Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

Byun, Yong-Soo; Tibrewal, Sapna; Kim, Eunjae; Yco, Lisette; Sarkar, Joy; Ivanir, Yair; Liu, Chia-Yang; Sano, Cecile M.; Jain, Sandeep

doi:10.1371/journal.pone.0112781

Cited by 11 publications

(9 citation statements)

References 43 publications

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“…This suggests that there was only partial differentiation of the cells to the keratocyte phenotype, in all media. In many instances, in vitro keratocyte differentiation has only been demonstrated by increased expression of keratocyte markers, but has not been confirmed by a simultaneous decrease in fibroblast markers such as CD90 and vimentin (Berryhill et al ., ; Byun et al ., ; Foster et al ., ; Gouveia and Connon ; Park et al ., ). The only differences seen between the differentiation media in this study were a downregulation of NT5E in media containing RA and differences in α‐SMA expression; α‐SMA fibre staining was not seen in cultures supplemented with FGF‐2.…”

Section: Discussionmentioning

confidence: 99%

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

Sidney

Hopkinson

2017

J Tissue Eng Regen Med

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. (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.

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Section: Discussionmentioning

confidence: 99%

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

Sidney

Hopkinson

2017

J Tissue Eng Regen Med

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“…ASCs, UCSCs, and BMMSCs have been shown to improve corneal opacity in vivo with differentiation of the MSCs into keratocytes after they have been applied to the injured or scarred cornea 20–22. Studies have shown that human MSCs derived from different tissues can differentiate toward the keratocyte lineage in vitro, and these MSC types include ASCs,52,58–60 BMMSCs,21 periodontal ligament stem cells,27,29 dental pulp stem cells,26 UCSCs,28 turbinate-derived MSCs,23 and CSSCs 8,25,61. However, there is no direct comparative study of keratocyte differentiation of MSCs derived from different tissue types.…”

Section: Discussionmentioning

confidence: 99%

Differentiation Capacity of Human Mesenchymal Stem Cells into Keratocyte Lineage

Santos

Balayan

Funderburgh

et al. 2019

Invest. Ophthalmol. Vis. Sci.

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Purpose Mesenchymal stem cells (MSCs) have been extensively studied for their capacity to enhance wound healing and represent a promising research field for generating cell therapies for corneal scars. In the present study, we investigated MSCs from different tissues and their potential to differentiate toward corneal keratocytes. Methods Adipose-derived stem cells, bone marrow MSCs, umbilical cord stem cells, and corneal stromal stem cells (CSSCs) were characterized by their expression of surface markers CD105, CD90, and CD73, and their multilineage differentiation capacity into adipocytes, osteoblasts, and chondrocytes. MSCs were also evaluated for their potential to differentiate toward keratocytes, and for upregulation of the anti-inflammatory protein TNFα-stimulated gene-6 (TNFAIP6) after simulation by IFN-γ and TNF-α. Results Keratocyte lineage induction was achieved in all MSCs as indicated by the upregulated expression of keratocyte markers, including keratocan, lumican, and carbohydrate sulfotransferase. TNFAIP6 response to inflammatory stimulation was observed only in CSSCs; increasing by 3-fold compared with the control ( P < 0.05). Conclusions Based on our findings, CSSCs appeared to have the greatest differentiation potential toward the keratocyte lineage and the greatest anti-inflammatory properties in vitro.

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“…Human and mice Co-MSCs were isolated and expanded as described before. 1 , 2 , 14 , 15 Briefly, the corneoscleral button from healthy cadaver eyes (provided by Eversight Eye Bank, Ann Arbor, MI, USA) or freshly enucleated mouse eyes were washed five times with PBS containing 2X Antibiotic-Antimycotic and 2X penicillin- streptomycin (both from Thermo Fisher Scientific, Waltham, MA, USA). After removing the central button with a trephine, the limbus was cut into three segments that were placed in 2.4 IU of protease (Dispase II; Thermo Fisher Scientific) for 1 hour at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Corneal Mesenchymal Stromal Cells Are Directly Antiangiogenic via PEDF and sFLT-1

Eslani

Putra

Shen

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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PurposeTo evaluate the angiogenic properties of corneal derived mesenchymal stromal cells (Co-MSC).MethodsCo-MSCs were extracted from human cadaver, and wild-type (C57BL/6J) and SERPINF1−/− mice corneas. The MSC secretome was collected in a serum-free medium. Human umbilical vein endothelial cell (HUVEC) tube formation and fibrin gel bead assay (FIBA) sprout formation were used to assess the angiogenic properties of Co-MSC secretome. Complete corneal epithelial debridement was used to induce corneal neovascularization in wild-type mice. Co-MSCs embedded in fibrin gel was applied over the debrided cornea to evaluate the angiogenic effects of Co-MSCs in vivo. Immunoprecipitation was used to remove soluble fms-like tyrosine kinase-1 (sFLT-1) and pigment epithelium-derived factor (PEDF, SERPINF1 gene) from the Co-MSC secretome.ResultsCo-MSC secretome significantly inhibited HUVECs tube and sprout formation. Co-MSCs from different donors consistently contained high levels of antiangiogenic factors including sFLT-1 and PEDF; and low levels of the angiogenic factor VEGF-A. In vivo, application of Co-MSCs to mouse corneas after injury prevented the development of corneal neovascularization. Removing PEDF or sFLT-1 from the secretome significantly diminished the antiangiogenic effects of Co-MSCs. Co-MSCs isolated from SERPINF1−/− mice had significantly reduced antiangiogenic effects compared to SERPINF1+/+ (wild-type) Co-MSCs.ConclusionsThese results illustrate the direct antiangiogenic properties of Co-MSCs, the importance of sFLT-1 and PEDF, and their potential clinical application for preventing pathologic corneal neovascularization.

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Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

Cited by 11 publications

References 43 publications

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

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

Differentiation Capacity of Human Mesenchymal Stem Cells into Keratocyte Lineage

Corneal Mesenchymal Stromal Cells Are Directly Antiangiogenic via PEDF and sFLT-1

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