scholarly journals Bevacizumab Induces Upregulation of Keratin 3 and VEGFA in Human Limbal Epithelial Cells in Vitro

2019 ◽  
Vol 8 (11) ◽  
pp. 1925 ◽  
Author(s):  
Maria Notara ◽  
Anna Lentzsch ◽  
Thomas Clahsen ◽  
Sara Behboudifard ◽  
Gabriele Braun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Wound Closure ◽  
Antiangiogenic Therapy ◽  
Protein Quantification ◽  
Epithelial Stem Cells ◽  
Scratch Wound ◽  
Forming Efficiency ◽  
Limbal Epithelial Stem Cells

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation.

333 GENERATION OF OOCYTE-LIKE STRUCTURE FROM OVARIAN SURFACE EPITHELIAL STEM CELLS OF GOAT

2015 ◽  
Vol 27 (1) ◽  
pp. 255 ◽  
Author(s):  
S. Fatima ◽  
V. Sharma ◽  
S. Saini ◽  
S. Saugandhika ◽  
H. N. Malik ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endangered Species ◽  
Epithelial Cells ◽  
Ovarian Surface Epithelium ◽  
Surface Epithelium ◽  
Epithelial Stem Cells ◽  
Rt Pcr ◽  
Ovarian Surface ◽  
Ovarian Surface Epithelial

Stem cells have potential for therapeutic application. Continuous repair of ovarian surface epithelium following folliculogenesis and ovarian carcinoma suggests the presence of stem cells in ovarian epithelial cells. In vitro gametogenesis in livestock will result in large numbers of oocytes production from a single ovary, resulting in faster multiplication of superior germplasm of livestock species, treatment of infertile animals, and conservation of endangered species. The present study was conducted with the objective of in vitro differentiation of putative ovarian surface epithelial stem cells into oocyte-like structures in goat model. Ovary samples of 1- to 2-year-old goats were collected from slaughterhouse. The surface of the ovary was gently scraped using sterile blunt scraper to isolate ovarian surface epithelial stem cells. These scraped cells were cultured in DMEM/F12 supplemented with 20% FBS for 3 weeks in 5% CO2 at 37°C with maximum humidity. The cultured stem cells were characterised for stemness by RT-PCR and immunostaining for Oct4, Sox2, and Nanog genes after 3 weeks. These putative stem cells were in vitro differentiated spontaneously to oocyte-like structures in DMEM/F12 medium and characterised for premeiotic markers by RT-PCR and immunostaining for VASA, DAZL, and STELLA genes. Results of this study provide evidence for the presence of putative stem cells with pluripotent characteristics in the ovarian surface epithelium. The cultured cells were found to be round in shape, with a high nucleus to cytoplasm ratio under inverted microscope, and found positive for stem cell markers of Oct4, Sox2, and Nanog genes. A total of 66 oocyte-like structures were produced from 12 ovaries. These oocyte-like structures were nearly similar to oocytes produced in vivo, both morphologically and in molecular gene expression. The oocyte-like structures were also found positive for premeiotic markers of VASA, DAZL, and STELLA genes by RT-PCR and immunostaining. From this study, we concluded that the ovarian surface epithelial cells have putative stem cells which can be in vitro differentiated into oocyte-like structures in goat. These oocyte-like structures need further characterisation of their surface membrane, more molecular markers, and following their developmental potential. These oocytes can help for multiplication of elite germplasm, curing infertile animals, and saving endangered species.

Collagen IV synthesis is restricted to the enteroendocrine pathway during multilineage differentiation of human colorectal epithelial stem cells

2001 ◽  
Vol 114 (11) ◽  
pp. 2055-2064
Author(s):  
Susan C. Kirkland ◽  
Karen Henderson
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Collagen Iv ◽  
Model System ◽  
Enteroendocrine Cells ◽  
Multilineage Differentiation ◽  
Epithelial Stem Cells ◽  
Enteroendocrine Cell ◽  
Cell Matrix

The human large intestine is lined by a rapidly renewing epithelial monolayer where cell loss is precisely balanced with cell production. The continuous supply of new cells is produced by undifferentiated multipotent stem cells via a coordinated program of proliferation and differentiation yielding three epithelial lineages: absorptive, goblet and enteroendocrine. Cell-matrix interactions have been suggested to be regulators of the multilineage differentiation program of the colorectal crypt but the expression of matrix proteins or their receptors does not appear to have the subtlety expected for this task. We have developed an in vitro model system of intestinal epithelial stem cells to facilitate the direct analysis of stem cells undergoing lineage commitment and differentiation. Using this culture system, we can now directly investigate the role of cell-matrix signalling in stem-cell decisions. In this study, collagen-IV synthesis has been followed in monolayers of multipotent cells that have been induced to differentiate into absorptive, goblet and enteroendocrine cells. Our experiments demonstrate that commitment to the enteroendocrine lineage is specifically accompanied by the expression of type-IV collagen that remains enteroendocrine-cell associated. Undifferentiated cells, absorptive cells and goblet cells do not express collagen IV. To confirm that the differential lineage-specific expression of collagen IV observed in the model system was representative of the in vivo situation, collagen-IV synthesis was analysed in isolated human colorectal crypts and tissue sections using immunocytochemistry and in situ hybridisation. These studies confirmed the in vitro findings, in that implementation of the enteroendocrine differentiation program involves synthesis and accumulation of a collagen-IV matrix. Thus, human colorectal enteroendocrine cells are unique in the colorectal crypt in that they assemble a cell-associated collagen-IV-rich matrix not observed on other colorectal epithelial cells. This study provides the first evidence for differential matrix synthesis between colorectal epithelial lineages in human colorectal epithelium. The specialised pericellular environment of the enteroendocrine cells might explain some of the unique phenotypic characteristics of this cell lineage. Furthermore, these findings suggest a potential mechanism whereby individual epithelial cells could modulate their cell-matrix signalling even while rapidly migrating in heterogeneous sheets over a shared basement membrane.

scholarly journals Comparative Analysis of KnockOut™Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Shaokun Zhang ◽  
Zaoxia Liu ◽  
Guanfang Su ◽  
Hong Wu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Epithelial Stem Cells ◽  
Fetal Bovine ◽  
Limbal Epithelial Stem Cells

The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.

scholarly journals Limbal Niche Cells in Three-Dimensional Matrigel Induced Dedifferentiation of Mature Corneal Epithelial Cells toward a Progenitor State

2020 ◽  
Author(s):  
Hui Zhu ◽  
Wei Wang ◽  
Lingjuan Xu ◽  
Menglin Jiang ◽  
Yongyao Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Single Cells ◽  
Three Dimensional ◽  
Stem Cell Marker ◽  
Epithelial Stem Cells ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Epithelial Marker

ABSTRACTPurposeTo investigate the possibility and the key factors of stably committed mature corneal epithelial cells dedifferentiate into corneal epithelial stem cells in vitro.MethodsMature cornea epithelia cell (MCEC) sheets or limbal epithelial progenitor cell (LEPC) sheets were isolated from central corneas or limbal segments by Dispase II and further digested with 0.25% trypsin/1 mM EDTA (T/E) to yield single cells. Limbal niche cells (LNC) were isolated from the limbal stroma by collagenase A and expanded on 5% Matrigel coated plastic. Single MCECs were seeded on 50% Matrigel with or without LNC culturing for 10 days, regarding as three-dimensional MCEC (3D-MCEC) group or three-dimensional MCEC+LNC (3D-MCEC+LNC) group. Expression of CK12, p63α, PCK, Vimentin were analyzed with immunofluorescence staining.ResultsThe expression of mature cornea epithelial marker (CK12) in MCEC was higher than that in LEPC (P=0.020) but epithelial stem cell marker (p63α) was lower than that in LEPC (P=0.000). When seeded in 3D Matrigel, single MCEC cells could form spheres within 72 hours, and the expression of CK12 reduced (P=0.005) and the expression of p63α also reduced to zero (P=0.000) compared to MCEC. Serial passages of LNC which were expanded in coated Matrigel could form spheres in 3D Matrigel. After mixing MCECs with LNC, rounder spheres emerged within 24 hours which consisted of both epithelia cells (PCK+/Vim-) and LNC (PCK-/Vim+). Moreover, epithelia cells in 3D-MCEC+LNC group expressed less CK12 and more p63α than those in MCEC group (P=0.043, 0.000). Besides, the diameter of spheres in 3D-MCEC+LNC group were larger than that in 3D-MCEC group (P=0.000).ConclusionHuman LNC and three-dimensional Matrigel could induce the dedifferentiation of mature corneal epithelial cells into corneal epithelial stem cells.

scholarly journals Peripheral (not central) corneal epithelia contribute to the closure of an annular debridement injury

2019 ◽  
Vol 116 (52) ◽  
pp. 26633-26643 ◽  
Author(s):  
Mijeong Park ◽  
Alexander Richardson ◽  
Elvis Pandzic ◽  
Erwin P. Lobo ◽  
J. Guy Lyons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Essential Role ◽  
Wound Closure ◽  
Cell Behavior ◽  
Epithelial Stem Cells ◽  
Self Renewal ◽  
Central Cornea ◽  
Limbal Epithelial Stem Cells

Corneal epithelia have limited self-renewal and therefore reparative capacity. They are continuously replaced by transient amplifying cells which spawn from stem cells and migrate from the periphery. Because this view has recently been challenged, our goal was to resolve the conflict by giving mice annular injuries in different locations within the corneolimbal epithelium, then spatiotemporally fate-mapping cell behavior during healing. Under these conditions, elevated proliferation was observed in the periphery but not the center, and wounds predominantly resolved by centripetally migrating limbal epithelia. After wound closure, the central corneal epithelium was completely replaced by K14+limbal-derived clones, an observation supported by high-resolution fluorescence imaging of genetically marked cells in organ-cultured corneas and via computational modeling. These results solidify the essential role of K14+limbal epithelial stem cells for wound healing and refute the notion that stem cells exist within the central cornea and that their progeny have the capacity to migrate centrifugally.

scholarly journals Paracrine activity of adipose derived stem cells on limbal epithelial stem cells

2021 ◽  
Author(s):  
Bartosz Sikora ◽  
Aleksandra Skubis-Sikora ◽  
Agnieszka Prusek ◽  
Joanna Gola
Keyword(s):  
Stem Cells ◽  
Culture Cell ◽  
Elisa Test ◽  
Inflammatory Factors ◽  
Epithelial Stem Cells ◽  
Limbal Stem Cells ◽  
Paracrine Effect ◽  
Expression Of Genes ◽  
Limbal Epithelial Stem Cells

Abstract Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of people for the cornea transplantation all over the world and the list of waiting patients is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and mitigates the adverse impact of induced inflammation.

scholarly journals Paracrine activity of adipose derived stem cells on limbal epithelial stem cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bartosz Sikora ◽  
Aleksandra Skubis-Sikora ◽  
Agnieszka Prusek ◽  
Joanna Gola
Keyword(s):  
Stem Cells ◽  
Culture Cell ◽  
Elisa Test ◽  
In Vitro Assays ◽  
Inflammatory Factors ◽  
Epithelial Stem Cells ◽  
Limbal Stem Cells ◽  
Paracrine Effect ◽  
Limbal Epithelial Stem Cells

AbstractLimbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of patients for the cornea transplantation all over the world and the list is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and has a potential role in mitigation of the adverse impact of inflammation induced by lipopolysaccharide.

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