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The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factor ?3.

ABSTRACT: Background: Corneal 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.

Methods: Primary 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-? and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor ?3 (hTGF?3) and hTGF?1 were examined by immunofluorescence and qPCR. TGF?3 knockdown for >?70% in hCSSC [hCSSC-TGF?3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGF?3(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.

Results: hTGF?3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGF?3 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 ?-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGF?3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring.

Conclusion: This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGF?3, inducing a scar-free tissue response.

SUBMITTER: Weng L

PROVIDER: S-EPMC7607765 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factor β3.

Weng Lin L Funderburgh James L JL Khandaker Irona I Geary Moira L ML Yang Tianbing T Basu Rohan R Funderburgh Martha L ML Du Yiqin Y Yam Gary Hin-Fai GH

Eye and vision (London, England) 20201103 1

<h4>Background</h4>Corneal 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.<h4>Methods</h4>Primary 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-i ...[more]

PMID: 33292650

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Project description:BackgroundMesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.MethodsA total of 24 donor corneal rims with informed consent were used-18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC's conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.ResultsBy comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters-stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)-were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.ConclusionsWe established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC-in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Dataset Information

The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factor ?3.

Publications

The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factor β3.

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