Citation: Toda M, Ueno M, Hiraga A, et al. Production of homogeneous cultured human corneal endothelial cells indispensable for innovative cell therapy. Invest Ophthalmol Vis Sci. 2017;58:201158: -202058: . DOI:10.1167 PURPOSE. Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. The aim of this study was to establish proper culture protocols to successfully obtain a reproducibly homogeneous subpopulation (SP) with matured cHCEC functions and devoid of cell-state transition suitable for cell-injection therapy.METHODS. The presence of SPs in cHCECs was investigated in terms of surface cluster-ofdifferentiation (CD) marker expression level by flow cytometry, as combined analysis of CD markers can definitively specify the SP (effector cells) conceivably the most suitable for cell therapy among diverse SPs. The culture conditions were evaluated by flow cytometry in terms of the proportion (E-ratio) of effector cells designated by CD markers. RESULTS. Flow cytometry analysis identifying CD44effector cells proved convenient and reliable for standardizing the culture procedures. To ascertain the reproducible production of cHCECs with E-ratios of more than 90% and with no karyotype abnormality, the preferred donor age was younger than 29 years. The continuous presence of Rho-associated protein kinase (ROCK)-inhibitor Y-27632 greatly increased the Eratios, whereas the presence of transforming growth factor-beta/Smad-inhibitor SB431542 greatly reduced the number of recovered cHCECs. The seeding cell density during culture passages proved vital for maintaining a high E-ratio for extended passages. The continuous presence of ROCK-inhibitor Y-27632 throughout the cultures greatly improved the E-ratio.CONCLUSIONS. Our findings elucidated the culture conditions needed to obtain reproducible cHCECs with high E-ratios, thus ensuring homogeneous cHCECs with matured functions for the treatment of corneal endothelial dysfunction.

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.

PURPOSE. To clarify whether cultured human corneal endothelial cells (cHCECs), heterogeneous in their differentiation state, exhibit distinctive energy metabolism with the aim to develop a reliable method to sort cHCECs applicable for regenerative medicine.METHODS. The presence of cHCEC subpopulations (SPs) was verified via surface cluster-ofdifferentiation (CD) marker expression. Cultured HCEC metabolic extracts or corresponding culture supernatants with distinctive cellular phenotypes in regard to energy-metabolismrelated functional markers c-Myc and CD44 were prepared and analyzed via capillary electrophoresis-tandem mass spectrometry. The metabolic requirements of heterogeneous SPs of cHCECs were also investigated. RESULTS. After successfully discriminating SPs, as verified via surface CD markers in terms of their secretory metabolites, we found that the CD44þþþ SP with cell-state transition (CST) exhibited disposition for anaerobic glycolysis, whereas the CD44 À SP without CST was disposed to mitochondria-dependent oxidative phosphorylation (OXPHOS). These results raised the possibility of establishing effective culture conditions to selectively expand mature cHCECs with a hexagonal cobblestone shape and inclination for mitochondria-dependent OXPHOS.CONCLUSIONS. The findings of this study open a pathway for monitoring the disposition of cHCECs via their energy metabolism, thus leading to safe and stable regenerative medicine by use of metabolically defined cHCECs in cell-suspension form.

Cultured HCECs sharing a CD44- phenotype of matured HCECs may be discriminated by measuring the amount of miRNAs or exosome in CS. Thus, miRNA in CS may serve as a tool to qualify cHCECs. Future detailed analysis of cell-to-cell communication via these EVs might open novel pathways for a better understanding of CST in HCEC cultures.

PURPOSE. To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy.METHODS. The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen).RESULTS. Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44 À SP from SPs with CD44 þþ~C D44 þþþ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. CONCLUSIONS. The specified cultured SPs sharing the CD44À surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44 þþþ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.Keywords: heterogeneity of cultured corneal endothelial cells, CD44, miR, gutatta, epithelium-mesenchymal transition (EMT) A lthough human corneal endothelial cells (HCECs) have the ability to proliferate in vitro, 1 culturing HCECs for an extended period of time is known to be extremely difficult.2 To date, most researchers have conceptualized cultured HCECs (cHCECs) only from the aspect that they are derived from corneal endothelium tissue, and disregarded details pertaining to the refinement of the biochemical features. In fact, cHCECs are known to be heterogeneous from culture to culture in their morphology and in their surface markers, such as cluster-ofdifferentiation (CD) antigens.3-5 Studies, either directly or indirectly, and including those from Joyce et al., [6][7][8][9][10][11][12][13] have shown that heterogeneity is present in HCEC cultures. Of particular and striking interest is the finding by Miyai et al.3 of the presence of frequent chromosomal aneuploidy in cHCECs, directly indicating the presence of heterogeneous subpopulations (SPs) with or without aneuploidy.Cultured HCECs have an inclination toward cell-state transition (CST) into a senescence phenotype, epitheliummesenchymal transition (EMT), and fibroblastic cell morphology. In addition, the plasticity of metabolic profiles of cH...