SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation

Hematopoiesis in most vertebrate species occurs in two distinct phases, primitive and definitive, which diverge from FLK1(+)VE-cadherin(-) mesoderm and FLK1(+)VE-cadherin(+) endothelial cells (EC), respectively. This study aimed at determining the stage at which hematopoietic lineage fate is determined by manipulating the SCL/tal-1 expression that is known to be essential for the early development of the primitive and definitive hematopoietic systems. We established SCL-null ES cell lines in which SCL expression is rescued by tamoxifen-inducible Cre recombinase-loxP site-mediated recombination. While no hematopoietic cells (HPC) were detected in SCL-null ES cell differentiation cultures, SCL gene reactivation from day 2 to day 4 after initiation of differentiation could rescue both primitive and definitive hematopoiesis. SCL reactivation at later phases was ineffective. Moreover, generation of VE-cadherin(+) EC that can give rise to definitive HPC required SCL reactivation prior to VE-cadherin expression. These results indicated that the competence to become HPC is acquired at the mesodermal stage by a SCL-dependent process that takes place independently of determination of endothelial fate.     

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.     

Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

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.     

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.     

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.     

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.     

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 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.     

Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands

Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event.