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.
视网膜色素上皮细胞（RPE）是一种单层有色极性细胞，它在维持光感受器和潜在血管系统的健康和功能中起关键作用。氧化应激会造成RPE数量的下降及老化，从而有损视网膜的健康。目前，RPE老化的分子机制尚不清楚。多能干细胞是一种具有分化潜力的细胞，理论上讲，它可以在体外条件下被诱导成任何一种细胞类型。为了获得与天然RPE更为接近的诱导RPE细胞，我们刻画了多能干细胞诱导的RPE在培养1个月及12个月后的转录组情况。我们对以上两个时间点的RPE进行单细胞RNA测序，共获得16576个细胞的数据。我们的结果表明，在培养过程中，未出现RPE向干细胞转化的不稳定现象，同时RPE的占比较为稳固。基因功能富集分析表明，随着培养时间推移，RPE上调基因富集了金属结合与抗氧化功能。这可能反应了随着细胞发育越来越成熟，细胞的抗氧化能力得到提升。与天然RPE细胞数据相类似，培养12月的诱导RPE细胞展现出成熟的RPE转录特征。我们的工作描述了多能干细胞诱导培养RPE随时间发展的转录组图谱，为天然样本或病理样本提供参考。





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