Endodermal growth factors promote endocardial precursor cell formation from precardiac mesoderm

AbstractWe previously demonstrated that the initial emergence of endocardial precursor cells (endocardial angioblasts) occurred within the precardiac mesoderm and that the endodermal secretory products promoted delamination of cells from the precardiac mesoderm and expression of endothelial lineage markers [Dev. Biol. 175 (1996), 66]. In this study, we sought to extend our original study to the identification of candidate molecules derived from the endoderm that might have induced endocardial precursor cell formation. We have detected expression of transforming growth factors β (TGFβ) 2, 3, and 4 in anterior endoderm at Hamburger and Hamilton (H-H) stage 5 by RT-PCR. To address the role of growth factors known to be present in the endoderm, precardiac mesodermal explants were isolated from H-H stage 5 quail embryos and cultured on the surface of collagen gels with serum-free defined medium 199. Similar to the effect of explants cocultured with anterior endoderm, when cultured with TGFβs 1–3 (3 ng/ml each), explants formed QH-1 (anti-quail endothelial marker)-positive mesenchymal cells, which invaded the gel and expressed the extracellular marker, cytotactin (tenascin). Another member of the TGFβ superfamily, bone morphogenetic protein-2 (BMP-2; 100 ng/ml), did not induce QH-1-positive mesenchymal cell formation but promoted formation of an epithelial monolayer on the surface of the collagen gel; this monolayer did not express QH-1. Explants treated with vascular endothelial growth factor (VEGF165, 100 ng/ml) also did not invade the gel but formed an epithelial-like outgrowth on the surface of the gel. However, this monolayer did express the QH-1 marker. Fibroblast growth factor-2 (FGF-2; 250 ng/ml)-treated explants expressed QH-1 and exhibited separation of the cells on the surface of the gel. Finally, a combination of TGFβs and VEGF enhanced formation of QH-1-positive cord-like structures within the gel from mesenchyme that had previously invaded the gel. Luminization of the cords, however, was not clearly evident. These findings suggest that TGFβs, among the growth factors tested, mediate the initial step of endocardial formation, i.e., delamination of endothelial precursor cells from precardiac mesoderm, whereas VEGF may primarily effect early vasculogenesis (cord-like structure formation)

Formation and Early Morphogenesis of Endocardial Endothelial Precursor Cells and the Role of Endoderm

AbstractThe formation of endocardial endothelium in quail embryos was investigated usingin vivoandin vitrosystems. Based on the expression of an quail endothelial marker, QH-1, the initial emergence of endothelial precursor cells in the embryo occurs at stage 7+(two somites) in the posterior parts of the bilateral heart forming regions. Cells that expressed the QH-1 antigen were mesenchymal and positioned between the mesodermal epithelium of the heart region and the endoderm. By confocal microscopy, an asymmetrical distribution of QH-1 positive cells was observed between the two heart regions: specifically between 7+and 8−, more precursor cells were seen in the right region than the left. Endothelial precursor cells did not appear outside of the heart forming regions until stage 8−(three somites). Free, mesenchymal-like endothelial precursor cells intrinsic to the heart regions also expressed two extracellular antigens, JB3, a fibrillin-like protein, and cytotactin, both associated with segments of the primary heart tube where endothelial cells “re-transform” back to a mesenchymal phenotype during cardiac cushion tissue formation. Between stages 8 and 9 (four to seven somites), (1) QH-1 positive cells within the heart forming region established vascular-like connections with QH-1 positive cells located outside of the heart region, as initially shown by Coffin and Poole (1988), (2) after fusion of the heart regions, a plexus of QH-1 positive cells was formed ventral to the foregut, and (3) the definitive endocardial lining of the primary heart tube formed directly from the ventral plexus of endothelial precursor cells. Because the QH-1 positive, endothelial precursor cells of each heart forming region were always in close association with anterior endoderm, we sought to determine if the endoderm mediated the formation of precursor cells committed to a cardiac endothelial lineage as reflected by their expression of QH-1, JB3 antigen, and cytotactin. To test this hypothesis, precardiac mesodermal explants were isolated from stage 5 heart forming regions prior to their expressing of either endocardial or myocardial markers and cultured on the surface of collagen gels in the presence or absence of endoderm. In the absence of endoderm, precardiac mesoderm of each stage 5 explant remained epithelial, formed contractile tissue, but did not exhibit any QH-1 positive cells or mesenchymal cells. Conversely, when cocultured with endoderm or endoderm conditioned medium, in addition to the formation of contractile tissue, the explant formed mesenchymal cells. The latter invaded the gel lattice and, asin vivo,expressed QH-1 antigen, JB3 antigen, and cytotactin. These findings suggest that endoderm induces mesoderm of the heart fields to undergo an epithelial to mesenchyme transformation that results in the segregation of myocardial and endocardial precursor cells

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.We thank the Leducq Fondation for supporting Tui Neri, and funding this research under the framework of the MITRAL network and for generously awarding us for the equipment of our cell imaging facility in the frame of their program “Equipement de Recherche et Plateformes Technologiques” (ERPT to M.P.), the Genopole at Evry and the Fondation de la recherche Medicale (grant DEQ20100318280) for supporting the laboratory of Michel Puceat. Part of this work in South Carolina University was conducted in a facility constructed with support from the National Institutes of Health, Grant Number C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources. Other funding sources: National Heart Lung and Blood Institute: RO1-HL33756 (R.R.M.), COBRE P20RR016434–07 (R.R.M., R.A. N.), P20RR016434–09S1 (R.R.M. and R.A.N.); American Heart Association: 11SDG5270006 (R.A.N.); National Science Foundation: EPS-0902795 (R.R.M. and R.A. N.); American Heart Association: 10SDG2630130 (A.C.Z.), NIH: P01HD032573 (A.C. Z.), NIH: U54 HL108460 (A.C.Z), NCATS: UL1TR000100 (A.C.Z.); EH was supported by a fellowship of the Ministere de la recherche et de l’éducation in France.TM-M was supported by a fellowship from the Fondation Foulon Delalande and the Leducq Foundation. P.v.V. was sponsored by a UC San Diego Cardiovascular Scholarship Award and a Postdoctoral Fellowship from the California Institute for Regenerative Medicine (CIRM) Interdisciplinary Stem Cell Training Program II. S.M.E. was funded by a grant from the National Heart, Lung, and Blood Institute (HL-117649). A.T. is supported by the National Heart, Lung, and Blood Institute (R01-HL134664).S

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

<div><p>Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of <i>versican</i> and <i>Has2</i> by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of <i>versican</i> and <i>Has2</i>. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions. </p> </div

BMP-2 treatment increases versican expression by AV endocardial cushion mesenchymal cells (CMCs).

<p>(A-F) Versican immunostaining in HH stage-24 AV CMC aggregate cultures on collagen gels. The CMC aggregates were cultured in Medium 199 alone (control) (A), or with BMP-2, 20 ng/ml (B), BMP-2, 50 ng/ml (C), BMP-2, 200 ng/ml (D), BMP-2 200 ng/ml plus noggin, 500 ng/ml (E) or noggin, 500 ng/ml (F), dissolved in Medium 199. As low as 20 ng/ml of BMP-2 promoted versican immunostaining in cultured CMC aggregates. (G) Quantitative evaluation of versican immunointensity in AV CMC aggregate cultures. Immunointensity of the CMC aggregates treated with concentrations as low as 20 ng/ml of BMP-2 was significantly higher than the control (**<i>p</i><0.01). Conversely, immunointensity in CMC aggregates cultured with noggin was significantly lower than the control (*<i>p</i><0.05). (H) Quantitative evaluation of <i>versican</i> mRNA expression in CMC aggregate cultures. QRT-PCR data revealed an approximately 7-fold increase of <i>versican</i> expression in AV CMC aggregate cultures incubated with BMP-2 over the control cultures (**<i>p</i><0.01). The potential of BMP-2 to stimulate <i>versican</i> expression was abolished when noggin was added to the cultures (*<i>p</i><0.05). Vertical bars indicate ± SD of the mean. *<i>p</i><0.05 ; **<i>p</i><0.01 as compared to the control (M199).</p

Collagen gel images of HH stage-24 AV CMC aggregate cultures treated with versican siRNA.

<p>Upper panels show the surface of the gel and the lower panels show a depth of 240 µm within the gel. Higher magnification views shown in the insets in Figures e, f, h and k indicate CMCs in the focal plane at 240µm below the surface of the gels (arrows in e, f, h and k). Note that there are no CMCs in the focal plane at 240 µm below the surface of the gels in Figures g and l. CMCs treated with BMP-2 (200 ng/ml) migrated deeper into the collagen gels (b and arrows in f) than the control cultures (M199, a and arrows in e). Versican siRNA treatment (100 nM) significantly reduced cell migration (j, l), whereas, treatments with control RNA (scrambled RNA, 100 nM) did not alter CMC migration (i and arrows in k). BMP-2-stimulated cell migration was significantly reduced when versican siRNA was added to the medium (c and g), whereas control RNA did not alter BMP-2-stimulated cell migration (d and arrows in h). </p