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Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions.

ABSTRACT: The avascular cornea is a uniquely-isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose in the behaviour of human corneal keratocytes has been overlooked. As such, we sought to investigate the effects of low-glucose formulations on the phenotype of human corneal stromal cells. Cells cultured in low-glucose were able to survive for extended periods when compared to high-glucose, serum-free conditions. Furthermore, low-glucose enhanced their reversal to a keratocyte-characteristic phenotype. Specifically, cells within low-glucose medium assumed dendritic morphologies, with bean-shaped condensed nuclei, absence of alpha-smooth muscle actin or stress fibres, and a corresponding reduction in migratory and contractile activities when compared with high-glucose, serum-free conditions. Moreover, cells within low-glucose uniquely recovered the ability to express a robust keratocyte-characteristic marker, CD34, while still expressing elevated levels of other representative phenotypic markers such as keratocan, lumican, ALDH1A1, and ALDH3A1. These results indicate that low-glucose enhances keratocyte-characteristic phenotype above and beyond established media formulations and thus has important implications for corneal biology in health and disease.

SUBMITTER: Foster JW

PROVIDER: S-EPMC4650697 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions.

Foster James W JW Gouveia Ricardo M RM Connon Che J CJ

Scientific reports 20150603

The avascular cornea is a uniquely-isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose in the behaviour of human corneal keratocytes has been overlooked. As such, we sought to investigate the effects of low-glucose formulations on the phenot ...[more]

PMID: 26039975

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Project description:In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.

Project description:Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

Dataset Information

Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions.

Publications

Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions.

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