scholarly journals Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3424-3431 ◽  
Author(s):  
D Vittet ◽  
MH Prandini ◽  
R Berthier ◽  
A Schweitzer ◽  
H Martin-Sisteron ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Vascular Endothelial ◽  
Endothelial Cell Differentiation ◽  
Kinetics Of

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

Modulation of hematopoietic and endothelial cell differentiation from mouse embryonic stem cells by different culture conditions

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 111-114 ◽  
Author(s):  
Wen Jie Zhang ◽  
Changwon Park ◽  
Elizabeth Arentson ◽  
Kyunghee Choi
Keyword(s):  
Endothelial Cell ◽  
Cell Differentiation ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Embryoid Bodies ◽  
Vascular Endothelial ◽  
Hematopoietic Progenitors ◽  
Endothelial Cell Differentiation

Abstract Embryonic stem (ES) cells can differentiate into many different somatic cells in culture. To better correlate hematopoietic and endothelial cell differentiation of ES cells in currently available protocols, we compared fetal liver kinase-1 (Flk-1)–, stem cell leukemia (Scl)–, and vascular endothelial–cadherin (VE-cadherin)–expressing cells generated in embryoid bodies (EBs) and on OP9 cells. We report that the kinetics of Scl and Flk-1 expression were similar in EBs and OP9 cells, although Flk-1 expression was extended on OP9 cells. CD45+ and Ter-119+ cells developed more efficiently in EBs, whereas VE-cadherin+ cells developed largely on OP9 cells. Cell sorting and replating studies showed that Scl+ cells, not Flk-1+ or VE-cadherin+ cells, were enriched for primitive and definitive hematopoietic progenitors. Our studies indicate that optimal hematopoietic and endothelial cell differentiation occur in EBs and on OP9 cells, respectively. Regardless of the culture systems used, Scl is the most relevant marker for enriching primitive and definitive hematopoietic progenitors.

scholarly journals Vascular Endothelial Growth Factor Induces Nephrogenesis and Vasculogenesis

1999 ◽  
Vol 10 (10) ◽  
pp. 2125-2134
Author(s):  
ALDA TUFRO ◽  
VICTORIA F. NORWOOD ◽  
ROBERT M. CAREY ◽  
R. ARIEL GOMEZ
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Blot Analysis ◽  
Nuclear Antigen ◽  
Vascular Endothelial ◽  
Endothelial Cell Differentiation ◽  
Endothelial Growth Factor

Abstract. The expression of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 in the rat kidney was examined during ontogeny using Northern blot analysis and immunocytochemistry. In prevascular embryonic kidneys (embryonic day 14 [E14]), immunoreactive Flt-1 and Flk-1 were observed in isolated angioblasts, whereas VEGF was not detected. Angioblasts aligned forming cords before morphologically differentiating into endothelial cells. In late fetal kidneys (E19), immunoreactive VEGF was detected in glomerular epithelial and tubular cells, whereas Flt-1 and Flk-1 were expressed in contiguous endothelial cells. To determine whether VEGF induces endothelial cell differentiation and vascular development in the kidney, the effect of recombinant human VEGF (5 ng/ml) was examined on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 d in culture in serum-free, defined media, metanephric kidney growth and morphology were assessed. DNA content was higher in VEGF-treated explants (1.9 ± 0.17 μg/kidney, n = 9) than in paired control explants (1.4 ± 0.10 μg/kidney, n = 9) (P < 0.05). VEGF induced proliferation of tubular epithelial cells, as indicated by an increased number of tubules and tubular proliferating cell nuclear antigen-containing cells. VEGF induced upregulation of Flk-1 and Flt-1 expression, as assessed by Western blot analysis. Developing endothelial cells were identified and localized using immunocytochemistry and electron microscopy. Flt-1, Flk-1, and angiotensin-converting enzyme-containing cells were detected in VEGF-treated explants, whereas control explants were negative. These studies confirmed previous reports indicating that the expression of VEGF and its receptors is temporally and spatially associated with kidney vascularization and identified angioblasts expressing Flt-1 and Flk-1 in prevascular embryonic kidneys. The data indicate that VEGF expression is downregulated in standard culture conditions and that VEGF stimulates growth of embryonic kidney explants by expanding both endothelium and epithelium, resulting in vasculogenesis and enhanced tubulogenesis. These data suggest that VEGF plays a critical role in renal development by promoting endothelial cell differentiation, capillary formation, and proliferation of tubular epithelia.

scholarly journals Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1253-1263 ◽  
Author(s):  
Masanori Hirashima ◽  
Hiroshi Kataoka ◽  
Satomi Nishikawa ◽  
Norihisa Matsuyoshi ◽  
Shin-Ichi Nishikawa
Keyword(s):  
Cell Adhesion ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Vascular Endothelial ◽  
Cell Cell

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34−to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

scholarly journals SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 440-450 ◽  
Author(s):  
Xuan Ou ◽  
Hee-Don Chae ◽  
Rui-Hong Wang ◽  
William C. Shelley ◽  
Scott Cooper ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Histone Deacetylases ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors ◽  
Endothelial Cell Differentiation ◽  
Delay Defects

Abstract SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis.

scholarly journals Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kelly Mulfaul ◽  
Joseph C. Giacalone ◽  
Andrew P. Voigt ◽  
Megan J. Riker ◽  
Dalyz Ochoa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Lentiviral Vector ◽  
Tissue Growth ◽  
Embryoid Bodies ◽  
Age Related Macular Degeneration ◽  
Human Ipscs ◽  
Induced Pluripotent

Abstract Background Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically. Methods We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function. Results We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors. Conclusion ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

scholarly journals Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1253-1263 ◽  
Author(s):  
Masanori Hirashima ◽  
Hiroshi Kataoka ◽  
Satomi Nishikawa ◽  
Norihisa Matsuyoshi ◽  
Shin-Ichi Nishikawa
Keyword(s):  
Cell Adhesion ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Vascular Endothelial ◽  
Cell Cell

Abstract A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34−to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

scholarly journals Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3720-3730 ◽  
Author(s):  
M Bielinska ◽  
N Narita ◽  
M Heikinheimo ◽  
SB Porter ◽  
DB Wilson
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Cell Stage ◽  
Primitive Hematopoiesis

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/-embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/-ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/-ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/-ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.

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