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.

Association of Upregulated Angiogenic Cytokines With Choroidal Abnormalities in Chronic Central Serous Chorioretinopathy.

Purpose: To clarify the distinct molecular pathogenesis of central serous chorioretinopathy (CSC) and pachychoroid neovasculopathy (PNV). Methods: Aqueous humor (AH) was collected from 18 acute CSC, 20 chronic CSC, and 20 PNV patients. Concentrations of 30 cytokines in the AH were analyzed using a multiplex bead immunoassay, and the cytokine profiles were compared among these three groups of patients. The areas of choroidal vascular hyperpermeability (CVH) and choroidal thickness (CT), including measurement of the vascular layers, were investigated to analyze the features of choroidal abnormality in acute CSC, chronic CSC, and PNV. Additionally, associations between cytokine profiles and choroidal abnormalities were analyzed. Results: Proinflammatory cytokines, IL-6 and IL-8 were significantly upregulated in the chronic CSC group compared with the acute CSC or PNV groups. Angiogenic cytokines and VEGF-A were upregulated at levels that almost reached significance along with disease progression from acute to chronic CSC, whereas the upregulation was not significant from chronic CSC to PNV. In the chronic CSC group, strong positive correlations were confirmed between VEGF-A and placental growth factor (PlGF) (r = 0.75, P < 0.001) and IL-6 and VEGF-A (r = 0.74, P < 0.001), and angiogenesis-related cytokines were positively correlated with the typical choroidal abnormalities, areas of CVH, mean CT, and mean large choroidal vessel layer thickness. However, there was no association between these choroidal abnormality parameters and AH cytokines in the PNV group. Conclusions: The results suggest that choroidal abnormalities in chronic CSC may be associated with upregulated angiogenesis.

Distinct Aqueous Humour Cytokine Profiles of Patients with Pachychoroid Neovasculopathy and Neovascular Age-related Macular Degeneration.

This study investigated the pathophysiological features of pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD) by analysing and comparing cytokine profiles in aqueous humour (AH) collected from 18 PNV, 18 nAMD and 11 control patients. Responses to intravitreal injection of aflibercept were also analysed in the PNV and nAMD groups. In the PNV group, vascular endothelial growth factor (VEGF)-A was significantly lower than in the nAMD group (p = 0.03) but was almost identical to that in the control group (p = 0.86). The nAMD group showed positive correlations between interleukin (IL)-6 and IL-8 (r = 0.78, p < 0.001), IL-6 and monocyte chemoattractant protein (MCP)-1 (r = 0.68, p = 0.002) and IL-8 and MCP-1 (r = 0.68, p = 0.002). In the nAMD group, eyes with dry maculae one month after the first aflibercept injection showed significantly lower VEGF-A and placental growth factor (PlGF) at baseline than those with wet maculae (p = 0.02 for both). However, there was no significant difference between dry and wet maculae in the PNV group. The results suggest that angiogenic factors and proinflammatory cytokines may play the distinct roles in the pathogenesis of PNV and nAMD.

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.

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.

The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet's Membrane Components.

PURPOSE: To clarify the adherent properties of cultured human corneal endothelial cell (cHCEC) subpopulations (SPs). METHODS: Each SP was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell-surface markers. Binding abilities of HCEC SPs were examined by adding the cells to culture plates immobilized with collagens, laminins, or proteoglycans, and then centrifuging the plates. Adhered cells were then evaluated by phase-contrast microscopy. RESULTS: The cHCECs were bound to laminin-511, laminin-411, and Type-IV collagen in a concentration-dependent manner, yet weakly bound to Perlecan, Agrin, and TSP-1. Comparison of the influence of cell-suspension vehicles on cHCEC attachment showed that cells suspended in Opti-MEM-I or Opeguard-MA were bound to laminin, yet no binding was observed in cells suspended in BSS-Plus. Next, we compared the adherent properties of HCEC SPs. Both the fully differentiated, mature cHCEC SP and the epithelial-to-mesenchymal-transitioned (EMT)-phenotype SP were found to attach to laminin- or collagen-coated plates. Interestingly, the binding properties to laminins differed among those SPs. Although the level of cells adhered to the laminin-411-coated plate was the same among the cHCEC SPs, the fully differentiated, mature cHCEC SP was significantly more tightly bound to laminin-511 than was the EMT-phenotype SP. CONCLUSIONS: The findings of this study suggest that the binding ability of cHCECs to major Descemet's membrane components is distinct among cHCEC SPs, and that Opti-MEM-I and Opeguard-MA are useful cell-suspension vehicles for cell-injection therapy.

A common sequence motif involved in selection of transcription start sites of Arabidopsis and budding yeast tRNA genes.

The transcription start site (TSS) is useful to predict gene and to understand transcription initiation. Although vast data on mRNA TSSs are available, little is known about tRNA genes because of rapid processing. Using a tobacco in vitro transcription system under conditions of impaired 5' end processing, TSSs were determined for 64 Arabidopsis tRNA genes. This analysis revealed multiple TSSs distributed in a region from 10 to 2bp upstream of the mature tRNA coding sequence (-10 to -2). We also analyzed 31 Saccharomyces cerevisiae tRNA genes that showed a smaller number but a broader distribution (-13 to -1) of TSSs. In both cases, transcription was initiated preferentially at adenosine, and a common 'TCAACA' sequence was found spanning the TSSs. In plant, this motif caused multiple TSSs to converge at one site and enhanced transcription. The TATA-like sequence upstream of Arabidopsis tRNA genes also contributed to TSS selection.