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.     

Differentiation stage-specific analysis of gene function with inducible short hair-pin RNA in differentiating embryonic stem cells

Cell differentiation is regulated by spatial and temporal coordination of gene expressions. Previously, we have established an embryonic stem (ES) cell differentiation system that can trace early cardiovascular developmental process in vitro. Here we show that tetracycline-induced short hair-pin RNA (shRNA) expression in differentiating ES cells successfully suppressed stage-specific genes for differentiation and modified cell fates. We established ES cell lines carrying shRNA gene driven by tRNA(val) promoter with tetracycline operator sequences (tet-ON system). When expression of vascular endothelial growth factor receptor-2 (VEGFR2) gene, a vascular progenitor and mesoderm marker and an essential gene for endothelial cell (EC) differentiation, was suppressed by shRNA in early ES cell differentiation, appearance of VEGFR2(+) mesoderm cells was substantially reduced. Suppression of VEGFR2 expression at mesoderm stage almost completely inhibited EC differentiation from VEGFR2(+) mesoderm cells. This novel experimental system, thus, can selectively determine stage-specific roles of genes in differentiation in vitro.     

SHB and angiogenic factors promote ES cell differentiation to insulin-producing cells

The potential use of embryonic stem (ES) cells for cell therapy of diabetes requires improved methods for differentiation and isolation of insulin-producing beta-cells. The signal transduction protein SHB may be involved in both angiogenesis and beta-cell development. Here we show that cells expressing the pancreatic endodermal marker PDX-1 appear in the vicinity of vascular structures in ES cell-derived embryoid bodies (EBs) cultured in vitro. Moreover, overexpression of SHB as well as culture of EBs in presence of the angiogenic growth factors PDGF or VEGF enhanced the expression of PDX-1 and/or insulin mRNA. Finally, expression of GFP under control of the PDX-1 promoter in EBs allowed for the enrichment by FACS of cells expressing PDX-1, C-peptide, and insulin as determined by immunofluorescence. It is concluded that SHB and angiogenic factors promote the development of cells expressing PDX-1 and insulin in EBs and that such cells can be separated by FACS.     

Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells

In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRα), c-Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post-gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1⁺ mesodermal cells could be further classified by the level of c-Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1⁺ mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supported in vitro hematopoiesis from ES cells. Flk1⁺ cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1⁺ cells will be required for in vitro ES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos or in vitro cultured ES cells.     

Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta signaling

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.     

Induction of cells expressing vascular endothelium markers from undifferentiated Xenopus presumptive ectoderm by co-treatment with activin and angiopoietin-2

Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegfl7. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as Xl-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.     

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.     

The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.     

Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies

Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation.     

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.     

Fibroblast growth factor-10 promotes cardiomyocyte differentiation from embryonic and induced pluripotent stem cells

Background

The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.

Methodology/Principal Findings

We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.

Conclusion/Significance

FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.