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Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

ABSTRACT: Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined cell-based treatment using CSSC and CSK in a mouse model of anterior stromal injury. We hypothesize that the immuno-regulatory nature of CSSC is effective to control tissue inflammation and delay the onset of fibrosis, and a subsequent intrastromal CSK treatment deposited collagens and stromal specific proteoglycans to recover a native stromal matrix. Using optimized cell doses, our results showed that the effect of CSSC treatment for suppressing corneal opacities was augmented by an additional intrastromal CSK injection, resulting in better corneal clarity. These in vivo effects were substantiated by a further downregulated expression of stromal fibrosis genes and the restoration of stromal fibrillar organization and regularity. Hence, a combined treatment of CSSC and CSK could achieve a higher clinical efficacy and restore corneal transparency, when compared to a single CSSC treatment.

SUBMITTER: Jhanji V

PROVIDER: S-EPMC9267074 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

Jhanji Vishal V Santra Mithun M Riau Andri K AK Geary Moira L ML Yang Tianbing T Rubin Elizabeth E Yusoff Nur Zahirah Binte M NZBM Dhaliwal Deepinder K DK Mehta Jodhbir S JS Yam Gary Hin-Fai GH

International journal of molecular sciences 20220623 13

Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined ...[more]

PMID: 35805991

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Project description:BackgroundCorneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect.MethodsPrimary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor β3 (hTGFβ3) and hTGFβ1 were examined by immunofluorescence and qPCR. TGFβ3 knockdown for > 70% in hCSSC [hCSSC-TGFβ3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFβ3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined.ResultshTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFβ3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring.ConclusionThis study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3, inducing a scar-free tissue response.

Project description:IntroductionCorneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects.ObjectivesTo examine miRNA profiles in EVs from human CSSCs showing "healing" versus "non-healing" effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications.MethodsSmall RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in "healing" EVs (P < 0.05) were subject to enriched gene ontology (GO) term analysis. MiRNA groups with predictive regulation on inflammatory and fibrotic signalling were studied by mimic transfection to (1) mouse macrophages (RAW264.7) for M1 phenotype assay; (2) human corneal keratocytes for cytokine-induced fibrosis, and (3) human CSSCs for corneal scar prevention in vivo. The expression of miR-29a was screened in additional CSSC batches and the anti-scarring effect of cells was validated in mouse corneal wounds.ResultsTwenty-one miRNAs were significantly expressed in "healing" CSSC-EVs and 9 miRNA groups were predicted to associate with inflammatory and fibrotic responses, and tissue regeneration (P <10-6). Overexpression of miR-29a and 381-5p significantly prevented M1 phenotype transition in RAW264.7 cells after lipopolysaccharide treatment, suppressed transforming growth factor β1-induced fibrosis marker expression in keratocytes, and reduced scarring after corneal injury. High miR-29a expression in EV fractions distinguished human CSSCs with strong healing potency, which inhibited corneal scarring in vivo.ConclusionWe characterized the anti-inflammatory and fibrotic roles of miR-29a and 381-5p in CSSCs, contributing to scar prevention. MiR-29a expression in EVs distinguished CSSCs with anti-scarring quality, identifying good quality cells for a scarless corneal healing.

Dataset Information

Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

Publications

Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

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