Stemness and Regenerative Potential of Corneal Stromal Stem Cells and Their Secretome After Long-Term Storage: Implications for Ocular Regeneration.

Purpose: To assess the stemness and regenerative potential of cryopreserved corneal stromal stem cells (cryo-CSSCs) after long-term storage. We also used the secretome from these cells to observe the effect on wound-healing capacity of corneal fibroblasts and on the expression of fibrotic markers during wound healing. Methods: CSSCs were obtained from three donors and stored in liquid nitrogen for approximately 10 years. Post thaw, cryo-CSSCs were characterized for stemness using phenotypic and genotypic markers along with colony-forming efficiency and three-dimensional spheroid formation. Multilineage differentiation was observed by differentiation into osteocytes, adipocytes, neural cells, and keratocytes. Secretome was harvested by culturing cryo-CSSCs in log phase. Wound-healing capacity was observed by live-cell time-lapse microscopy. Statistical analysis was done using 1-way ANOVA and Tukey posttest. Results: CSSCs displayed good viability post thaw and showed >90% expression of stem cell markers CD90, CD73, CD105, STRO1, and CD166. cryo-CSSCs also expressed stem cell genes OCT4, KLF4, and ABCG2, and could also form colonies and three-dimensional spheroids. Multipotency assessment showed that all three cryo-CSSCs could differentiate into osteocytes, adipocytes, neural cells, as shown by β-III tubulin and neurofilament antibody staining and corneal keratocytes as observed by staining for Kera C, J19, and collagen V antibodies. The secretome derived from these three populations could promote the wound healing of corneal fibroblasts and reduce the expression of fibrotic markers SPARC and fibronectin. Conclusions: CSSCs maintained their stemness and multipotency after long-term storage, and secretome derived from these cells can be of paramount importance for corneal regeneration and prevention of fibrosis.