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

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab333 ◽

2015 ◽

Vol 27 (1) ◽

pp. 255 ◽

Cited By ~ 1

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.

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

10.1101/2020.06.01.128629 ◽

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.

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

Journal of Clinical Medicine ◽

10.3390/jcm8111925 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1925 ◽

Cited By ~ 1

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.

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EXPANSION OF HEMATOPOIETIC STEM CELLS IN VITRO AS A MODEL SYSTEM FOR HUMAN TISSUE ENGINEERING

Orthopedic Clinics of North America ◽

10.1016/s0030-5898(05)70167-x ◽

2000 ◽

Vol 31 (3) ◽

pp. 499-509 ◽

Cited By ~ 3

Author(s):

Joel S. Greenberger ◽

Julie P. Goff ◽

Jason Bush ◽

Alfred Bahnson ◽

Douglas Koebler ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Human Tissue ◽

Model System ◽

Hematopoietic Stem

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CP30, a Cysteine Proteinase Involved inTrichomonas vaginalis Cytoadherence

Infection and Immunity ◽

10.1128/iai.68.9.4907-4912.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 4907-4912 ◽

Cited By ~ 82

Author(s):

M. Remedios Mendoza-López ◽

Cecilia Becerril-Garcia ◽

Loriz V. Fattel-Facenda ◽

Leticia Avila-Gonzalez ◽

Martha E. Ruíz-Tachiquín ◽

...

Keyword(s):

Epithelial Cells ◽

Hela Cell ◽

Trichomonas Vaginalis ◽

Cysteine Proteinase ◽

Collagen Iv ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cell Monolayers ◽

Thiol Proteinase

ABSTRACT We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

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Porphyromonas gingivalis lipopolysaccharide stimulation in human periodontal ligament stem cells: role of epigenetic modifications to the inflammation

European Journal of Histochemistry ◽

10.4081/ejh.2017.2826 ◽

2017 ◽

Vol 61 (3) ◽

Cited By ~ 8

Author(s):

Francesca Diomede ◽

Soundara Rajan Thangavelu ◽

Ilaria Merciaro ◽

Monica D'Orazio ◽

Placido Bramanti ◽

...

Keyword(s):

Stem Cells ◽

Porphyromonas Gingivalis ◽

Inflammatory Disease ◽

Periodontal Ligament ◽

Model System ◽

Epigenetic Mechanism ◽

Periodontal Ligament Stem Cells ◽

Porphyromonas Gingivalis Lipopolysaccharide

Periodontitis is a chronic oral inflammatory disease produced by bacteria. Gingival retraction and bone and connective tissues resorption are the hallmarks of this disease. Chronic periodontitis may contribute to the risk of onset or progression of neuroinflammatory pathological conditions, such as Alzheimer’s disease. The main goal of the present study was to investigate if the role of epigenetic modulations is involved in periodontitis using human periodontal ligament stem cells (hPDLSCs) as an in vitro model system. hPDLSCs were treated with lipopolysaccharide of Porphyromonas gingivalis and the expression of proteins associated with DNA methylation and histone acetylation, such as DNMT1 and p300, respectively, and inflammatory transcription factor NF-kB, were examined. Immunofluorescence, Western blot and next generation sequencing results demonstrated that P. gingivalis lipopolysaccharide significantly reduced DNA methylase DNMT1, while it markedly upregulated the level of histone acetyltransferase p300 and NF-kB in hPDLSCs. Our results showed that P. gingivalis lipopolysaccharide markedly regulate the genes involved in epigenetic mechanism, which may result in inflammation induction. We propose that P. gingivalis lipopolysaccharide-treated hPDLSCs could be a potential in vitro model system to study epigenetics modulations associated with periodontitis, which might be helpful to identify novel biomarkers linked to this oral inflammatory disease.

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Low Oxygen Tension Potentiates Proliferation and Stemness but Not Multilineage Differentiation of Caprine Male Germline Stem Cells

10.21203/rs.3.rs-420611/v1 ◽

2021 ◽

Author(s):

Shiva Pratap Singh ◽

Suresh Dinkar Kharche ◽

Manisha Pathak ◽

Ravi Ranjan ◽

Yogesh Kumar Soni ◽

...

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Growth Characteristics ◽

Population Doubling ◽

Population Doubling Time ◽

Germline Stem Cells ◽

Multilineage Differentiation ◽

Low Oxygen ◽

Differentiation Potential

Abstract The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O2) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O2). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O2) and normoxic (21% O2) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

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