Corneal Endothelial Expansion Promoted by Human Bone Marrow Mesenchymal Stem Cell-Derived Conditioned Medium

Abstract

Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs), even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM) obtained from human bone marrow-derived mesenchymal stem cells (MSCs) (MSC-CM) for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na+/K+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary for the endothelial functions.

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells

“…Mesenchymal stem cell-conditioned medium was prepared as previously described. 28 The HCECs were cultured by using MSC-conditioned medium at 378C in a humidified atmosphere containing 5% CO 2 , and the culture medium was changed twice per week. The HCECs were passaged at ratios of 1:3 by using 10x TrypLE Select (Life Technologies) at 378C for 12 minutes when they reached confluence.…”

Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Corneal Endothelial Cell Proliferation

“…16 An additional complication is that the proliferative ability of HCECs is limited even under ideal culture conditions, so multiple procedures have been developed to promote cell proliferation. [29][30][31][32] For instance, we reported that the use of a ROCK inhibitor, 13 conditioned medium derived from mesenchymal stem cells, 14 and laminin 511 fragment as a culture substrate 17 enhances cell proliferation. In the current study, we screened several low-molecular-weight compounds and found that the p38 MAPK inhibitor, SB203580, promotes cell proliferation while maintaining cell density and functional properties.…”

“…Corneal endothelial growth medium was prepared by conditioning basal medium mix (OptiMEM-I, 8% fetal bovine serum [ 14,16 Briefly, confluent 3T3 fibroblasts were incubated with 4 lg/mL mitomycin C (MMC) (Kyowa Hakkko Kirin Co., Ltd., Tokyo, Japan) for 2 hours, seeded on plastic dishes at a cell density of 2 3 10 4 cells/cm 2 , and cultured with DMEM (Life Technologies Corp., Grand Island, NY, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 lg/mL streptomycin (Nacalai Tesque, Inc., Kyoto, Japan). The 3T3 fibroblasts were washed 3 times with PBS and cultured with basal medium mix for an additional 24 hours.…”

Stem Cell-Based Therapy of Corneal Epithelial and Endothelial Diseases

“…It is estimated that the adult primary CECs can be passaged around four times, and there is little improvement even with modified medium and supplemented cytokines [43]. Yet, several protocols are developed to promote the proliferation of CECs, including the use of human bone marrow mesenchymal stem-cell-derived conditioned medium [44], or human amniotic epithelial-cell-derived conditional medium [45] (Table 2). In addition, telomerase or Cdk4R24C (constitutively active mutant form of Cdk4) and CyclinD1 transduction into CECs showed in vitro pump function [46,47].…”

“…[40][41][42][43][44][45] Establishment of CEC cell lines CEC lines were generated by transduction of telomerase or Cdk4R24C (constitutively active mutant form of Cdk4) and CyclinD1 gene. [46,47] Fetal human CEC Fetal CEC culture Protocol of primary culture, passage and freezing fetal CECs.…”