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

Abstract

Citation: Hamuro J, Toda M, Asada K, et al. Cell homogeneity indispensable for regenerative medicine by cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2016;57:4749-4761. DOI:10.1167/ iovs.16-19770 PURPOSE. To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy.METHODS. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na þ /K þ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. RESULTS. Flow cytometry analysis identified the effector cell expressing CD166 and the presence of other SPs with CD166þþþ (CD26 and CD24, either þ or À) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Naþ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200~220 lm 2 , and the density of cHCECs exceeded 2500 cells/mm 2 . CONCLUSIONS.A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

Production of Homogeneous Cultured Human Corneal Endothelial Cells Indispensable for Innovative Cell Therapy

“…As described elsewhere, CD44 expression decreased in accordance with the differentiation of cHCECs to matured cHCECs. 22 The presence of SPs expressing either CD24 or CD26 in some cultures quickly raised the alarm of the presence of some SPs with remarkable karyotype abnormality, such as sex chromosome loss, trisomy, or translocation, because we confirmed the presence of karyotype abnormality in cHCEC SPs expressing CD24 or CD26. 18 Thus, most of those cells are not suitable for transplantation in the clinical setting.…”

“…At first observation, it quickly became evident that the cHCECs contained a variety of SPs, as we reported in a previous study. 22 It is also of note that those cHCECs elicited a strikingly distinct proportion of SPs defined by cell-surface CD markers, even when cultured along the lines of the protocol proposed by Okumura et al 15 The E-ratio defined by the proportion of SPs with CD44 Table 1). As described elsewhere, CD44 expression decreased in accordance with the differentiation of cHCECs to matured cHCECs.…”

“…In consideration of the above-mentioned observations, the culture protocol of HCECs to obtain the utmost homogeneity for clinical use was tentatively fixed as follows: (1) optimal donor age being between 7 and 29 years, 22 (2) seeding cell density after passage 1 greater than 750 cells/mm 2 (note: further detailed investigation is needed to ultimately determine the recommended seeding cell density with regard to the Eratio of cHCECs of each passage), and (3) no addition of SB431542 (a selective inhibitor of the TGF-b receptor) to use the beneficial effect of TGF-b signaling. In addition, we further revised the protocols proposed by Okumura et al 29 The addition of ROCK-inhibitor Y-27632 was changed from the addition only at the beginning of new passages to every 3 days throughout the entire culture period to produce an efficient differentiation to CD44 À SP.…”

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells

“…15 This finding makes it possible to adapt this SP in the clinical setting, and the combination of CD markers defined in this study was found to be the most appropriate for quality control to ensure the functional characteristics of cHCECs. However, the flow cytometry application impedes cultured cells by scraping out cells for analysis, forcing us to seek a new noninvasive way to discriminate the quality of heterogeneous cHCEC SPs.…”

“…Combined analysis of CD markers clearly specified the SPs (effector cells) adaptable for cell-based therapy among diverse SPs, and we successfully elucidated a method to quantitate the proportion of effector SPs in cultures (E-ratio). 15 The E-ratio is the ratio of the effector cells contained in cHCECs and suitable for cellinjection therapy. We defined the SP with CD133, CD105, CD90, CD44, CD26, CD24, and HLA-DR negative and CD166, HLA-ABC, and PD-L1 positive as effector cells, with complete absence of karyotype aneuploidy and with a CD marker profile consistent with that of HCECs in fresh corneal tissues.…”