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Series GSE78740 Query DataSets for GSE78740
Status Public on Dec 01, 2016
Title Restoration of mesenchymal RPE by transcription factor-mediated reprogramming
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary TGFbeta-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelial (RPE) differentiation after periods of sustained sub-confluent culture or repetitive passage. Inhibition of TGFbeta signaling using receptor kinase inhibitors forestalls the onset of passage-dependent EMT and can restore the capacity to differentiate to cells that previously underwent the mesenchymal switch [Radeke et al., 2015, Genome Med. 7:58]. However, even with the sustained inhibition of mesenchymal gene expression using TGFbeta signaling inhibitors the cells eventually lose the capacity to attain a characteristic phenotype. This suggests that there are additional mechanisms at play that contributeto the prevention of RPE differentiation after protracted periods of wound stimulus and mitosis. In this study we investigate the non-TGFbeta-mediated processes that contribute to the demise of the RPE phenotype after extended periods of proliferative wound response. Using comparative transcriptomics, we show that with increasing passage there is a downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential that cannot be prevented or reversed by inhibition of TGFbeta signaling using the TGFbeta receptor kinase inhibitor A-83-01. Importantly, among the RPE genes with decreased expression are several transcription factors known to be critical for RPE development. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 treatment restored the ability of passage 7 RPE to differentiate. Taken together, these results demonstrate that the loss of capacity to differentiate as a result of chronic wound stimulus is a product of both TGFbeta pathway-dependent increases in mesenchymal gene expression and a TGFbeta pathway-independent loss of RPE programming.
Overall design PolyA+ RNA-Seq analysis of primary fetal RPE donor lines as a function of passage with and without treatment with A-83-01 or after transcription factor-mediated reprogramming were done in triplicate using cells obtained from different donors. PolyA+ RNA-Seq analysis of the adult RPE cell line ARPE-19 was carried out in triplicate using independent experimental replicates obtained on different days. In two instances the reads from duplicate sequencing runs were carried out on the same seqeuncing libraries and the sequence files were combined prior to analysis.
Contributor(s) Radeke MJ, Shih Y, Radeke CM, Coffey P
Citation(s) 28118667
Submission date Feb 29, 2016
Last update date May 15, 2019
Contact name Monte J. Radeke
Phone 805-893-3695
Organization name University of California, Santa Barbara
Department Neuroscience Research Institute
Lab Center for the Study of Macular Degeneration
Street address Neuroscience Research Institiute
City Santa Barbara
State/province CA
ZIP/Postal code 93106-5060
Country USA
Platforms (2)
GPL17301 Ion Torrent PGM (Homo sapiens)
GPL17303 Ion Torrent Proton (Homo sapiens)
Samples (23)
GSM2075268 RPEA_P0_D32 [re-analysis]
GSM2075269 RPEB_P0_D32 [re-analysis]
GSM2075270 RPEC_P0_D32_1 [re-analysis]
BioProject PRJNA313410
SRA SRP070938

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SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE78740_ARPE19-2_D32_7dA83.txt.gz 490.5 Kb (ftp)(http) TXT
GSE78740_Mesenchymal-RPE_Reprogramming_matrix.txt.gz 1.0 Mb (ftp)(http) TXT
GSE78740_RAW.tar 9.1 Mb (http)(custom) TAR (of TXT)
GSE78740_RPEC_P0_D32.txt.gz 489.1 Kb (ftp)(http) TXT
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Raw data are available in SRA
Processed data provided as supplementary file
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