Human pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of diseases associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate time points of retinal pigment epithelium (RPE) cultures that reflect native counterparts, we characterised the transcriptomic profiles of the hPSC-derived RPE cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single-cell RNA-sequencing of a total of 16,576 cells to assess the molecular changes of the RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial–mesenchymal transition, and with the maturing populations of the RPE observed with time in culture. Assessment of Gene Ontology pathways revealed that as the cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirms a maturing transcriptional profile of RPE cells in culture. These results suggest that long-term in vitro culture of RPE cells allows the modelling of specific phenotypes observed in native mature tissues. Our work highlights the transcriptional landscape of hPSC-derived RPE cells as they age in culture, which provides a reference for native and patient samples to be benchmarked against.

人类多能干细胞(hPSC) 衍生的后代是细胞的未成熟版本，对与成熟或年龄相关的疾病的准确建模存在潜在限制。因此，重要的是要表征培养中使用的细胞与其天然对应物的相似程度。为了选择反映原生对应物的视网膜色素上皮(RPE) 培养物的适当时间点，我们对来自 1 个月和 12 个月培养物的 hPSC 衍生 RPE 细胞的转录组谱进行了表征。我们分化出人类胚胎干细胞H9 线进入 RPE 细胞，对总共 16,576 个细胞进行单细胞 RNA 测序，以评估 RPE 细胞在这两个培养时间点的分子变化。我们的结果表明 RPE 转录组特征的稳定性，没有上皮 - 间质转化的证据，并且随着培养时间的推移观察到 RPE 的成熟群体。基因本体通路的评估表明，随着培养物年龄的增长，RPE 细胞上调了与金属结合和抗氧化功能相关的基因的表达。这可能反映了随着细胞成熟处理氧化应激的能力增加。与天然人类 RPE 数据的比较证实了培养中 RPE 细胞的成熟转录谱。这些结果表明，在体外长期RPE 细胞的培养允许对在天然成熟组织中观察到的特定表型进行建模。我们的工作突出了 hPSC 衍生的 RPE 细胞在培养中老化时的转录景观，这为本地和患者样本进行基准测试提供了参考。