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Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring.

ABSTRACT: Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound-healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor corneas were embedded in soluble rat-tendon collagen, gelled at 37°C, and partially dehydrated to a thickness of 100 µm by passive absorption. The CCG disks were dimensionally stable, easy to handle, and could be adhered securely to de-epithelialized mouse cornea with fibrin-based adhesive. CSSC in CCG maintained >80% viability for >1 week in culture media and could be cryopreserved in 20% fetal bovine serum-10%DMSO in liquid nitrogen. CCG containing as few as 500 CSSC effectively prevented visible scarring and suppressed expression of fibrotic Col3a1 mRNA. CSSC in CCG were more effective at blocking scarring on a per-cell basis than CSSC delivered directly in a fibrin gel as previously described. Collagen-embedded cells retained the ability to suppress corneal scarring after conventional cryopreservation. This study demonstrates use of a common biomaterial that can facilitate storage and handling of stem cells in a manner that may provide off-the-shelf delivery of stem cells as a therapy for corneal scarring. Stem Cells Translational Medicine 2018;7:487-494.

SUBMITTER: Shojaati G

PROVIDER: S-EPMC5980128 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring.

Shojaati Golnar G Khandaker Irona I Sylakowski Kyle K Funderburgh Martha L ML Du Yiqin Y Funderburgh James L JL

Stem cells translational medicine 20180414 6

Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound-healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor corneas were embedded in soluble rat-tendon collagen, gelled at 37°C, and partially dehydrated to a thickness of 100 µm by passive absorption. The CCG disks ...[more]

PMID: 29654654

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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.

Project description: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.

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Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring.

Publications

Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring.

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