The Interplay Between Metabolites and MicroRNAs in Aqueous Humor to Coordinate Corneal Endothelium Integrity.

Purpose: The purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity. Design: Prospective, comparative, observational study. Participants: A total of 55 patients with BK and 31 patients with cataract (Cat) as control. Methods: A biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n = 18) and Cat (n = 8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in AqH specimens from 2 disease groups, BK (total n = 55) and Cat (total n = 31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of interacting metabolites and miRs in AqH attenuating HCE degeneration. Results: The metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P = 0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P = 0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells. Conclusions: Metabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Cellular Interplay Through Extracellular Vesicle miR-184 Alleviates Corneal Endothelium Degeneration.

Objective: The objective of the study was to reveal the presence of cellular interplay through extracellular vesicle (EV) microRNAs (miRs), to dampen the vicious cycle to degenerate human corneal endothelium (HCE) tissues. Design: Prospective, comparative, observational study. Methods: The miR levels in neonate-derived corneal tissues, in the aqueous humor (AqH) of bullous keratoplasty and cataract patients, as well as in the culture supernatant (CS) and EV of cultured human corneal endothelial cells (hCECs), were determined using 3D-Gene human miR chips and then validated using the real-time polymerase chain reaction. The extracellularly released miRs were profiled after the forced downregulation of cellular miR-34a, either by an miR-34a inhibitor or exposure to H2O2. The senescence-associated secretory phenotypes and mitochondrial membrane potential (MMP) were assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of functional miRs attenuating HCE degeneration. Results: The miRs in AqH were classified into 2 groups: expression in 1 group was significantly reduced in neonate-derived tissues, whereas that in the other group remained almost constant, independent of aging. The miR-34a and -29 families were typical in the former group, whereas miR-184 and -24-3p were typical in the latter. Additionally, a larger amount of the latter miRs was detected in AqH compared with those of the former miRs. There was also a greater abundance of miR-184 and -24-3p in hCECs, EV, and CS in fully mature CD44-/dull hCEC, leading to sufficient clinical tissue regenerative capacity in cell injection therapy. The repression of cellular miR-34a, either due to miR-34a inhibitors or exposure to oxidative stress, unexpectedly resulted in the elevated release of miR-184 and -24-3p. Secretions of VEGF, interleukin 6, monocyte chemotactic protein-1, and MMP were all repressed in both mature CD44-/dull and degenerated CD44+++ hCEC, transfected with an miR-184 mimic. Conclusions: The elevated release of miR-184 into AqH may constitute cellular interplay that prevents the aggravation of HCE degeneration induced by oxidative stress, thereby sustaining tissue homeostasis in HCE.

Quiescent innate and adaptive immune responses maintain the long-term integrity of corneal endothelium reconstituted through allogeneic cell injection therapy.

This study aims to clarify the immunogenicity in acquired and innate immune responses of cultured human corneal endothelial cells (hCECs) applied for cell injection therapy, a newly established modality for corneal endothelium failures. Thirty-four patients with corneal endothelial failure received injection of allogeneic hCEC suspension into anterior chamber. No sign of immunological rejection was observed in all 34 patients during the 5-8 years postoperative follow-up period. Cell injection therapy was successful in 2 patients treated for endothelial failure after penetrating keratoplasty and one patient with Descemet membrane stripping automated endothelial keratoplasty failure. ELISPOT assays performed in allo-mixed lymphocyte reaction to the alloantigen identical to that on the injected hCECs, elicited sparse IFN-γ-specific spots in the peripheral blood mononuclear cells of patients who received hCEC injection. The therapy generated simple and smooth graft-host junctions without wound stress. The injection of C57BL/6 CECs into the anterior chamber of BALB/c mice, which rejected C57BL/6 corneas 6 weeks ago, induced no sign of inflammatory reactions after the second challenge of alloantigen. Collectively, injection of the hCEC cell suspension in the aqueous humor induces immune tolerance that contributes to the survival of the reconstituted endothelium.

Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure.

Purpose: To reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber. Methods: The varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis. Results: P53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H2O2) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma. Conclusions: The upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.

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

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-of-differentiation (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 CD44-CD166+CD133-CD105-CD24-CD26- effector 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 E-ratios, 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.

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells.

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- SP from SPs with CD44++∼CD44+++ 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 CD44- surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44+++ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.

Metabolic Plasticity in Cell State Homeostasis and Differentiation of Cultured Human Corneal Endothelial Cells.

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-of-differentiation (CD) marker expression. Cultured HCEC metabolic extracts or corresponding culture supernatants with distinctive cellular phenotypes in regard to energy-metabolism-related 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 CD44+++ SP with cell-state transition (CST) exhibited disposition for anaerobic glycolysis, whereas the CD44-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.

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

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+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. RESULTS: Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ 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 µm2, and the density of cHCECs exceeded 2500 cells/mm2. 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.

Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells.

PURPOSE: We elucidate a method to use secreted miRNA profiles to qualify cultured human corneal endothelial cells (cHCECs) adaptable for cell-injection therapy. METHODS: The variations of cHCECs in their composites of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers. Integrated analysis of micro RNA (miRNA) profiles in culture supernatants (CS) were investigated by 3D-Gene Human microRNA Chips. To validate 3D-Gene results, quantitative real-time PCR was done from numerous cultures with distinct morphology and SP composition. Exosomes and miRNAs in CS also were analyzed. RESULTS: Secreted miRNA profiles among morphologically-diverse cHCEC SPs proved useful for individual distinction. Candidate miRNAs to discriminate CD44- SPs from those with CD44++ ∼ CD44+++ phenotypes were miRs 221-3p, 1246, 1915-3p, and 4732-5p. The levels of the latter-three miRs decreased dramatically in cHCEC CS without cell-state transition (CST) compared to those of control medium, whereas those from cHCECs with senescence-like CST showed an increase. MicroR184 decreased inversely in parallel with the upregulation of CD44 on cHCECs. CD9+ exosomes were more elevated in cHCEC CS with senescence-like CST than those without CST, indicating the possible import of these extracellular vesicles (EVs) into cHCECs without CST. CONCLUSIONS: 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.

Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells.

PURPOSE: To develop a method to qualify the function of cultured human corneal endothelial cells (cHCECs) applicable for clinical settings. METHODS: The diversified gene and microRNA (miRNA) signatures in HCECs from a variety of tissue donors were confirmed by three-dimensional (3D) gene human miRNA profiling. These were compared with those of more than 20 cHCECs distinct in their cell morphology or culture lots. Candidate genes were selected after quantitative (q)RT-PCR validation, and gene products were assayed by ELISA. After three additional screening steps, final candidate cytokines for qualification were selected. RESULTS: Gene and miRNA signatures among distinct cHCEC lots were greatly diversified compared with those among fresh tissues from different age donors. By comparing more than 20 lots of cultures, 32 candidate genes were assigned to be seemingly linked to distinct cHCEC morphologic features. The validation of candidate genes by qRT-PCR revealed the genes, either upregulated or downregulated, corresponding to morphologic variances in cHCECs (e.g., epithelial-mesenchymal transition or cell senescence). Further adding the ELISA results by Bio-Plex Human Cytokine 27-Plex Panel, 11 candidate cytokines suitable to qualify cHCEC function were selected. In consideration of the presence of these cytokines in the anterior chamber, IL-8, tissue inhibitors of metalloproteinases 1 (TIMP-1), monocyte chemotactic protein-1 (MCP-1), and platelet-derived growth factor-BB (PDGF-BB) were ultimately selected and applied in practice for the qualification of cHCECs actually used in our clinical cell-injection studies. CONCLUSIONS: The specified cytokines properly discriminating the functional features of cHCECs indicates a correlation between profiling signatures and cell morphology.