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

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-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. Results hTGFβ3 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. Conclusion This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3, inducing a scar-free tissue response.

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

“…The stromal injection of human CSSC into normal mouse corneas and thin corneas of lumican null mice showed an accumulation of human keratocan, indicating cell differentiation to keratocyte lineage in vivo [16]. Similar to in vitro studies, the possible autocrine action of TGFβ3 produced by CSSC could drive the keratocyte differentiation and stromal ECM remodeling, improving corneal clarity and thickness [18]. Treatment with CSSC with TGFβ3 knockdown showed a loss of such effects.…”

“…The intrastromal injection of CSK reduced corneal haze in a rat model of corneal injury induced by laser photo-ablation [14,15]. Stromal injection or topical application of human corneal stromal stem cells (CSSC) has been shown to block corneal tissue scarring in mice with anterior stromal injury [13,[16][17][18]. CSSC are located in the anterior limbal stroma and are the mesenchymal stem cell type of progenitors that can differentiate into keratocytes.…”

“…CSSC are located in the anterior limbal stroma and are the mesenchymal stem cell type of progenitors that can differentiate into keratocytes. They were reported to control corneal tissue inflammation and fibrosis [17][18][19]. Our recent study has identified that 2 specific miRNAs (miR-29a and 381-5p), delivered via CSSC-derived EV, could downregulate stromal inflammation and fibrotic onset after corneal injury in mice, leading to a scarless stromal ECM remodeling [20].…”

Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells—Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing

“…These cells are capable of differentiating into the native keratocyte phenotype [ 22 , 23 ]. Recent studies by Orozco Morales et al [ 70 ], Hertsenberg et al [ 76 ], Weng et al [ 77 ] Chameettachal et al [ 78 ] and Chandru et al [ 79 ] have shown the potential of these cells in healing the cornea both in vitro and in vivo in animal models. However, the underlying mechanisms of how these cells achieve the scarless wound healing is not clearly studied.…”

Cell-Free Biological Approach for Corneal Stromal Wound Healing

“…Among them, TGFβ1 and β2 isoforms promote fibrosis and scarring, whereas TGFβ3 inhibits scarring and drives scar-free healing effects (Penn et al, 2012;Gilbert et al, 2016). Our group has reported that human CSSC produce TGFβ3 when they were applied to acute corneal wounds, hence restoring clear cornea tissues (Weng et al, 2020). CSSC with TGFβ3 knockdown by small interfering RNA method lost this scar-reducing effect.…”