Frausto Ricardo F, Le Derek J, Aldave Anthony J
The Jules Stein Eye Institute, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA, USA.
Cell Transplant. 2016;25(6):1159-76. doi: 10.3727/096368915X688948. Epub 2015 Sep 2.
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 endothelial cells (evHCEnC) with that of primary HCEnC (pHCEnC) 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 data sets 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 pHCEnC most closely resemble evHCEnC and thus represent the most viable cell culture-based therapeutic option for managing corneal endothelial cell dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of their potential clinical utility for the management of corneal endothelial cell failure.
角膜内皮在维持角膜稳态和透明度方面起主要作用，一旦出现严重影响视力的功能障碍，必须用同种异体供体角膜内皮进行手术置换。然而，全球范围内供体角膜组织短缺，促使人们寻找可移植组织的替代来源。在动物模型的角膜内皮功能障碍实验模型中，培养的人角膜内皮细胞（HCEnC）已被证明能恢复角膜透明度，但对培养的HCEnC的特性描述仍不完整。为此，我们利用下一代RNA测序技术，比较体外人角膜内皮细胞（evHCEnC）与原代HCEnC（pHCEnC）和HCEnC系的转录组图谱，并确定培养的和永生化的角膜内皮细胞作为体内角膜内皮模型的效用。对转录组数据集的多维分析表明，原代HCEnC与evHCEnC的关系比永生化HCEnC更密切。随后的分析表明，与evHCEnC相比，在培养细胞中，大多数在HCEnC中特异性表达（在体外角膜上皮或成纤维细胞中不表达）的基因表现出明显的表达变异性。此外，还研究了与角膜内皮细胞功能或角膜内皮营养不良相关的基因。除AGBL1和LOXHD1（RNA测序或qPCR未检测到）外，与evHCEnC相比，在所测试的每个基因的培养细胞中均观察到基因表达和蛋白质水平的显著差异。我们的转录组分析表明，在分子水平上，pHCEnC与evHCEnC最相似，因此是治疗角膜内皮细胞功能障碍最可行的基于细胞培养的治疗选择。我们的研究结果还表明，研究人员在确定培养的HCEnC对角膜内皮细胞衰竭的潜在临床效用之前，应评估其整个转录组。
