ABSTRACT
The corneal endothelium is essential for maintaining corneal transparency; therefore, corneal endothelial dysfunction causes serious vision loss. Tissue engineering-based therapy is potentially a less invasive and more effective therapeutic modality. We recently started a first-in-man clinical trial of cell-based therapy for treating corneal endothelial dysfunction in Japan. However, the senescence of corneal endothelial cells (CECs) during the serial passage culture needed to obtain massive quantities of cells for clinical use is a serious technical obstacle preventing the push of this regenerative therapy to clinical settings. Here, we show evidence from an animal model confirming that senescent cells are less effective in cell therapy. In addition, we propose that density-gradient centrifugation can eliminate the senescent cells and purify high potency CECs for clinical use. This simple technique might be applicable for other types of cells in the settings of regenerative medicine.
Subject(s)
Cell- and Tissue-Based Therapy , Cellular Senescence , Centrifugation/methods , Corneal Diseases/therapy , Endothelium, Corneal/transplantation , Regenerative Medicine , Tissue Engineering/methods , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Cells, Cultured , Disease Models, Animal , Endothelium, Corneal/cytology , Female , Humans , Male , RabbitsABSTRACT
PURPOSE: Cultured human corneal endothelial cells (HCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, corneal endothelial cells (CECs) tend to exhibit fibroblastic transformation, thereby losing their functional phenotype when cultured. The purpose of this study was to investigate the usefulness of surface markers of CECs displaying fibroblastic phenotypes as a means of cell characterization. METHODS: The expression levels of 242 cell surface antigens were screened in cultured human and monkey CECs using flow cytometry. An expression intensity ratio of nonfibroblastic/fibroblastic CECs > 2 and of fibroblastic/nonfibroblastic CECs > 2 were selected as indicating nonfibroblastic and fibroblastic markers, respectively. Nonfibroblastic and fibroblastic CECs were mixed, and CD73-positive and -negative cells were sorted using flow cytometry and further cultured. The functional phenotype of the sorted cells was evaluated according to morphology and the expression of function-related (Na(+)/K(+)-ATPase and ZO-1) and fibroblastic (type I collagen and fibronectin) markers. RESULTS: Flow cytometry analysis demonstrated that CD98, CD166, and CD340 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are markers of fibroblastic phenotype HCECs. The CECs that sorted as CD73-negative exhibited normal hexagonal morphology and expressed functional markers, whereas CECs that sorted as CD73-positive exhibited the fibroblastic phenotype. CONCLUSIONS: These markers will be useful for quality control to characterize the phenotype of cells destined for tissue engineering-based therapy. In addition, this selection protocol will provide a novel method for purification of functional cells.
