, there is no conclusive evidence showing unequivocally the initiating role of the YS in murine adult hematopoiesis. Recently, we have demonstrated the important role of embryo body tissues in the development of CFU-S before the establishment of definitive hematopoiesis in the fetal liver. This finding suggests that the early development of the hematopoietic system in the mouse is more complex than has been previously proposed and we consider here the early hematopoietic events in the developing mouse embryo.     

Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Blood islands' of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper. These lineages arise from a layer of extraembryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules (Indian hedgehog) is produced by visceral endoderm, can induce formation of blood and endothelial cells in explant cultures and can reprogram prospective neurectoderm along hematopoietic and endothelial cell lineages. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. These findings may have important implications for regulating hematopoiesis and vascular development for therapeutic purposes in humans and for the development of new sources of stem cells for transplantation and gene therapy.     

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.     

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.     

内皮祖细胞(EPCs)研究进展

组织工程血管以及组织工程化组织的血管化因目前内皮种子细胞扩增能力和生物活力的不足而受到限制。EPCs(内皮祖细胞)是内皮细胞的前体细胞。在胚胎期,内皮细胞系与造血细胞系来源于血岛内共同的祖先细胞;出生后,EPCs存在于骨髓,并可被转移至外周血,参与缺血组织的血管重建和血管的内膜化。因此EPCs有望成为今后组织工程内皮种子细胞的重要来源。     

Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos

The hemangioblast in the mesoderm gives rise to both angioblasts and hematopoietic stem cells. The movement of hemangioblast precursor cells in the fetal trunk is a critical event in early embryogenesis. Vascular endothelial growth factor (VEGF) signaling is likely involved in this migration given the partial disturbance of VEGF receptor (VEGFR)-positive cell accumulation and migration in VEGFR2 null mice or mice with a truncated VEGFR1. However, it is not clear how the VEGF system regulates this migration or its direction. We show here that the expression of VEGF-A is dominant in the anterior portion of the embryo, whereas VEGFR1 and VEGFR2 are expressed in the posterior portion of the embryo. An inhibitor of VEGFR kinase blocked the migration of VEGFR-positive cells in a whole-embryo culture system. In addition, VEGFR-positive cells migrated toward a VEGFR1- or VEGFR2-specific ligand in vitro. Furthermore, VEGFR-positive cells derived from wild-type or VEGFR2(+/-) mice moved rapidly anteriorly, whereas cells derived from VEGFR2(+/-) mice carrying a truncated VEGFR1 [VEGFR1(TM-TK)(-/-)] migrated little when injected into wild-type mice. These results suggest that the VEGF-A protein concentrated in the anterior region plays an important role in the guidance of VEGFR-positive cells from the posterior portion to the head region by interacting with VEGFR in the mouse embryo.     

Production of hematopoietic cells from ES cells

Murine embryonic stem (ES) cells are cell lines established from blastocyst which can contribute to all adult tissues, including the germ-cell lineage, after reincorporation into the normal embryo. ES cell pluripotentiality is preserved in culture in the presence of LIF. LIF withdrawal induces ES cell differentiation to nervous, myocardial, endothelial and hematopoietic tissues. The model of murine ES cell hematopoietic differentiation is of major interest because ES cells are non transformed cell lines and the consequences of genomic manipulations of these cells are directly measurable on a hierarchy of synchronized in vitro ES cell-derived hematopoietic cell populations. These include the putative hemangioblast (which represents the emergence of both hematopoietic and endothelial tissues during development), myeloid progenitors and mature stages of myeloid lineages. Human ES cell lines have been recently derived from human blastocyst in the USA. Their manipulation in vitro should be authorized in France in a near future with the possibility of developing a model of human hematopoietic differentiation. This allows to envisage in the future the use of ES cells as a source of human hematopoietic cells.     

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.     

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.     

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.     

The notch ligand delta-like 4 regulates multiple stages of early hemato-vascular development

Background

In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis.

Methodology/Principal Findings

Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4^+/− and Dll4^−/− embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis.

Conclusions/Significance

This study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis.     

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.     

Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression

Accumulating studies support the idea that a common progenitor, termed the hemangioblast, generates both hematopoietic and endothelial cell lineages. To better define the relationship between these cell lineages, we have generated knock-in embryonic stem (ES) cells carrying a non-functional human CD4 at the Scl locus. By using in vitro differentiated Scl(+/CD4) ES cells, we demonstrate that FLK1 and SCL are molecular determinants of the hemangioblast. Furthermore, our studies demonstrate that hematopoietic and endothelial cells develop via distinct, sequential generation of FLK1 and SCL-expressing cells. FLK1(+)CD4(-) cells first arise in developing embryoid bodies. The Scl gene is turned on within FLK1(+)CD4(-) cells to give rise to FLK1(+)CD4(+) cells. Alternatively, a subpopulation of the initial FLK1(+)CD4(-) cells remains as SCL negative. Within the FLK1(+)CD4(+) cells, FLK1 is down regulated to generate FILK1(-)CD4(+) cells. Replating studies demonstrate that hematopoietic progenitors are enriched within FLK1(+)CD4(+) and FLK1(-)CD4(+) cells, while endothelial cells develop from FLK1(+)CD4(+) and FLK1(+)CD4(-) cell populations.     

Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2

The ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2-mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1^highPDGFRα⁻. Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2-dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability.