title: 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 p27(Kip1) 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.
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Nakahara
authors.forename: Naoki
authors.initials: N
authors.lastname: Okumura
authors.forename: EunDuck P
authors.initials: EP
authors.lastname: Kay
authors.forename: Michio
authors.initials: M
authors.lastname: Hagiya
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Imagawa
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Hosoda
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.forename: Noriko
authors.initials: N
authors.lastname: Koizumi
mesh_terms: D015415:Biomarkers; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D016039:Corneal Transplantation; D017077:Culture Media, Conditioned; D004728:Endothelium, Corneal / Q000166:cytology*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism
chemical_list: D015415:Biomarkers; D018797:Cell Cycle Proteins; D017077:Culture Media, Conditioned
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