Corneal keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with fibroblast growth factor-2, transforming growth factor-β3 and retinoic acid

Abstract

. (2016) Corneal keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with FGF-2, TGF-ß3 and retinoic acid. Journal of Tissue Engineering and Regenerative Medicine . ISSN 1932-7005 Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/39574/1/Author%27s%20manuscript.pdf Copyright and reuse:The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. This article is made available under the University of Nottingham End User licence and may be reused according to the conditions of the licence. For more details see: http://eprints.nottingham.ac.uk/end_user_agreement.pdf A note on versions:The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. For more information, please contact eprints@nottingham.ac.ukThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/term.2316 This article is protected by copyright. All rights reserved. Immunofluorescence and RT-qPCR demonstrated in vivo keratocyte expression of ALDH3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not keratocyte markers. Differentiating the cMSC back to a keratocyte phenotype using non-supplemented, serum-free medium restored keratocyte markers but did not maintain cell viability or support corneal extracellular matrix (ECM) production.Supplementing the differentiation medium with combinations of fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and retinoic acid (RA) maintained viability, restored expression of CD34, ALDH3A1 and keratocan, and facilitated production of abundant ECM as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with RA, FGF-2 and TGF-β3 can enhance this effect. This is relevant for development of corneal regenerative strategies that require the production of a keratocyte phenotype.

In vitro evaluation of electrospun blends of gelatin and PCL for application as a partial thickness corneal graft

“…Experiments on 2D controls of the same formulations demonstrated that chemistry was the dominant effect on cell adhesion in these studies (rather than fiber morphology). ALDH3A1 and CD34 are important markers of keratocytes in vivo alongside keratocan expression . The expression of CD90 (gene name THY1 ) and α‐SMA (gene name ACTA2 ) are characteristic of a less desirable myofibroblastic phenotype that leads to scarring .…”

“…Previous work published by our laboratories have compared similar isolated cells cultured in vitro to those resident in the native cornea. Compared to the native cornea, ALDH3A1, CD34, and ACTA2 were significantly downregulated and THY1 was significantly upregulated . This suggests that when cultured in vitro , the whole population of cells do not differentiate to the keratocyte phenotype warranting further optimization of scaffold morphology and in vitro culture conditions.…”

“…ALDH3A1 33,34 and CD34 [35][36][37] are important markers of keratocytes in vivo alongside keratocan expression. 38,39 The expression of CD90 (gene name THY1) and α-SMA (gene name ACTA2) are characteristic of a less desirable myofibroblastic phenotype that leads to scarring. 38 Our data suggested that 50:50 and 25:75 scaffolds offer a substrate more supportive of the keratocyte phenotype, with decreased α-SMA and increased CD34 and ALDH3A1 expression.…”

“…38,39 The expression of CD90 (gene name THY1) and α-SMA (gene name ACTA2) are characteristic of a less desirable myofibroblastic phenotype that leads to scarring. 38 Our data suggested that 50:50 and 25:75 scaffolds offer a substrate more supportive of the keratocyte phenotype, with decreased α-SMA and increased CD34 and ALDH3A1 expression. Previous work published by our laboratories have compared similar isolated cells cultured in vitro to those resident in the native cornea.…”

“…Compared to the native cornea, ALDH3A1, CD34, and ACTA2 were significantly downregulated and THY1 was significantly upregulated. 38 This suggests that when cultured in vitro, the whole population of cells do not differentiate to the keratocyte phenotype warranting further optimization of scaffold morphology and in vitro culture conditions. In addition, comparison of the expression of these markers from in vitro cultured cells with those expressed in vivo is further required.…”