EC Differentiation Research Articles

Aquaporins enriched in endothelial vacuole membrane regulate the diameters of microvasculature in hyperglycaemia.

In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. Yet, the regulatory mechanism and effective treatment approach remain elusive. Our initial findings revealed a notable decrease in the expression of human AQP1 in both diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Subsequently, our investigations unveiled a reduction in vascular diameter and compromised perfusion within zebrafish embryos subjected to high glucose treatment. Further analysis indicated a significant down-regulation of two aquaporins, aqp1a.1 and aqp8a.1, which are highly enriched in ECs and are notably responsive to hyperglycaemic conditions. Intriguingly, the loss of function of aqp1a.1 and/or aqp8a.1 resulted in a reduction of intersegmental vessel diameters, effectively mirroring the phenotype observed in the hyperglycaemic zebrafish model. The overexpression of aqp1a.1/aqp8a.1 in zebrafish ECs led to notable enlargement of microvascular diameters. Moreover, the reduced vessel diameters resulting from high-glucose treatment were effectively rescued by the overexpression of these aquaporins. Additionally, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and the loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Notably, while the loss of AQP1 did not impact EC differentiation from human stem cells, it significantly inhibited vascular formation in differentiated ECs. EC-enriched aquaporins regulate the diameter of blood vessels through an intracellular vacuole-mediated process under hyperglycaemic conditions. These findings collectively suggest that aquaporins expressed in ECs hold significant promise as potential targets for gene therapy aimed at addressing vascular perfusion defects associated with diabetes.

GhRCD1 regulates cotton somatic embryogenesis by modulating the GhMYC3-GhMYB44-GhLBD18 transcriptional cascade.

Plant somatic embryogenesis (SE) is a multifactorial developmental process where embryos that can develop into whole plants are produced from somatic cells rather than through the fusion of gametes. The molecular regulation of plant SE, which involves the fate transition of somatic cells into embryogenic cells, is intriguing yet remains elusive. We deciphered the molecular mechanisms by which GhRCD1 interacts with GhMYC3 to regulate cell fate transitions during SE in cotton. While silencing of GhMYC3 had no discernible effect on SE, its overexpression accelerated callus formation, and proliferation. We identified two of GhMYC3 downstream SE regulators, GhMYB44 and GhLBD18. GhMYB44 overexpression was unconducive to callus growth but bolstered EC differentiation. However, GhLBD18 can be triggered by GhMYC3 but inhibited by GhMYB44, which positively regulates callus growth. On top of the regulatory cascade, GhRCD1 antagonistically interacts with GhMYC3 to inhibit the transcriptional function of GhMYC3 on GhMYB44 and GhLBD18, whereby a CRISPR-mediated rcd1 mutation expedites cell fate transition, resembling the effects of GhMYC3 overexpression. Furthermore, we showed that reactive oxygen species (ROS) are involved in SE regulation. Our findings elucidated that SE homeostasis is maintained by the tetrapartite module, GhRCD1-GhMYC3-GhMYB44-GhLBD18, which acts to modulate intracellular ROS in a temporal manner.

Blood–brain barrier microfluidic chips and their applications

As a natural dynamic barrier separating blood from brain parenchyma, the blood–brain barrier (BBB) is mainly composed of brain microvascular endothelial cells (BMECs), pericytes, astrocytes, and a variety of neurons. The BBB regulates the highly selective transport of various substances between the brain and blood and maintains the stability of the central nervous system (CNS). Owing to this tight control, the BBB represents a formidable challenge for the delivery of drugs and other exogenous compounds into the CNS, which has bottlenecked the development of many drugs for CNS diseases. Therefore, efficient and precise in vitro models of the BBB are needed to assess the efficacy and toxicity of drugs targeting the CNS to inform drug design and to improve the success rate of agents that enter clinical evaluation. In vitro BBB models have rapidly advanced from the early two-dimensional (2D) static models to the current three-dimensional (3D) dynamic microfluidic chips. Although the commonly used, static, in vitro BBB models are simple to construct and TEER values are convenient to detect, the static models do not provide an ideal (i.e., accurate) BBB environment, since they lack the correct physiological size/scale and hemodynamic shear stress, both of which play substantial roles in promoting and maintaining EC differentiation into a specific BBB phenotype. Compared with traditional static models, 3D microfluidic models thus enable cells to react in a manner more closely resembling in vivo behavior by simulating a microenvironment with more natural signal transduction. As a result, the dynamic 3D BBB model can more accurately recapitulate the structure and function of the human BBB. Here we summarize the recent progress in in vitro microfluidic BBB chips and their research applications as well as discuss the prospects and challenges for where the technology is headed.

In Vitro Angiogenesis Inhibition and Endothelial Cell Growth and Morphology.

A co-culture assay with human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) was used to study whether selected angiogenesis inhibitors were able to inhibit differentiation and network formation of HUVECs in vitro. The effect of the inhibitors was determined by the morphology and the calculated percentage area covered by HUVECs. Neutralizing VEGF with avastin and polyclonal goat anti-VEGF antibody and inhibiting VEGFR2 with sorafenib and vatalanib resulted in the formation of HUVEC clusters of variable sizes as a result of inhibited EC differentiation. Furthermore, numerous inhibitors of the VEGF signaling pathways were tested for their effect on the growth and differentiation of HUVECs. The effects of these inhibitors did not reveal a cluster morphology, either individually or when combined to block VEGFR2 downstream pathways. Only the addition of N-methyl-p-bromolevamisole revealed a similar morphology as when targeting VEGF and VEGFR2, meaning it may have an inhibitory influence directly on VEGFR signaling. Additionally, several nuclear receptor ligands and miscellaneous compounds that might affect EC growth and differentiation were tested, but only dexamethasone gave rise to cluster formation similarly to VEGF-neutralizing compounds. These results point to a link between angiogenesis, HUVEC differentiation and glucocorticoid receptor activation.

Metformin Regulates TET2 Expression to Inhibit Endometrial Carcinoma Proliferation: A New Mechanism.

ObjectivesTo investigate the relationship between TET2 expression and endometrial cancer’s clinicopathological features and prognosis, and the effect of metformin on TET2 and 5hmC levels in endometrial cancer cells.MethodsThe clinical significance of TET2 expression in endometrial carcinoma was analyzed from TCGA public database. Eighty-eight patients with endometrial cancer and 20 patients with normal proliferative endometrium were enrolled in this study. TET2 and 5hmC were respectively detected by Immunohistochemistry and ELISA in endometrial tissues. Kaplan-Meier and Cox proportional hazard regression models were used to analyze relationships between TET2 and 5hmC and the overall survival of EC patients. Endometrial cell proliferation was assessed after TET2 gene knockdown. Western blotting and real-time PCR were used to detect the effect of metformin on TET2 expression and to explore whether AMPK is involved in metformin-mediated TET2 regulation.ResultsThe clinical significance of expression of TET2 in endometrial cancer from TCGA public database confirmed that TET2 expression was significantly down-regulated in cancer samples and TET2 expression was also significantly different among different histopathological samples and TET2 is down-regulated in advanced, high-grade, and relapsed endometrial carcinoma tissues(P<0.05). Immunohistochemical analysis showed that TET2 and 5hmC levels were significantly lower in endometrial adenocarcinoma(P<0.05). TET2 expression was correlated with the degree of EC differentiation (P < 0.05). 5hmC levels were associated with clinical stage, differentiation, the depth of myometrial invasion, and lymph node metastasis (P < 0.05). The mean survival time of patients with negative staining for TET2 and 5hmC was shorter than that of patients with positive staining for both markers (P<0.05). Multivariate Cox regression analysis showed that TET2 expression was an independent risk factor for prognosis in patients with endometrial adenocarcinoma (HR = 14.520, 95% CI was 1.From 060 to 198.843, P = 0.045). siRNA-mediated TET2 knockdown increased the proliferation of EC cells. Metformin increased the levels of TET2 and 5hmC in EC cells. AMPK was involved in the regulation of TET2 by metformin.ConclusionsTET2 may play an important role in EC development and may be a prognostic marker. Moreover, TET2 may be involved in a novel mechanism by which metformin inhibits EC cell proliferation.

Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells.

Background: Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. They have recently emerged as important regulators of angiogenesis. To identify novel lncRNAs that may be involved in the regulation of angiogenesis, we detected the mRNA of 84 lncRNAs in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia for 24h. One of these, rhabdomyosarcoma 2-associated transcript (RMST), is significantly upregulated by hypoxia. Little is known about the presence and roles of RMST in EC function.Objective: The main objective of the study was to investigate the regulation of RMST in ECs and to determine its role in EC survival, proliferation, migration, and differentiation.Methods: Using qPCR, basal mRNA levels of 10 RMST isoforms in HUVECs were measured. Levels were then measured in response to 24h of hypoxia, 7days of differentiation in a co-culture assay, and exposure to four different angiogenesis factors. Functional roles of RMST in EC survival, migration, and differentiation were quantified by using a loss-of-function approach (transfection with single-stranded antisense LNA GapmeRs). EC survival was measured using cell counts and crystal violet assays. Cell migration and differentiation were measured using scratch wound healing and Matrigel® differentiation assays, respectively.Results: Five RMST isoforms (RMST-202, -203, -204, -206, and -207) were detected in HUVECs and human microvascular endothelial cells (HMEC-1s). Other types of vascular cells, including human aortic valve interstitial cells and human aortic smooth muscle cells, did not display this expression profile. RMST was significantly upregulated in response to 24h of hypoxia and in response to 7days of HUVEC co-culture with human lung fibroblasts. RMST was significantly downregulated by angiopoietin-2 (Ang-2), but not by VEGF, FGF-2, or angiopoietin-1 (Ang-1). Selective knockdown of RMST demonstrated that it promotes EC survival in response to serum deprivation. It is also required for VEGF- and Ang-1-induced EC survival and migration, but not for differentiation.Conclusion: We conclude that RMST is expressed in human ECs and that this expression is upregulated in response to hypoxia and during differentiation into capillary-like structures. We also conclude that RMST plays important roles in EC survival and migration.

Abstract 722: Unveiling Ccbe1 Role as a Modulator of Cardiomyocyte Differentiation

Chronic heart failure is a major unmet clinical need arising from the loss of viable and functional cardiac muscle, representing a major cause of mortality worldwide. There is a need to identify key molecules and signaling pathways acting on the coronary vasculature system towards regenerative therapies. Human induced pluripotent stem cells (hiPSC) are an attractive cell source to understand the regulatory networks involved in cardiac commitment and cardiomyocyte (CM) differentiation. Particularly, CCBE1 (collagen and calcium-EGF biding domain 1) has been studied as a secreted protein critical for lymphatic/cardiac vascular development and recent reports have proposed that CCBE1 may potentially be used to restore cardiac tissue upon heart injury, through CCBE1-mediated cardiac commitment and/or augmentation of lymphangiogenesis. Therefore, the goal of our work is to understand the molecular pathways underlying CCBE1-based cardiac commitment in loss-of-function studies. By exploring gene editing and “omics” tools we aim to unveil the molecular basis of CCBE1-induced cardiogenesis and provide novel insights towards the development of CCBE1-mediated therapeutic strategies for cardiac regenerative medicine. To selectively knock-down (KD) CCBE1 expression along hiPSC cardiac differentiation, we used modified hiPSC line with CRISPR interference technology (CRISPRi-hiPSC). The CCBE1 KD led to a reduction on the expression of cardiac troponin marker TNNT2 and on the ratios of MYH7:MYH6 and TNNI3:TNNI1. The CCBE1 KD-derived CMs also presented an immature-related ultrastructure, suggesting that CCBE1 may modulate the CM phenotype. On the other hand, the EC differentiation was not impaired by CCBE1 KD. A comprehensive and integrated characterization of the transcriptome and proteome along the differentiation in the presence or absence of CCBE1 is being pursued to identify the key players at cardiac mesoderm and cardiac progenitors stages and their interactions with CCBE1. This work opens new avenues for the identification of CCBE1-modulated proteins/pathways in cardiac commitment, which may contribute for novel cell-based or cell-free approaches towards more efficacious cardiac regenerative therapies.

Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling.

The mortality rate for (cardio)‐vascular disease is one of the highest in the world, so a healthy functional endothelium is of outmost importance against vascular disease. In this study, human induced pluripotent stem (iPS) cells were reprogrammed from 1 ml blood of healthy donors and subsequently differentiated into endothelial cells (iPS‐ECs) with typical EC characteristics. This research combined iPS cell technologies and next‐generation sequencing to acquire an insight into the transcriptional regulation of iPS‐ECs. We identified endothelial cell‐specific molecule 1 (ESM1) as one of the highest expressed genes during EC differentiation, playing a key role in EC enrichment and function by regulating connexin 40 (CX40) and eNOS. Importantly, ESM1 enhanced the iPS‐ECs potential to improve angiogenesis and neovascularisation in in vivo models of angiogenesis and hind limb ischemia. These findings demonstrated for the first time that enriched functional ECs are derived through cell reprogramming and ESM1 signaling, opening the horizon for drug screening and cell‐based therapies for vascular diseases. Therefore, this study showcases a new approach for enriching and enhancing the function of induced pluripotent stem (iPS) cell‐derived ECs from a very small amount of blood through ESM1 signaling, which greatly enhances their functionality and increases their therapeutic potential. Stem Cells 2019;37:226–239

Abstract 472: Large-Scale Single-Cell RNA-Seq Identifies Heterogeneous Populations of Human Primary and Induced Pluripotent Stem Cell-Derived Endothelial Cells

Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation and of primary endothelial cells isolated from human coronary arteries. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs sorted by CD144 antibody-conjugated magnetic sorting exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. These findings were then cross-analyzed with the primary EC data. Conclusions: Massively parallel scRNA-seq allowed analysis of thousands of human iPSCs subjected to EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity. Cross-analysis with scRNA-seq data of human coronary artery ECs reveals transcriptional similarities and dissimilarities of iPSC-ECs to the primary ECs that will be addressed in subsequent studies.

Abstract 97: Epigenetic Signatures Contribute to the Superior Endothelial Cell Identity in Human Induced Pluripotent Stem Cells Derived From Endothelial Cells

Human induced pluripotent stem cells (iPSCs) can be derived from multiple types of somatic cells by transient overexpression of four Yamanaka factors. Epigenetic memory of the tissue of origin is seen in early passage iPSCs, which may interfere the directed differentiation towards target lineages in disease modeling and drug discovery. Here we derived human iPSC from three types of somatic cells of the same individuals: fibroblast (FB-iPSCs), endothelial cells (EC-iPSCs) and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into endothelial cells by using sequential administration of Activin, BMP4, bFGF and vEGF. EC-iPSCs show higher EC differentiation propensity and EC-specific markers (PECAM1 and NOS3) gene expression in early passage iPSCs than FB-iPSCs and CPC-iPSCs. In vivo, EC-iPSC-ECs display significantly greater revascularization capacity than those of FB-iPSCs and CPC-iPSCs when transplanted to the hindlimb ischemic mice. In addition, transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31+ population and higher EC-specific markers (PECAM1, KDR and ICAM) gene expression by using single cell qPCR. In vitro, EC-iPSC-ECs exhibit better endothelial cell character maintenance along with extensive culturing and passaging. Several chromatin signatures, including H3K27ac, H3K4me1 and p300 were found highly enriched in ECs and EC-iPSCs, but not in human embryonic stem cells (ESCs). Gene ontology analysis indicates that the differentially enriched regions are primarily associated with angiogenesis and vascular development, reflecting the residual epigenetic signatures in EC-iPSCs. Finally EC-specific enhancer markers undergo dynamic changes during the process of EC fate commitment and differentiation, though the majority of them sustain conserved pattern in EC-iPSCs, CPC-iPSCs and FB-iPSCs. In conclusion, these results highlight that the residual epigenetic signatures of tissue of origin may affect lineage differentiation propensity in early-passage human iPSCs.

Dpp/Gbb signaling is required for normal intestinal regeneration during infection

Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues with a high intrinsic turnover, such as the intestinal epithelium, must launch a rapid response to infections while simultaneously coordinating cell proliferation and differentiation decisions. In this study, we searched for genes required for regeneration of the Drosophila intestine, and thereby affecting overall organism survival after infection with pathogenic bacteria. We found that Dpp/Gbb (BMP) signaling is essential for normal midgut regeneration, and that infection induces the BMP signaling ligands Dpp and Gbb. We demonstrate that Dpp is induced in visceral muscle and required for signaling activation. Subsequently, Gbb is induced in enterocytes after oral infection. Loss-of Dpp signaling in ISCs and transient committed progenitors called enteroblasts (EBs), or in EBs alone, led to a blockage in EC differentiation or maturation. Furthermore, our data show that down-regulation of Dpp signaling in the precursor cells including EBs also resulted in an increased number of abnormally small Pdm1-positive cells, suggesting a role of Dpp/Gbb signaling in EC growth. In addition, we show that Dpp/Gbb signaling acted downstream or in parallel to the Notch pathway to promote EC differentiation and growth. Our results suggest that Dpp/BMP signaling plays an important role in EBs to maintain tissue integrity and homeostasis during pathogenic infections.

Investigating in vitro angiogenesis by computer-assisted image analysis and computational simulation.

In vitro assays that stimulate the formation of capillary-like structures by EC have become increasingly popular, because they allow the study of the EC's intrinsic ability to self-organize to form vascular-like patterns. Here we describe a widely applied protocol involving the use of basement membrane matrix (Matrigel) as a suitable environment to induce an angiogenic phenotype in cultured EC. EC differentiation on basement membrane matrix is a highly specific process, which recapitulates many steps in blood vessel formation and for this reason it is presently considered as a reliable in vitro tool to identify factors with potential antiangiogenic or pro-angiogenic properties. The morphological features of the obtained cell patterns can also be accurately quantified by computer-assisted image analysis and the main steps of such a procedure will be here outlined and discussed. The dynamics of in vitro EC self-organization is a complex biological process, involving a network of interactions between a high number of cells. For this reason, the combined use of in vitro experiments and computational modeling can represent a key approach to unravel how mechanical and chemical signaling by EC coordinates their organization into capillary-like tubes. Thus, a particularly helpful approach to modeling is also briefly described together with examples of its application.

Extracellular matrix‐mediated endothelial differentiation of human induced pluripotent stem cells (1152.4)

Over 8 million Americans are suffering from Peripheral Arterial Disease (PAD), which is due to atherosclerotic occlusion of the peripheral arteries of the limbs. Recent studies suggest that human induced pluripotent stem cell (iPSC)‐derived endothelial cells (iPSC‐ECs) can serve as a promising source for treatment of PAD. However, the low differentiation efficiency of iPSC‐ECs is a major limitation to their clinical use. The purpose of this study was to investigate the role of extracellular matrix (ECM) and cell‐ECM interactions in endothelial differentiation of iPSCs utilizing high throughput ECM microarray technique. We developed a high‐throughput ECM microarray that contains circular printed areas of ECMs on glass slides using a robotic DNA microarrayer. Multi‐component mixtures of purified ECMs components (gelatin, fibronectin, laminin, heparin sulfate proteoglycans, collagen IV, and matrigel) were deposited onto glass slides at fixed distances. A total of 280 spotted matrix features were fabricated with multi‐component mixtures of the ECM components. After dissociating and culturing iPSCs onto ECM microarrays, differentiation was induced by adding VEGF (50ng/ml) for 5 days. EC differentiation was confirmed with CD31 staining, whose expression was scored semi‐quantitatively and averaged among the six replicates on the slide. A greater abundance of CD31 with higher score was observed in ECM features composed of gelatin + laminin + fibronectin tri‐component mixtures comparing to other conditions. Gene expressions of various integrin subunits were compared between iPSCs and iPSC‐ECs to elucidate the role of cell‐ECM interactions. The expression of integrins α1 through α6 and β1 through β4 were elevated in iPSC‐ECs comparing to iPSCs. On the other hand, β5 shows a significant decrease in iPSC‐ECs. In total, this data suggested that ECM differentially promote iPSC differentiation into EC, possibly through binding to various types of integrins.Grant Funding Source: NIH, NSF, Stanford CVI

O186 Nucleosome Assembly Protein 1-Like 1 Knockdown Promotes Cardiomyocytes Differentiation By Notch Signaling In Mouse Induced Pluripotent Stem Cells

miR-199b overexpression. Futhermore, we found that the effect of Jagged-1 on iPS cell-EC differentiation can be contract by miR-199b inhibitor. meanwhile, we also found that miR199b can regulate the ips differentiation into ECs through VEGF signalling pathway. Conclusion: we conclude that miR-199b plays a critical role in the determination of EC fate during iPS cell–EC differentiation by target JAG-1, which may indicate that miR-199b has a role to promote EC differentiation. Disclosure of Interest: None Declared

The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells

Current EC differentiation protocols are inefficient, and the phenotypes of the differentiated ECs are only briefly stable, which significantly inhibits their utility for basic science research. Here, a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively, these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and, furthermore, the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks in vitro.

Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells.

While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models.A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01).Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

Abstract 222: Dkk1 and Msx2-Wnt7b Signaling Reciprocally Regulate the Endothelial-Mesenchymal Transition in Aortic Endothelial Cells

Objective Endothelial cells (ECs) can undergo an endothelial-mesenchymal transition (EndMT) during tissue fibrosis. Wnt- and Msx2-regulated signals participate in arteriosclerotic calcification and fibrosis. We studied the impact of Wnt7, Msx2, and Dkk1 (Wnt7 antagonist) on EndMT in primary aortic endothelial cells (AoECs). Methods and Results Transduction of AoECs with vectors expressing Dkk1 suppressed EC differentiation and induced a mineralizing myofibroblast phenotype. Dkk1 suppressed claudin 5, PECAM, cadherin 5 (Cdh5), Tie1 and Tie2. Dkk1 converted the cuboidal cell monolayer into a spindle-shaped multilayer and inhibited EC cord formation. Myofibrogenic and osteogenic markers - e.g., SM22, type I collagen, Osx, Runx2, alkaline phosphatase – were upregulated by Dkk1 via activin-like kinase / Smad pathways. Dkk1 increased fibrosis and mineralization of AoECs cultured under osteogenic conditions - the opposite of mesenchymal cell responses. Msx2 and Wnt7b maintained the “cobblestone” morphology of differentiated ECs and promoted EC marker expression. Deleting EC Wnt7b with the Cdh5-Cre transgene in Wnt7b(fl/fl);LDLR-/- mice upregulated aortic osteogenic genes (Osx, Sox9, Runx2, Msx2) and nuclear pSmad1/5, and increased collagen accumulation. Conclusions Dkk1 enhances EndMT in AoECs, while Msx2-Wnt7 signals stabilize EC phenotype. EC responses to Dkk1, Wnt7b, and Msx2 are the opposite of mesenchymal cell responses, coupling EC phenotypic stability with osteofibrogenic predilection during arteriosclerosis.

High-level Shear Stress Stimulates Endothelial Differentiation and VEGF Secretion by Human Mesenchymal Stem Cells

Growing experimental evidence suggests that mechanical stimulation play important roles in determining the proliferation, migration, and apoptosis of human mesenchymal stem cells (hMSCs). Here, we show that shear stress stimulates hMSCs toward an EC phenotype in the absence of chemical induction. Most importantly, fluorescence microscopy clearly demonstrated for the first time that the distributions of endothelial-specific markers, vascular endothelial (VE)-cadherin and CD31, in hMSCs were similar to those of ECs at cell–cell adhesion sites after exposing hMSCs to a shear stress of 2 Pa for 2 days with subsequent static culture for 5 days. Western blot analysis proved that shear stress of 2 Pa significantly induced protein expression of von Willebrand factor (vWF), VE–cadherin, and CD31. However, an unclear expression of the endothelial-specific markers was observed in the 0.2 Pa shear stress group. In addition, there was a cumulative production of vascular endothelial growth factor (VEGF), which is known to induce endothelial differentiation of MSCs. By exerting shear stress of 2 Pa on hMSCs for 2 days with subsequent culture for 5 days, the production level of approximately 2-fold compared with that of the control group was achieved. Our findings suggest that high-level shear stress can induce VEGF production and EC differentiation from hMSCs. This may provide a means for addressing the cell sourcing issue for effective tissue engineering.

Suicide Gene Reveals the Myocardial Neovascularization Role of Mesenchymal Stem Cells Overexpressing CXCR4 (MSCCXCR4)

BackgroundOur previous studies indicated that MSCCXCR4 improved cardiac function after myocardial infarction (MI). This study was aimed to investigate the specific role of MSCCXCR4 in neovascularization of infarcted myocardium using a suicide gene approach.MethodsMSCs were transduced with either lentivirus-null vector/GFP (MSCNull as control) or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC) differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSCNull or MSCCXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV) or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging.ResultsThe expression of VEGF-A and HIF-1α was significantly higher in MSCCXCR4 as compared to MSCNull under hypoxia. Additionally, MSCCXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSCCXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor). In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSCCXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSCCXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function.ConclusionThe transplanted MSCCXCR4 enhanced neovascularization after MI by boosting release of angiogenic factors and increasing the potential of endothelial differentiation.

One year stored corneas: is it possible?

AbstractPurpose To evaluate the endothelial characteristics after very long term organ culture (OC) of human corneas.Methods Human corneas with initial endothelial cell density (ECD) &gt;2000 cells/mm2 were stored at 31°C in sealed flasks containing 100mL of a commercial medium with 2% foetal calf serum (CorneaMax, Eurobio, France): 6 for 3‐6 months (M), 7 for 6‐12 M and 5 for more than 12 M. The medium was renewed every 2 M. Transparency was quantified by analysis of modulation and contrast transfer functions. On one half of each cornea, ECD and EC morphology were analysed by image analysis (CellP‐Olympus, and SambaCornea‐TribVn) after alizarin red staining and flat mounting. On the other half of the cornea, EC differentiation status (NaK ATPase, ZO‐1, JAM‐1) and proliferative capacity (Ki67, Click‐it EdU) were studied by immunostaining in flat mounted corneas (He, MolVis2011;17:3494 and He, StemCells2012 in press). Epithelial cells and stroma were observed by histology on cross sections stained with Hematein/Eosin/Safran and by transmission electron microscopyResults Corneal transparency and ECD decreased after very long‐term OC, but were partially preserved. ECD was about 1200, 800 and 400 cell/mm2 after respectively 3, 6 and 12 months. Morphology and differentiation of residual EC remained nevertheless almost preserved. The epithelium lost its multilayered organizationConclusion Corneal cells, particularly EC, can survive for a very long time in the rustic environment of OC, suggesting that they are well equipped against environmental stress and cell death. The improvement of storage process and/or storage medium could potentially allow very long term storage without significant attrition of graft quality and therefore provide corneas suitable for graft