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 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.

Endothelial cell integrin alpha5beta1 expression is modulated by cytokines and during migration in vitro

1999 ◽  
Vol 112 (4) ◽  
pp. 569-578 ◽  
Author(s):  
G. Collo ◽  
M.S. Pepper
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Integrin Subunit ◽  
Subunit Expression ◽  
Synergistic Induction ◽  
Co Addition ◽  
Extracellular Matrix Interactions

Alterations in endothelial cell-extracellular matrix interactions are central to the process of angiogenesis. We have investigated the effect of wound-induced two-dimensional migration, basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1) and leukemia inhibitory factor (LIF) on expression of the alpha5beta1 integrin in endothelial cells. In multiple-wounded monolayers of bovine microvascular endothelial (BME) cells, an increase in mRNA and total protein for both alpha5 and beta1 subunits was observed, and this could be correlated with a reduction in cell density but not proliferation, both of which are induced following wounding. Although as previously reported, the alpha5 subunit was increased when cells were exposed to TGF-beta1 alone, co-addition of bFGF and TGF-beta1 resulted in a striking synergistic induction of alpha5, with no significant changes in the expression of beta1. In contrast, the alpha5 subunit was decreased by LIF in bovine aortic endothelial but not in BME cells. These findings suggest that quantitative alterations in alpha5 and beta1 integrin subunit expression modulate the adhesive and migratory properties of endothelial cells during angiogenesis.

scholarly journals Inhibiting the NLRP3 inflammasome with MCC950 ameliorates retinal neovascularization and leakage by reversing the IL-1β/IL-18 activation pattern in an oxygen-induced ischemic retinopathy mouse model

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Ailing Sui ◽  
Xiuping Chen ◽  
Jikui Shen ◽  
Anna M. Demetriades ◽  
Yiyun Yao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Nlrp3 Inflammasome ◽  
Retinal Neovascularization ◽  
Platelet Derived Growth Factor ◽  
Vegf Receptor ◽  
Activation Pattern ◽  
Ischemic Retinopathy

Abstract Activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays an important role in ocular neovascularization. In our study, we found that the expression and activation levels of NLRP3 inflammasome components, including NLRP3, an apoptosis-associated speck-like protein (ASC) containing caspase activation and recruitment domain (CARD) and caspase-1 (CAS1), were significantly upregulated. In addition, we found interleukin (IL)-1β activity increased while IL-18 activity decreased in the retinas of oxygen-induced ischemic retinopathy (OIR) mice. MCC950, an inhibitor of NLRP3, reversed the IL-1β/IL-18 activation pattern, inhibited the formation of retinal neovascularization (RNV), decreased the number of acellular capillaries and reduced leakage of retinal vessels. Moreover, MCC950 could regulate the expression of endothelial cell- and pericyte function-associated molecules, such as vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1, VEGFR2, matrix metalloproteinase (MMP)2, MMP9, tissue inhibitor of metalloproteinases (TIMP)1, TIMP2, platelet-derived growth factor receptor-β (PDGFR-β), platelet-derived growth factor-B (PDGF-B), and angiopoietin2 (Ang2). In vitro, recombinant human (r)IL-18 and rIL-1β regulated the expression of endothelial cell- and pericyte function-associated molecules and the proliferation and migration of endothelial cells and pericytes. We therefore determined that inhibiting the NLRP3 inflammasome with MCC950 can regulate the function of endothelial cells and pericytes by reversing the IL-1β/IL-18 activation pattern to ameliorate RNV and leakage; thereby opening new avenues to treat RNV-associated ocular diseases.

scholarly journals Induced Pluripotent Stem Cells as Vasculature Forming Entities

2019 ◽  
Vol 8 (11) ◽  
pp. 1782 ◽  
Author(s):  
Antonio Palladino ◽  
Isabella Mavaro ◽  
Carmela Pizzoleo ◽  
Elena De Felice ◽  
Carla Lucini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Tissue Growth ◽  
Safety Issues ◽  
Induced Pluripotent

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaffold. In this context, one of the most effective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are affected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most effective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an efficient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine.

Role of Connective Tissue Growth Factor In Endothelin-Mediated Endothelial Cell Activation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2107-2107
Author(s):  
Anna J Hernandez ◽  
Sonia Henriquez ◽  
Enrique R Maldonado ◽  
Rodeler Youte ◽  
Gregory N Prado ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factors ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Connective Tissue ◽  
Cell Activation ◽  
Fold Increase ◽  
Tissue Growth ◽  
Vegf Expression ◽  
Endothelial Cell Activation

Abstract Abstract 2107 Endothelial cell activation and elevated levels of circulating Endothelin-1 (ET-1) have been reported in patients with atherosclerosis and sickle cell disease (SCD). ET-1 is a well-described vasoconstrictor, mitogen and regulator of endothelial cells migration that has been shown to promote structural changes in blood vessels. ET-1 is produced in response to increases in vasoactive hormones, growth factors, hypoxia, shear stress and free radicals, events that are commonly observed in patients with SCD. Endothelial cell activation is in part characterized by increases of cytokines such as monocyte chemotactic protein-1 (MCP-1) and growth factors that are important in vascular maintenance and fibrogenesis such as connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF). CTGF and VEGF are important for blood vessel remodeling, fibrogenesis and angiogenesis. Indeed there is evidence that incubation of smooth muscle cells with ET-1 leads to increases in CTGF and VEGF levels. However, the relationship between ET-1 and CTGF in endothelial cell activation is unclear. We hypothesize that increasing ET-1 would stimulate CTGF production and endothelial cell activation. We studied the effects of ET-1 on the human endothelial cell line, EA.hy926 (EA), as well as in primary cultures of mouse aortic endothelial cells (MAEC). We performed gene expression time course experiments (0, 2, 4, 8, 16, 24 Hr) on EA cells following incubation with 100nM ET-1 using quantitative RT-PCR with Taqman chemistries and GAPDH and beta-actin as endogenous controls. We observed increases of CTGF and VEGF expression between 4 and 8 hr for CTGF (1.74 fold increase vs time 0, n=6, P<0.03) and 4 hr for VEGF (2.14 fold increase vs time 0, n=3, P<0.04). Additional experiments on EA cells showed that incubation with 100nM ET-1 for 4 hr in the presence of BQ123 and BQ788, two inhibitors of ET-1 type A and B receptors, respectively, blocked the ET-1 stimulated rises in CTGF and VEGF as well as MCP-1 expression. We then performed western blot analyses (Abcam-CTGF antibody ab6992; Abcam VEGF antibody ab1316) and showed increases in cell associated CTGF protein levels following incubation of EA cells with 100nM ET-1 for 24 hr. The ET-1 stimulated rise in CTGF levels were significantly blunted by pre-incubation of EA cells with both BQ788 and BQ123. To study whether the effects of ET-1 were unique to EA cells, we also analyzed the effects of ET-1 on early cultures of MAEC isolated from C57BLJ mice. Consistent with our observations in human endothelial cells, incubation of MAEC with 100nM ET-1 for 4 hr were associated with increases of CTGF and VEGF expression (1.86 fold vs vehicle, n=3, P<0.03; 1.73 fold vs vehicle, n=3 P<0.04 respectively). Furthermore, ET-1 stimulated rises in CTGF and VEGF expressions were likewise blocked by pre-incubation with BQ123 andBQ788. We conclude that addition of ET-1 leads to activation of endothelial cells and increases in CTGF and VEGF from human and mouse endothelial cells. Thus we suggest that therapies designed to block ET-1 receptors will reduce endothelial cell activation in part by reducing CTGF production leading to alterations in cellular and tissue architecture. This work was supported by NIH R01HL090632 to AR and R01HL096518 to JRR. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fisp12/Mouse Connective Tissue Growth Factor Mediates Endothelial Cell Adhesion and Migration through Integrin αvβ3, Promotes Endothelial Cell Survival, and Induces Angiogenesis In Vivo

1999 ◽  
Vol 19 (4) ◽  
pp. 2958-2966 ◽  
Author(s):  
Alexander M. Babic ◽  
Chih-Chiun Chen ◽  
Lester F. Lau
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Connective Tissue ◽  
Tissue Growth ◽  
Microvascular Endothelial Cells ◽  
Endothelial Cell Survival ◽  
Vessel Growth

ABSTRACT Fisp12 was first identified as a secreted protein encoded by a growth factor-inducible immediate-early gene in mouse fibroblasts, whereas its human ortholog, CTGF (connective tissue growth factor), was identified as a mitogenic activity in conditioned media of human umbilical vein endothelial cells. Fisp12/CTGF is a member of a family of secreted proteins that includes CYR61, Nov, Elm-1, Cop-1/WISP-2, and WISP-3. Fisp12/CTGF has been shown to promote cell adhesion and mitogenesis in both fibroblasts and endothelial cells and to stimulate cell migration in fibroblasts. These findings, together with the localization of Fisp12/CTGF in angiogenic tissues, as well as in atherosclerotic plaques, suggest a possible role for Fisp12/CTGF in the regulation of vessel growth during development, wound healing, and vascular disease. In this study, we show that purified Fisp12 (mCTGF) protein promotes the adhesion of microvascular endothelial cells through the integrin receptor αvβ3. Furthermore, Fisp12 stimulates the migration of microvascular endothelial cells in culture, also through an integrin-αvβ3-dependent mechanism. In addition, the presence of Fisp12 promotes endothelial cell survival when cells are plated on laminin and deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, Fisp12 induces neovascularization in rat corneal micropocket implants. These results demonstrate that Fisp12 is a novel angiogenic inducer and suggest a direct role for Fisp12 in the adhesion, migration, and survival of endothelial cells during blood vessel growth. Taken together with the recent finding that the related protein CYR61 also induces angiogenesis, we suggest that Fisp12/mCTGF and CYR61 comprise prototypes of a new family of angiogenic regulators that function, at least in part, through integrin-αvβ3-dependent pathways.

EXPRESSION OF THE PROANGIOGENIC PROTEIN HEPATOMA-DERIVED GROWTH FACTOR IN NEOVESSELS IN AN ARTERIAL VENOUS LOOP-BASED TISSUE ENGINEERING CHAMBER

2013 ◽  
Vol 25 (05) ◽  
pp. 1340007
Author(s):  
Elsa C. Chan ◽  
Ming-Hong Tai ◽  
Pei-Chang Wu ◽  
Hsiao-Mei Kuo ◽  
Fan Jiang ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Mrna Level ◽  
Inflammatory Cells ◽  
Tube Formation ◽  
Tissue Growth ◽  
Angiogenic Factor ◽  
In Vitro Proliferation

Boosting angiogenesis is a crucial process to enhance tissue growth in tissue engineering (TE). Hepatoma-derived growth factor (HDGF) has been identified as an angiogenic factor, but its involvement in angiogenesis in an arteriovenous loop-based TE chamber developed by the laboratory is unclear. In this study, the authors first examined the effects of HDGF on angiogenic responses in endothelial cells and in a corneal model of neovascularization, and then characterized the expression of HDGF in the TE chamber. HDGF (1–500 ng/mL) induced concentration-dependent angiogenic responses in human endothelial cells in vitro (proliferation, migration, and tube formation). Local application of HDGF stimulated neovascularization in a rat model of corneal angiogenesis. In the TE chamber, there was an increase in blood vessel volume from day 3 to day 14. Immunofluorescence microscopy revealed that HDGF is highly expressed in the neovessels in the chamber. Peak expression of HDGF (day 3) coincided with the infiltration of inflammatory cells, and the mRNA level of endogenous HDGF correlated with that of tumor necrosis factor α (TNFα). In vitro, TNFα stimulated HDGF expression in endothelial cells. The data suggest that HDGF may be involved in angiogenic responses in the TE chamber and the proinflammatory cytokine TNFα may have a pivotal role in stimulating HDGF expression. Enhancing HDGF signaling may be a new approach to extend vascularization for TE.

Abstract P092: Simplified Monolayer Differentiation of Human-Induced Pluripotent Stem Cells to Functional Cardiac Myocytes

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Jennifer K Lang ◽  
Stanley Fernandez ◽  
Thomas Cimato
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cardiac Myocytes ◽  
Pluripotent Stem Cells ◽  
Action Potentials ◽  
Cardiac Myocyte ◽  
Human Ipscs ◽  
Myocyte Differentiation ◽  
Induced Pluripotent

Background: Human induced pluripotent stem cells (hiPSCs) are an important model for cardiovascular research, drug discovery, and translational research applications. Commonly used methods to direct iPSCs to cardiac myocytes can be technically demanding. Prior studies have shown that both VEGF and endothelial cells promote differentiation of stem cells to cardiac myocytes. Furthermore, DMEM/F12 with 10% fetal calf serum (DMEM-FCS) has been shown to induce cardiac myocytes in an embryoid body (EB) system. The objective of this study was to determine if differentiation of hiPSCs using conditions that support endothelial cell differentiation would promote cardiac myocyte colony formation. Methods: Two hiPSC lines derived using non-genome integrating methods were maintained on Matrigel-coated surfaces under serum free conditions in mTeSR1 medium. We performed a comparison of monolayer myocyte differentiation efficiency using DMEM-FCS and endothelial cell medium (EC). Cells were maintained in iPSC medium (mTeSR1) as a negative control. The number of beating colonies derived under each growth condition was determined using phase microscopy at 4 weeks. Cardiac myocyte commitment was characterized using an α-MHC-GFP reporter vector and electrophysiologic action potentials on isolated beating colonies. Results: Differentiation of human iPSCs in EC medium induced substantial numbers of beating colonies 4 weeks after differentiation (2.29 ± 0.3 beating colonies/cm2 culture area, n=42). Unlike EB models of myocyte differentiation, no beating clusters were observed in our monolayer system with DMEM-FCS medium (n=14) (p<0.01). As expected, mTESR1 (n=12) did not induce any cardiac myocytes. All beating cell colonies expressed GFP driven by the cardiac specific α-MHC promoter. Electrophysiological studies confirmed the presence of action potentials with ventricular phenotypes. Conclusions: Differentiation of human iPSCs under monolayer conditions that support endothelial cells facilitates efficient induction of functional human cardiac myocytes. Our findings simplify the differentiation of iPSCs to cardiac myocytes, making research with human iPSCs more accessible to a broad range of cardiovascular investigators.

scholarly journals Generation of gene-corrected functional osteoclasts from osteopetrotic induced pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaojie Xian ◽  
Roksana Moraghebi ◽  
Henrik Löfvall ◽  
Anders Fasth ◽  
Kim Henriksen ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Lentiviral Vector ◽  
Fatal Outcome ◽  
Autosomal Recessive Disorder ◽  
Embryoid Bodies ◽  
Bovine Bone ◽  
Malignant Osteopetrosis ◽  
The Media ◽  
Induced Pluripotent

Abstract Background Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by non-functional osteoclasts and a fatal outcome early in childhood. About 50% of patients have mutations in the TCIRG1 gene. Methods IMO iPSCs were generated from a patient carrying a homozygous c.11279G>A (IVS18+1) mutation in TCIRG1 and transduced with a lentiviral vector expressing human TCIRG1. Embryoid bodies were generated and differentiated into monocytes. Non-adherent cells were harvested and further differentiated into osteoclasts on bovine bone slices. Results Release of the bone resorption biomarker CTX-I into the media of gene-corrected osteoclasts was 5-fold higher than that of the uncorrected osteoclasts and 35% of that of control osteoclasts. Bone resorption potential was confirmed by the presence of pits on the bones cultured with gene-corrected osteoclasts, absent in the uncorrected IMO osteoclasts. Conclusions The disease phenotype was partially corrected in vitro, providing a valuable resource for therapy development for this form of severe osteopetrosis.

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