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Hiroshi Tanaka, Morio Ueno, Munetoyo Toda, Junji Hamuro, Kengo Yoshii, Noriko Koizumi, Naoki Okumura, Shigeru Kinoshita, Monty Montoya, Bernie Iliakis; Investigation of the donor tissue information on the phenotypes of cultivated human corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1164.
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© ARVO (1962-2015); The Authors (2016-present)
We are developing a novel technology for corneal regenerative medicine for bullous keratopathy (BK) that involves the injection of cultivated human corneal endothelial cells (cHCECs) into the anterior chamber. One key for a successful corneal transplantation is the preparation of fully differentiated cHCECs with a cobblestone-like shape. However, mature cHCECs with a fully differentiated phenotype are damped under in vitro culture conditions by phenotypic transition; i.e., epithelial-mesenchymal transition (EMT), cell senescence, and fibrosis. The purpose of this present study was to clarify the relationship between this phenotypic transition and the characteristics of donor human corneal tissues.
Donor human corneal tissues were obtained from an eye bank (SightLife, Seattle, WA) and HCECs obtained from individual donor corneas were re-suspended. Basal growth medium containing OptiMEM®-I Reduced Serum Medium (Life Technologies), fetal bovine serum, epidermal growth factor, ascorbic acid, calcium chloride, chondroitin sulfate, and gentamicin was conditioned by cultivation of human bone marrow-derived mesenchymal stem cells (MSCs), and then recovered as culture medium for HCECs. All cells were cultured using the MSC-conditioned basal growth medium supplemented with Rho kinase inhibitor and TGF-β inhibitor. Proportions of fully differentiated cHCECs after primary culture or 2 serial passage culture were detected using flow cytometry according to a combination of several cell surface markers. We examined the relationship between the percentages of fully differentiated cHCECs and donor information including gender, age, race, cause of death, and CEC density.
This study included 39 donors (27 males, 12 females; mean age: 42.2±22.7 years). Mean CEC density was 3105.5±315.3/mm2. Races were grouped into Caucasian or other. Cause of death was classified into acute disease, chronic disease, or other. The percentages of differentiated cHCECs had relation to donor age (R=-0.60, p=0.0002) and CEC density (R=0.42, p=0.009). CEC density was also related to age (R=-0.44, p=0.007). The percentages of differentiated cHCECs had no relation to gender (p=0.81), race (p=0.76), and cause of death (p=0.16).
The percentage of mature cHCECs with fully differentiated phenotype is related to donor age and CEC density.
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