Corneal endothelial expansion using human umbilical cord mesenchymal stem cell‐derived conditioned medium

Abstract

Rabbit corneal endothelial cells are frequently used in pharmacological experiments and are useful for corneal transplant experiments. We performed the present study to analyze the effect of conditioned medium (CM) derived from human umbilical cord mesenchymal stem cells (HUMSCs) on the growth of rabbit corneal endothelial cells (RCECs) and to establish a program for expansion of RCECs in vitro. RCECs were cultured using a CM derived from HUMSCs (HUMSCs‐CM) in vitro. The proliferation ability of RCECs cultured in the presence of HUMSCs‐CM was evaluated by conducting 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide, colony formation, and scratch migration assays. The proliferation ability of RCECs maintained in HUMSCs‐CM was significantly enhanced as compared to RCECs cultivated in the control group. Immunofluorescence indicated that zonula occludens‐1 (ZO‐1) and N‐cadherin were located at intercellular junctions. Real‐time PCR and western blot analyses demonstrated that the CEC‐relative functional markers were expressed in RCECs maintained in HUMSCs‐CM. Flow cytometry analyses demonstrated that HUMSCs‐CM promoted the G0/G1 entrance to the S phase in RCECs. Our results demonstrated that HUMSCs‐CM induced the proliferation of RCECs in vitro and maintained the necessary characteristic phenotypes. The expanded RCECs may provide a promising cell source for experimental research and clinical therapy.

Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation

“…This result may partially explain why the treatment of rabbit CECs with 10 µM Y-27632 under in vitro culture conditions promoted cell adhesion and inhibited apoptosis but did not promote cell proliferation. Y-27632 treatment may have mediated a delay in the G1 to S phase transition [34,35]. In contrast, the Y-CM group showed the smallest change in the CDC25C gene, which drives the cell cycle process (Figure 8B).…”

“…Several reports have been published on MSC exosomes, such as bone marrow-derived endothelial progenitor cell-conditioned medium [17], bone marrow mesenchymal stem cellconditioned medium [38], mouse embryonic stem cell-conditioned medium [20], and human umbilical cord mesenchymal stem cell-derived conditioned medium [35]. Conditioned medium contains exosomes which stimulate rat CEC proliferation and maintain functional markers, such as ZO-1, aquaporin 1, and Na + /K + -ATPase [38][39][40][41].…”

Cell therapy in corneal endothelial disease

“…A dual-media approach with alternating stabilisation (low-mitogenic) and proliferation (high mitogenic) conditions has been shown to be effective [25,44,45]. More recently, human platelet lysate (HPL) [46][47][48][49], small molecules [50,51] and mesenchymal stem cell derived conditioned media [52,53] have been reported to be helpful.…”

In Vitro Expansion of Corneal Endothelial Cells for Clinical Application: Current Update

“…As an alternate source of growth stimulants and other factors, conditioned media derived from cultures of mouse embryonic stem cells, 35 human bone marrow-derived mesenchymal stem cells and mouse embryonic fibroblasts (NIH-3T3) 36 and human umbilical cord mesenchymal stem cells, 37 bone marrow-derived endothelial progenitor cells and corneal stromal cells, 38 orbital adipose-derived stem cells, 39 and Mesenchymal stem cells in specific have gained attention because of their immunomodulatory properties and their ability to encourage wound healing and tissue regeneration with the release of paracrine factors. 41,42 Studies have shown that the addition of conditioned medium (ranging from 10% to 70%) to the CE cells led to higher cell proliferation, reduced cell apoptosis, increased migration, compact and homogeneous cell size, and retention of the endothelial phenotype over several passages.…”

“…As an alternate source of growth stimulants and other factors, conditioned media derived from cultures of mouse embryonic stem cells, 35 human bone marrow–derived mesenchymal stem cells and mouse embryonic fibroblasts (NIH-3T3) 36 and human umbilical cord mesenchymal stem cells, 37 bone marrow–derived endothelial progenitor cells and corneal stromal cells, 38 orbital adipose-derived stem cells, 39 and amniotic fluid 40 have been used to expand CE cells. Mesenchymal stem cells in specific have gained attention because of their immunomodulatory properties and their ability to encourage wound healing and tissue regeneration with the release of paracrine factors.…”