Vascular Endothelial-like Cells Research Articles

Vascularized characteristics and functional regeneration of three-dimensional cell reconstruction of oral mucosa equivalents based on vascular homeostasis phenotypic modification

Our prior research has effectively developed tissue-engineered vascularized oral mucosa equivalents (VOME); however, challenges such as low repeatability and stability, as well as the inability to accurately replicate the complexity of real blood vessels, were encountered. Therefore, this study aimed to screen the VOME and native oral mucosa vascular homeostasis phenotypes by tandem mass tag-tagged proteomics associated with laser capture microdissection and human angiogenesis antibody array technology. Then, lentiviruses were constructed and stably transfected with vascular endothelial-like cells (VELCs) to detect angiogenic capacity. HE, EdU Apollo tracer staining, immunofluorescence staining, scanning electron microscopy, biomechanical testing, and a small animal ultrasound imaging system were used to analyze the characteristics of vascularization homeostasis and monitor functional regeneration of the vascularized homeostatic phenotypic oral mucosal equivalents (VHPOME). The results showed that PGAM1, COL5A1, ANG, and RNH1 are potential specific angiogenesis phenotypes. High expression of PGAM1, COL5A1, and ANG and/or low expression of RNH1 can promote the angiogenesis of VOME. ANG/shRNH1 has the most significant tube-like structure-formation ability. The expression of PGAM1, COL5A1, and ANG in the VHPOME group was higher than that of the control group, and the expression of RNH1 was lower than that of the control group. COL5A1/ANG can significantly improve the mechanical properties. The blood flow signal was most significant in the ANG/shRNH1 group. PGAM1, COL5A1, ANG, shRNH1, PGAM1/ANG, COL5A1/ANG, PGAM1/shRNH1, PGAM1/shRNH1, COL5A1/shRNH1, and ANG/shRNH1 may be the targets for establishing vascularization homeostasis and functional regeneration of oral mucosal equivalent genes (groups), and ANG/shRNH1 has the most significant effect.

Human endometrial perivascular stem cells exhibit a limited potential to regenerate endometrium after xenotransplantation.

What are the localization, characteristics and potential for tissue regeneration of two perivascular stem cells, namely CD34+ adventitial cells and CD146+ pericytes, in human endometrium? Human endometrial CD34+ adventitial cells (located in the outermost layer of blood vessels and mainly in the basal layer) and CD146+ pericytes showed mesenchymal stem cell (MSC) phenotypes in in vitro culture, but presented limited potential to regenerate endometrium. Periodic endometrial regeneration is considered to be maintained by MSCs. Blood vessel wall, regarded as stem cell niche, harbors a large reserve of progenitor cells that may be integral to the origin of MSCs. However, a lack of validated markers has hampered the isolation of putative endometrial MSCs. Currently, CD146+ pericytes and Sushi Domain Containing 2 (SUSD2) positive cells have been identified in the endometrial perivascular region as sharing MSCs characteristics. The locations of adventitial cells and pericytes in the human endometrium were identified by immunofluorescence staining (n = 4). After CD34+CD146-CD45-CD56-CD144- adventitial cells and CD146+CD34-CD45-CD56-CD144- pericytes were isolated from the endometrium of normal women (n = 6) by fluorescence-activated cell sorting, their characteristics were investigated in culture. Adventitial cells and pericytes were induced to differentiate, respectively, into vascular endothelial-like cells or endometrial stromal-like cells in vitro, with their potential explored by in vivo xenotransplantation (n = 2 in each group) and eutopic transplantation (n = 2 in each group). CD34+ adventitial cells and CD146+ pericytes were cultured in the inducing medium to differentiate into endothelial-like cells in vitro, and then analyzed for CD31, von Willebrand factor immunofluorescent staining and tube formation. They were also cultured to differentiate into endometrial stromal cells in vitro, with the expression of vimentin and CD13 being detected by western blot before and after induction, and the expression of prolactin and insulin-like growth factor-binding protein 1 being determined as well. Single dispersed CD34+ adventitial cells and CD146+ pericytes were respectively transplanted under the kidney capsule of NOG mice to investigate their differentiation potential in vivo. A eutopic transplantation model was constructed by grafting recellularized uterine matrix loaded up with CM-Dil labeled adventitial cells or pericytes into the injury region of nude rat's uterus. CD34+ adventitial cells were mainly located at the outmost layer of endometrial large vessels, while CD146+ pericytes were found surrounding the inner endothelial cells of microvessels. A small proportion of CD34+ adventitial cells expressed SUSD2. The number of adventitial cells was ∼40 times higher than that of pericytes in the endometrium. Both adventitial cells and pericytes showed MSC phenotypes after in vitro culture. After in vitro induction into endometrial endothelial-like cells and stromal-like cells, adventitial cells showed higher plasticity than pericytes and a closer correlation with stromal-like cells. In the mouse xenotransplantation model, vimentin+ cells, CD31+ endothelial-like cells and CD146+ pericyte-like cells could be observed after adventitial cells were transplanted. CM-Dil-labeled adventitial cells or pericytes could survive in the immunocompromised nude rats after eutopic transplantation, and vimentin+ cells were detected. In addition, CM-Dil-labeled adventitial cells or pericytes did not express α-smooth muscle actin or E-cadherin after transplantation. N/A. CD34 was chosen as a novel marker to isolate adventitial cells from human endometrium according to previous literature. The association of endometrial CD34+ adventitial cells and SUSD2+ MSCs should be further investigated. The decellularized uterine matrix model might be useful in endometrial stem cell therapy. L.D. is supported by grants from National Key Research and Development Program of China (2018YFC1004700), Nature Science Foundation of China (81871128, 81571391) and Nanjing Medical Science Development Project (ZKX16042). H.S. is supported by a grant from Jiangsu Province Social Development Project (BE2018602). X.Z. was supported by grants from the Postgraduate Innovative Project of Jiangsu Province (KYCX19-1177). The authors declare no conflict of interest.

Abstract PO-094: Human pluripotent stem cells acquire malignancy under tumor microenvironment

Abstract Cancer stem cell (CSC) theory suggests that cancer tissue contains a subpopulation of stem-like cells with self-renewal and differentiation potential as normal stem cells maintain organs by self-renewing and differentiation. Even in both cases of malignancy and normal the stemness is affected by the niche surrounding the stem cells. In the initial stage no stem cells should be related with cancer. The most important question is “How come the stem cells acquire malignancy?” Here in this study, using human induced pluripotent stem cells (iPSCs) we tried to generate cancer stem cells in the presence of the conditioned medium (CM) derived from human liver cancer cell line Hep 3B cells and human breast cancer cell line BT459 cells. The CM from both cells was collected when the cells became confluent and sterilized by 0.22 micrometer filter. The colonies of human iPSCs were detached from a mitomycin treated fibroblast feeder layer by collagenase and manually dissected into small pieces which were transferred onto ultra-low-attachment dishes. After 7 days the formed embryoid bodies (EBs) of human iPSCs were plated on Matrigel coated plates and maintained in Repro FF2 medium containing 5 ng/ml of basic FGF. Just after 24 hours from plating on Matrigel, the EBs of human iPSCs were treated with CM mixed with Repro FF2, in the ratio of 1:1. The medium was changed every two days with CM for 4 weeks. EBs cultured in the complete Repro FF2 medium without CM were prepared as the control but did not survive for 4 weeks. The survived cells were analyzed by RT-qPCR analysis and Immunofluorescence (IF) staining to confirm the expression of stemness and CSC markers and evaluated the conversion of iPSCs into CSCs. Distinct expression of self-renewal and CSC markers, such as Nanog, SOX2, CD44 and EPCAM, were observed in the survived cells. Sphere formation assay further confirmed the potential of self-renewal in the survived cells. The survived cells possessed differentiation potential into vascular endothelial-like cells. Thus, we concluded the iPSCs were converted into CSCs (hiPS-CSCs). Through these experiments, 2 different conditions derived from 2 independent cell lines Hep3B and BT549 cells were successful to hiPS-CSCs as hiPS-Hep3Bcm and hiPS-BT549cm cells. Furthermore, being treated with paclitaxel, the hiPS-CSCs exhibited potential of chemo-resistance maintaining the intact clonogenic morphology when compared to hiPSC. This study tries to summarize human cancer disease by utilizing the tumor microenvironment in the form of conditioned medium without any genetic manipulation. This should pave the way for the establishment of effective therapeutic agents. Citation Format: Said M. Afify, Ghmkin Hassan, Akimasa Seno, Yoshiaki Iwasaki, Masaharu Seno. Human pluripotent stem cells acquire malignancy under tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-094.

Study of tissue engineered vascularised oral mucosa-like structures based on ACVM-0.25% HLC-I scaffold in vitro and in vivo

Purpose To explore the feasibility of constructing tissue-engineered vascularised oral mucosa-like structures with rabbit ACVM-0.25% HLC-I scaffold and human gingival fibroblasts (HGFs), human gingival epithelial cells (HGECs) and vascular endothelial-like cells (VEC-like cells). Method Haematoxylin and Eosin (H&E) staining, immunohistochemical, immunofluorescence, 5-ethynyl-2′-deoxyuridine (EdU) staining and scanning electron microscope (SEM) were performed to detect the growth status of cells on the scaffold complex. After the scaffold complex implanted into nude mice for 28 days, tissues were harvested to observe the cell viability and morphology by the same method as above. Additionally, biomechanical experiments were used to assess the stability of composite scaffold. Results Immunofluorescence and Immunohistochemistry showed positive expression of Vimentin, S100A4 and CK, and the induced VEC-like cells had the ability to form tubule-like structures. In vitro observation results showed that HGFs, HGECs and VEC-like had good compatibility with ACVM-0.25% HLC-I and could be layered and grow in the scaffold. After implanted, the mice had no immune rejection and no obvious scar repair on the body surface. The biomfechanical test results showed that the composite scaffold has strong stability. Conclusion The tissue-engineered vascularised complexes constructed by HGFs, HGECs, VEC-like cells and ACVM-0.25% HLC-I has good biocompatibility and considerable strength.

Imbalanced Production of Reactive Oxygen Species and Mitochondrial Antioxidant SOD2 in Fabry Disease-Specific Human Induced Pluripotent Stem Cell-Differentiated Vascular Endothelial Cells

Fabry disease (FD) is an X-linked inherited lysosomal storage disease caused by α-galactosidase A (GLA) deficiency. Progressive intracellular accumulation of globotriaosylceramide (Gb3) is considered to be pathogenically responsible for the phenotype variability of FD that causes cardiovascular dysfunction; however, molecular mechanisms underlying the impairment of FD-associated cardiovascular tissues remain unclear. In this study, we reprogrammed human induced pluripotent stem cells (hiPSCs) from peripheral blood cells of patients with FD (FD-iPSCs); subsequently differentiated them into vascular endothelial-like cells (FD-ECs) expressing CD31, VE-cadherin, and vWF; and investigated their ability to form vascular tube-like structures. FD-ECs recapitulated the FD pathophysiological phenotype exhibiting intracellular Gb3 accumulation under a transmission electron microscope. Moreover, compared with healthy control iPSC-derived endothelial cells (NC-ECs), reactive oxygen species (ROS) production considerably increased in FD-ECs. Microarray analysis was performed to explore the possible mechanism underlying Gb3 accumulation-induced ROS production in FD-ECs. Our results revealed that superoxide dismutase 2 (SOD2), a mitochondrial antioxidant, was significantly downregulated in FD-ECs. Compared with NC-ECs, AMPK activity was significantly enhanced in FD-ECs. Furthermore, to investigate the role of Gb3 in these effects, human umbilical vein endothelial cells (HUVECs) were treated with Gb3. After Gb3 treatment, we observed that SOD2 expression was suppressed and AMPK activity was enhanced in a dose-dependent manner. Collectively, our results indicate that excess accumulation of Gb3 suppressed SOD2 expression, increased ROS production, enhanced AMPK activation, and finally caused vascular endothelial dysfunction. Our findings suggest that dysregulated mitochondrial ROS may be a potential target for treating FD.

Human gingival fibroblasts induced and differentiated into vascular endothelial-like cells.

A novel method for repair of vascular disease, mechanical damage, and tissue rebuilding is urgently required. Vascular endothelial cells (VECs) play an essential role in vascular rebuilding and vasotransplantation. In the present study, human gingival fibroblasts (HGFs) were cultured and induced into endothelial-like cells invitro in order to confirm that HGFs with stem cell properties possessed the potential for differentiation into endothelial-like cells. The epithelium was extracted from normal human gingiva consisting of epithelium and connective tissue, which was isolated from patients. The identification of HGFs and induced endothelial-like cells were confirmed by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunocytochemical stain (ICS), and immunofluorescence stain (ISA). The morphology of human gingival fibroblasts with 8ng/mL VEGF165 induced for different periods of days were observed by inverted microscope. Before induction, flow cytometry analysis showed that HGFs were positive for vimentin, but negative for CD31. RT-PCR, ICS, and ISA showed vimentin, S100A4, α-SMA, collagen III, and S100A4 were specifically expressed in these fibroblast cells. After induction, ICS showed induced vascular endothelial-like cells were positive for CD34 and CD31; ISA showed cells induced were positive for vWF and E-cadherin; RT-PCR results demonstrated that tie2 was specifically expressed in the cells induced. Flow cytometry analysis of the transformation efficiency from HGFs to endothelial-like cells. In conclusion, we found that HGFs possessed capacity for being induced and differentiated into vessel endothelial-like cells with typical and specific morphological, ultrastructural, and immunological characters of endothelial-like cells by induction with VEGF.

The New Role of CD163 in the Differentiation of Bone Marrow Stromal Cells into Vascular Endothelial-Like Cells.

Bone marrow stromal cells (BMSCs) can differentiate into vascular endothelial cells (VECs). It is regarded as an important solution to cure many diseases, such as ischemic diseases and diabetes. However, the mechanisms underlying BMSC differentiation into VECs are not well understood. Recent reports showed that CD163 expression was associated with angiogenesis. In this study, overexpression of CD163 in BMSCs elevated the protein level of the endothelial-associated markers CD31, Flk-1, eNOS, and VE-cadherin, significantly increased the proportion of Alexa Fluor 488-acetylated-LDL-positive VECs, and promoted angiogenesis on Matrigel. Furthermore, we demonstrated that CD163 acted downstream homeobox containing 1 (Hmbox1) and upstream fibroblast growth factor 2 (FGF-2). These data suggested that CD163 was involved in Hmbox1/CD163/FGF-2 signal pathway in BMSC differentiation into vascular endothelial-like cells. We found a new signal pathway and a novel target for further investigating the gene control of BMSC differentiation into a VEC lineage.

Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro

Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

Human basic fibroblast growth factor gene-modified rat bone marrow mesenchymal stem cells in vitro

Objective To investigate the capability of differentiation of human basic fibroblast growth factor (bFGF) gene-modified rat bone marrow mesenchvmal stem cells (BMSCs) into vascular endothelial-like cells in vitro.Methods BMSCs were separated from SD rat hone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface antigens of BMSCs in 3rd generation were examined by fluorecene activated cell sorter (FACS).BMSCs of the third generati were obtained and divided into three groups:normal group (general BMSCs group),blank control group (the GFP lentiviral vector without bFGF gene was transfected into BMSCs),experimental group (the GFP lentiviral vector containing bFGF target gene was transfected into MBSCs).Three groups were were cultured in the same way.[10％ FBS,50 μg/L vascular endothelial growth factor (VEGF)-DMEM].One week later,the level of bFGF was measured by using enzyme-linked immunosorhent assay (ELISA) in the supernatant of each group.Four weeks later,the morphological changes were observed,the expression levels of CD31,CD34 and the factor Ⅷ were detected by using immunocytochemistry,and the phagocytosis phenomenon of Dil-AC-LDL was examined.Results BMSCs could be separated and amplified in vitro.Cultured BMSCs at the third passage were positive for CD44 and CD90 [(97.87 ± 0.84) ％ and (96.85 ± 1.49) ％,respectively],but negative for CD11h/c and CD34 [(14.16 ± 0.72) ％ and (1.29 ± 0.46) ％,respectively].The bFGF level in experimental group [(5.50 ± 0.12) ng/L] was higher than in blank control group [(2.65 ± 0.40) ng/L] and normal group [(2.76 ± 0.25) ng/L] (both P ＜ 0.01).There was no significant difference in the morphological changes of BMSCs among three groups.The positive expression rate of CD31,CD34 and the factor Ⅷ in experimental group [(22.73 ±5.42)％,(26.32 ±3.95)％ and (30.34±6.62)％] was higher than in blank control group [(7.85 ±1.83)％,(8.93 ±1.37)％ and (10.23±1.21)％] and normal group [(7.57 ±1.21)％,(8.41 ±0.97)％ and (9.97 ±0.52)％](all P ＜ 0.01).After induction,the cells in experimental group could swallow Dil-AC-LD.The expression of mean absorbance value of the fluorescence in experimental group (0.52 ± 0.23) was significantly higher than in blank control group (0.05 ± 0.03) and normal group (0.07 ± 0.02) (both P ＜ 0.01).Conclusion The human bFGF gene-modified rat BMSCs can secrete bFGF.The gene-modified cells in the induction of VEGF can more effectively differentiate into endothelial-like cells in vitro. Key words: Basic fibroblast growth factor; Mesenchymal stem cells; Gene transfection; Differentiation

Cytosolic free Ca2+ concentration in canine aortic endothelial cells lining the polyester arterial prosthesis.

A rise in baseline cytosolic free Ca2+ in canine vascular endothelial-like cells (VEC) lining the luminal surface of the polyester arterial prosthesis is described. In one, three and six month implantation experiments we employed six adult mongrel dogs, polyester arterial prostheses Arteknit Ra K, fluorescent Ca2+ indicator Fura-2 and digital imaging microscopy to study cytosolic free Ca2+ in cultured VEC. The electron microscopy scanning of the luminal surface in different regions of the graft were also performed. A rise in cytosolic free Ca2+ in the VEC lining the luminal surface of the prosthesis is probably the result of the immunologic reaction and mechanical stress which stimulate the proliferation activity of the endothelial cells. It seems that the baseline cytosolic free Ca2+ reflects the course of the endothelization process on the polyester arterial prosthesis.