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

Abstract

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.

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells

“…Mitochondria and lysosome related proteins were enriched in HQ cHCECs, whereas cytosolic proteins, extracellular exosomes, stress fiber or actin cytoskeleton related proteins were enriched in LQ cHCECs. Some of the observations were fully consistent with our previous observation describing the metabolomic profiling segregated differentiated from dedifferentiated-cHCECs, 9 the linkage of the dedifferentiation state with epithelial-mesenchymal transition and transformed fibroblastic cell morphology, 7,8 and the observation that cHCECs SPs with CST secreted higher amount of exosomes than the mature differentiated cHCECs SPs. 30…”

“…The content of CD44-CD90-Downloaded from iovs.arvojournals.org on 10/02/2020 mature SPs in cHCECs corresponds to the higher Eratios in cHCECS, which had been designated in our previous publication. 7,8 This means that lactic acid and pyruvic acid in the CS negatively correlated with the E-ratios.…”

“…HCEC cells were collected from the culture dish by TrypLE Select treatment, as described above, and provided to Fluorescence Activated Cell Sorter (FACS) analysis according to the procedures described previously. 7,8 The antibodies used were as follows: FITC-conjugated anti-human CD90 mAb, phycoerythrin (PE)-conjugated anti-human CD166 mAb, PerCP-Cy 5.5 conjugated anti-human CD24 mAb, PE-Cy 7-conjugated anti-human CD44 and FITCconjugated antihuman CD90 mAb (all from BD Biosciences, San Jose, CA), and allophycocyanin APC-conjugated antihuman CD105 (eBioscience, Inc.). After washing with FACS buffer, the HCECs were analyzed by use of the BD FACSCanto II (BD Biosciences) fluidics-system flow cytometer.…”

“…1 Recently, we reported our development of a novel clinically effective therapeutic modality to regenerate monolayer CE tissues by injection into an anterior chamber of cultured human CE cells (cHCECs). [2][3][4][5][6][7][8] Recently, cHCECs have been recognized to be heterogeneous not only in their biochemical features but also in their key functions, including the metabolic plasticity. 9 Corneal endothelium is one of the most metabolically active tissues in the human body.…”

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion

“…Temporal changes in evCEnC-specific gene expression as a consequence of passaging allowed us to identify a novel cell surface marker, encoded by TMEM178A , as a potential positive selection marker for high quality cultured CEnC. In addition, after a reassessment of reported selection markers, we propose SLC4A11 (positive), TMEM178A (positive) and CD44 (negative) as the minimum for selection of high quality in vitro CEnC (Bartakova et al, 2018; Cheong et al, 2013; Chng et al, 2013; Ding et al, 2014; Frausto et al, 2016; Okumura et al, 2014a; Toda et al, 2017; Yoshihara et al, 2015). SLC4A11 is a highly expressed transporter protein in the corneal endothelium that plays a significant role in CEnC function, and biallelic mutation of this protein leads to congenital hereditary endothelial dystrophy (Aldave et al, 2013; Jiao et al, 2007; Vithana et al, 2006).…”