39 research outputs found
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A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells
Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes
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Differentiation Capacity of Human Mesenchymal Stem Cells into Keratocyte Lineage.
PurposeMesenchymal 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.MethodsAdipose-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.ResultsKeratocyte 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).ConclusionsBased 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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Differentiation Capacity of Human Mesenchymal Stem Cells into Keratocyte Lineage.
PurposeMesenchymal 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.MethodsAdipose-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.ResultsKeratocyte 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).ConclusionsBased on our findings, CSSCs appeared to have the greatest differentiation potential toward the keratocyte lineage and the greatest anti-inflammatory properties in vitro
Corneal stromal stem cells reduce corneal scarring by mediating neutrophil infiltration after wounding
<div><p>Corneal scarring limits vision for millions of individuals worldwide. Corneal transplantation (keratoplasty) is the standard of care for corneal opacity; however, it bears the risk of graft rejection and infection and is not universally available. Stem cell therapy holds promise as an alternative to keratoplasty. Stem cells from human corneal stroma (CSSC) induce regeneration of transparent corneal tissue in a mouse wound-healing model. In this study we investigated the mechanism by which CSSC prevent deposition of fibrotic tissue. Infiltration by CD11b<sup>+</sup>/Ly6G<sup>+</sup> neutrophils and myeloperoxidase expression were increased in corneas 24 hr after corneal wounding but were reduced in CSSC-treated wounds. Secretion of TSG-6, a protein known to regulate neutrophil migration, was up-regulated in CSSC in response to TNFα and as CSSC differentiate to keratocytes. In vivo, wounded mouse corneas treated with CSSC contained human TSG-6. Inhibition of neutrophil infiltration into cornea by CSSC was reversed when TSG-6 expression was knocked down using siRNA. Silencing of TSG-6 expression in CSSC reduced their ability to block scarring and the expression of mRNA for fibrosis-associated proteins collagen III, tenascin C, and smooth muscle actin in wounded corneas. Neutropenic mice exhibited a significant reduction in corneal scarring and fibrotic mRNA expression 2 weeks after wounding. These results support the conclusion that neutrophil infiltration is an essential event in the fibrotic response to corneal damage and that prevention of scarring by CSSC is mediated by secretion of TSG-6 by these cells.</p></div
TSG-6 accumulates in CSSC-treated wounds.
<p>Mouse corneas were treated immediately after a debridement wound with (<b>A-C</b>) CSSC in fibrinogen or (<b>D</b>) fibrinogen only as described under Materials and Methods. Corneas were fixed and cryosections were stained at (<b>A</b>) 2 days, <b>(B)</b> 14 days, and (<b>C, D</b>) 4 weeks after wounding using antibody specific for human TSG-6 (red) and DAPI to stain cell nuclei (blue).</p
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Mesenchymal Stem Cells Reduce Corneal Fibrosis and Inflammation via Extracellular Vesicle-Mediated Delivery of miRNA.
Mesenchymal stem cells from corneal stromal stem cells (CSSC) prevent fibrotic scarring and stimulate regeneration of transparent stromal tissue after corneal wounding in mice. These effects rely on the ability of CSSC to block neutrophil infiltration into the damaged cornea. The current study investigated the hypothesis that tissue regeneration by CSSC is mediated by secreted extracellular vesicles (EVs). CSSC produced EVs 130-150 nm in diameter with surface proteins that include CD63, CD81, and CD9. EVs from CSSC reduced visual scarring in murine corneal wounds as effectively as did live cells, but EVs from human embryonic kidney (HEK)293T cells had no regenerative properties. CSSC EV treatment of wounds decreased expression of fibrotic genes Col3a1 and Acta2, blocked neutrophil infiltration, and restored normal tissue morphology. CSSC EVs labeled with carboxyfluorescein succinimidyl ester dye, rapidly fused with corneal epithelial and stromal cells in culture, transferring microRNA (miRNA) to the target cells. Knockdown of mRNA for Alix, a component of the endosomal sorting complex required for transport, using siRNA, resulted in an 85% reduction of miRNA in the secreted EVs. The EVs with reduced miRNA were ineffective at blocking corneal scarring. Furthermore, CSSC with reduced Alix expression also lost their regenerative function, suggesting EVs as an obligate component in the delivery of miRNA. The results of these studies support an essential role for extracellular vesicles in the process by which CSSC cells block scarring and initiate regeneration of transparent corneal tissue after wounding. EVs appear to serve as a delivery vehicle for miRNA, which affects the regenerative action. Stem Cells Translational Medicine 2019;8:1192-1201