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Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.

ABSTRACT: The cornea is covered by a stratified epithelium that is renewed by stem cells located in the peripheral region of the cornea known as the limbus. This stroma of the limbus contains stromal keratocytes that, when expanded in culture, are termed limbal fibroblasts (LFs). It is thought that LFs exhibit similar characteristics to bone marrow mesenchymal stem cells (BM MSCs) and help maintain the epithelial stem cell phenotype in the limbal region. In this study, we aimed at reprogramming stage-specific embryonic antigen-4 (SSEA4+) LFs and BM MSCs into corneal epithelial lineage using a three-dimensional culture system and embryonic stem cell medium. After enrichment, SSEA4+ cells showed a higher level of stem cell marker expression such as Sox2, Oct4, Nanog, Rex1, ABCG2, and TRA-1-60, and colony-forming efficiency than did SSEA4- cells. SSEA4+, as compared to SSEA4- cells, had a greater propensity to form spheres that, in turn, were induced into ectodermal lineage and further differentiated into functional corneal epithelium. Results show that LFs were similar to BM MSCs in marker profiles, and together with the differences noted between SSEA4+ and SSEA4- cells, point to LFs' being tissue-specific MSCs. However, LFs showed a greater potential for differentiation into corneal epithelium, indicating the potential importance of tissue-specific adult progenitors in their reprogramming capacity into cells of interest. This study opens a new avenue for investigating the molecular mechanism involved in maintaining a limbal stem cell niche and thus a potentially important clinical application to treat corneal epithelial stem cell loss.

SUBMITTER: Katikireddy KR

PROVIDER: S-EPMC8262232 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PURPOSE: To investigate the modulatory effect of rat bone marrow mesenchymal stem cells (MSC) on human corneal epithelial cells (HCE-T) stimulated with pro-inflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in an in vitro co-cultured model. METHODS: HCE-T alone and co-cultured with MSC were stimulated with IFN-γ/TNF for 24 and 48 hours or left untreated. The expression of intracellular adhesion molecule (ICAM)-1, human leukocyte antigen ABC, DR and G (HLA-ABC, HLA-DR, HLA-G) were investigated by flow cytometry. Subcellular localization of nuclear factor-kappa B (NF-κB) and expression of indoleamine 2,3-dioxygenase (IDO) were assessed by immunofluorescence staining and western blot. The concentration of transforming growth factor beta 1 (TGF-β1) in the conditioned media from different cultures was evaluated by enzyme-linked immunosorbent assay. NF-κB and TGF-β1 signaling pathway blocking experiments were performed to analyze associations between the expression of cell surface molecules and the NF-κB transcription pathway, and the expression of IDO and TGF-β1 signaling pathway. RESULTS: IFN-γ/TNF treatment significantly up-regulated expression of ICAM-1, HLA-ABC, and induced de novo expression of HLA-DR and IDO on HCE-T cultured alone, while HLA-G expression remained unaffected. Up-regulation was significantly inhibited by co-culture with MSC. Increased TGF-β1 secretion was detected in 48 h IFN-γ/TNF-stimulated MSC monocultures and HCE-T/MSC co-cultures. MSC attenuated the activation of cytokine-induced NF-κB and IDO induction. Blockade of NF-κB transcription pathway by BMS-345541 significantly reduced the up-regulation of ICAM-1, HLA-ABC, HLA-DR and IDO expression, while blockade of TGF-β1 signaling pathways reversed the modulatory effect of MSC on IDO expression. CONCLUSIONS: MSC reduced the expression of adhesion and immunoregulatory molecules on pro-inflammatory cytokine-stimulated HCE-T via the NF-κB transcription pathway. MSC attenuated expression of IDO through both NF-κB transcription and TGF-β1 signaling pathways. Co-culture of HCEC with MSC therefore provides a useful in vitro model to study the anti-inflammatory properties of MSC on corneal epithelium.

Project description:ObjectiveMouse bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our previous studies demonstrated that BMSCs can differentiate in the direction of EECs when co-cultured with endometrial stromal cells in vitro. Here, we obtain and analyse differential proteins and their relevant pathways in the process of BMSCs differentiating into EECs by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis.MethodsA 0.4-μm pore size indirect co-culture system was established with female mice endometrial stromal cells (EStCs) restricted in the upper Transwell chamber and BMSCs in the lower well plate. After indirect co-culture for several days, the BMSCs were revealed to progressively differentiate towards EECs in vitro. Then, four groups were divided according to different co-culture days with single culture groups of BMSCs as controls. Proteins were detected using iTRAQ based on 2DLC-ESI-MS/MS and data were analysed by bioinformatics.ResultsA total number of 311 proteins were detected, of which 210 proteins were identified with relative quantitation. Among them, 107 proteins were differentially expressed with a 1.2-fold change as the benchmark, with 61 up-regulated and 46 down-regulated proteins. Differential proteins CK19 and CK8 were epithelial markers and upregulated. Stromal marker vimentin were downregulated. Top canonical pathways was "remodeling of epithelial adhesions junctions" and "actin cytoskeleton signaling". Top networks was "cell-to-cell signaling and interaction, tissue development and cellular movement" regulated by ERK/MAPK and α-catenin.ConclusionTo the best of our knowledge, this is the first preliminary study of differential protein expression in the differentiation process of BMSCs into EECs in vitro. We further elucidated BMSCs differentiated in the direction of EECs. In addition, ERK/MAPK and α-catenin played important roles by regulating core differential proteins in the "cell-to-cell signaling and interaction, tissue development and cellular movement" network.

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Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.

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