Transcriptomic analysis of cultured corneal endothelial cells as a validation for their use in cell-replacement therapy. Homo sapiens

The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelium (evHCEnC) with that of primary HCEnC and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level primary HCEnC most closely resemble evHCEC and thus represent a viable therapeutic option for managing corneal endothelial dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of the potential clinical utility of the cultured HCEnC for the management of corneal endothelial cell failure. Overall design: Transcriptomes from ex vivo corneal endothelium, primary cultures and three cell lines were compared. Three samples of each endothelial cell group were submitted for RNA sequencing for a total of 15 samples. The transcriptome for the ex vivo corneal endothelium was used as the reference (i.e., proxy for in vivo corneal endothelium). Transcript abundances for a subset of genes associated with corneal endothelial cell function or disease were validated with qPCR and western blot. Samples of ex vivo endothelium used for validation were independent replicates not used for RNA-sequencing.

角膜内皮细胞（corneal endothelium）在维持角膜稳态与透明度方面发挥核心作用，当出现显著影响视觉功能的功能障碍时，需通过手术替换为同种异体供体角膜内皮组织。然而全球范围内供体角膜组织短缺，促使研究者探寻可移植组织的替代来源。
培养的人角膜内皮细胞（cultured human corneal endothelial cells, HCEnC）已被证实可在动物角膜内皮功能障碍实验模型中恢复角膜透明度，但目前对培养HCEnC的特征描述仍不完整。
为此，本研究利用下一代RNA测序技术，比较离体人角膜内皮细胞（ex vivo human corneal endothelium, evHCEnC）与原代HCEnC及HCEnC细胞系的转录组谱，并评估培养及永生化角膜内皮细胞作为体内角膜内皮细胞模型的实用性。
对转录组数据集的多维分析显示，相较于永生化HCEnC，原代HCEnC与evHCEnC的转录组相似性更高。
后续分析表明，HCEnC特异性表达（即不在离体角膜上皮细胞或成纤维细胞中表达）的绝大多数基因，在培养细胞中相较于evHCEnC均表现出显著的表达差异。
此外，本研究还对与角膜内皮细胞功能或角膜内皮营养不良相关的基因进行了分析。除AGBL1和LOXHD1未被RNA测序或qPCR检测到外，其余所有检测基因在培养细胞与evHCEnC之间均观察到基因表达及蛋白水平的显著差异。
本研究的转录组分析表明，从分子层面而言，原代HCEnC与evHCEnC最为相似，因此可作为治疗角膜内皮功能障碍的可行方案。研究结果同时提示，研究者在评估培养HCEnC用于治疗角膜内皮细胞衰竭的潜在临床价值前，应先对培养HCEnC的完整转录组进行分析。
整体实验设计：本研究比较了离体角膜内皮细胞、原代培养细胞及三种细胞系的转录组。每个内皮细胞组设置3个生物学重复样本，总计提交15个样本进行RNA测序。以离体角膜内皮细胞的转录组作为参考（即体内角膜内皮细胞的替代模型）。针对部分与角膜内皮细胞功能或疾病相关的基因，通过qPCR及蛋白质印迹法验证其转录本丰度。用于验证的离体内皮细胞样本为独立重复样本，未参与RNA测序。