Human pluripotent stem cell(h PSC)-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 char...Human pluripotent stem cell(h PSC)-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 h PSC-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 themolecular 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 h PSC-derived RPE cells as they age in culture,which provides a reference for native and patient samples to be benchmarked against.展开更多
基金a National Health and Medical Research Council(NHMRC-Australia)Practitioner Fellowship(awarded to AWH)Career Development Fellowship(awarded to JEP)+10 种基金Senior Research Fellowship(Grant No.1154389,awarded to AP)an Australian Research Council Future Fellowship(Grant No.FT 140100047,awarded to AP)NHMRC project grants(Grant Nos.1138253 awarded to ELF and AP,as well as 1062820 and 1124812 awarded to SHN)a NHMRC synergy grant(Grant No.1181010 awarded to ELF and AP)grants from the Macular Disease Foundation Australia(awarded to AP,JEPAWH)the Jack Brockhoff Foundation(awarded to GEL)the DHB Foundation(awarded to GEL and AP)the Ophthalmic Research Institute of Australia(awarded to AP and AWH)Stem Cells Australia-the Australian Research Council Special Research Initiative in Stem Cell Science(awarded to SHN,AWH,JEP,and AP)the TMG Family Fund(awarded to AP and GEL)。
文摘Human pluripotent stem cell(h PSC)-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 h PSC-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 themolecular 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 h PSC-derived RPE cells as they age in culture,which provides a reference for native and patient samples to be benchmarked against.
