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
The compound eye evolved over 500 million years ago and enables mosaic vision in most arthropod species.The molecular regulation of the development of the compound eye has been primarily studied in the fruit fly Droso...The compound eye evolved over 500 million years ago and enables mosaic vision in most arthropod species.The molecular regulation of the development of the compound eye has been primarily studied in the fruit fly Drosophila melanogaster.However,due to the nature of holometabolous insects halting growth after their terminal metamorphosis into the adult form,they lack the capacity to regenerate.Crustaceans,unlike holometabolous insects,continue to grow during adulthood,achieved through regular shedding of their exoskeleton,in a cyclic process known as molting.This therefore offers crustaceans as a highly suitable model to study ocular regeneration in the adult arthropod eye.We have assessed the regenerative capacity of the retinal section of the Cherax quadricarinatus(red-claw crayfish)eye,following ablation and successive post-metamorphic molts.This work then provides a transcriptomic description of the outer,pigmented retinal tissue(the ommatidia and lamina ganglionaris)and the basal,non-pigmented neuroendocrine ocular tissue(the X-organ Sinus Gland complex,hemiellipsoid body and optic nerve).Using comparative analysis,we identified all the transcripts in the C.quadricarinatus ocular transcriptome that are known to function in compound eye development in D.melanogaster.Differentially and uniquely transcribed genes of the retina are described,suggesting proposed mechanisms that may regulate ocular regeneration in decapod Crustacea.This research exemplifies the application C.quadricarinatus holds as an optimal model to study the regulation of ocular regeneration.Further in-depth transcriptomic analyses are now required,sampled throughout the regeneration process to better define the regulatory mechanism.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.
基金This work was supported by funding from the Bright Focus Foundationa Ramaciotti Establishment Grant to AWH.
文摘The compound eye evolved over 500 million years ago and enables mosaic vision in most arthropod species.The molecular regulation of the development of the compound eye has been primarily studied in the fruit fly Drosophila melanogaster.However,due to the nature of holometabolous insects halting growth after their terminal metamorphosis into the adult form,they lack the capacity to regenerate.Crustaceans,unlike holometabolous insects,continue to grow during adulthood,achieved through regular shedding of their exoskeleton,in a cyclic process known as molting.This therefore offers crustaceans as a highly suitable model to study ocular regeneration in the adult arthropod eye.We have assessed the regenerative capacity of the retinal section of the Cherax quadricarinatus(red-claw crayfish)eye,following ablation and successive post-metamorphic molts.This work then provides a transcriptomic description of the outer,pigmented retinal tissue(the ommatidia and lamina ganglionaris)and the basal,non-pigmented neuroendocrine ocular tissue(the X-organ Sinus Gland complex,hemiellipsoid body and optic nerve).Using comparative analysis,we identified all the transcripts in the C.quadricarinatus ocular transcriptome that are known to function in compound eye development in D.melanogaster.Differentially and uniquely transcribed genes of the retina are described,suggesting proposed mechanisms that may regulate ocular regeneration in decapod Crustacea.This research exemplifies the application C.quadricarinatus holds as an optimal model to study the regulation of ocular regeneration.Further in-depth transcriptomic analyses are now required,sampled throughout the regeneration process to better define the regulatory mechanism.
