Comment[ArrayExpressAccession]	E-MTAB-960
Investigation Title	RPE Redifferentiation (miRNA)
Comment[Submitted Name]	RPE Redifferentiation (miRNA)
Experiment Description	Retinal Pigment Epithelial (RPE) cells are located behind the retina and are critical for photoreceptor survival. Loss of RPE is associated with several pathogenic conditions such as Age Related Macular Degeneration and Retinitis Pigmentosa. RPE derived from human embryonic stem cells (hESC) offer a potential source for producing these cells for therapy. Here we report the molecular and cellular characterization of RPE differentiated from hESC. hESC derived RPE are capable of proliferation and lose their epithelial characteristics before becoming confluent and re-differentiating back into their typical pigmented, cobblestoned appearance. During the proliferative phase, they adopt a mesenchymal morphology and express mesenchymal markers. Our results demonstrate that this apparent Epithelial-Mesenchymal Transition is not regulated by the classical EMT transcription factors SNAIL and SLUG. Furthermore, it is possible to regulate RPE de-differentiation and re-differentiation by modulating the Wnt and BMP pathway respectively. These findings further our understanding of the genesis and expansion of RPE which is essential for their therapeutic use.
Experimental Design	time_series_design	in_vitro_design	co-expression_design
Comment[AEExperimentType]	microRNA profiling by array
Comment[AEExperimentDisplayName]	microRNA profiling by array of human retinal pigment epithelial (RPE) cells to study the regulation of RPE redifferentiation
Experimental Factor Name	TIME
Experimental Factor Type	time
Quality Control Type	biological_replicate	technical_replicate
Public Release Date	2013-01-17
Person Last Name	Gutteridge
Person First Name	alex.gutteridge@pfizer.com
Person Mid Initials	Alex
Person Email	alex.gutteridge@pfizer.com
Person Phone	1304641571
Person Address	Granta Park, Cambridge, UK
Person Affiliation	Neusentis
Person Roles	submitter
Publication Author List	Parul Choudhary, Christos Michaelides, Alex Gutteridge, Paul Whiting
Publication Title	Characterization of hES derived RPE: RPE proliferation involves a non-classical EMT.
Publication Status	Submitted
Protocol Name	P-MTAB-25099	P-MTAB-25100	P-MTAB-25101	P-MTAB-25102	P-MTAB-21877	P-AFFY-6	P-MTAB-25103	P-MTAB-25104
Protocol Type	grow	specified_biomaterial_action	nucleic_acid_extraction	labeling	hybridization	scanning	bioassay_data_transformation	bioassay_data_transformation
Protocol Description	Shef1.3 hES cells were cultured as colonies either on inactivated mouse embryonic fibroblasts (MEF) in knockout DMEM (GIBCO) supplemented with 20% KSR (GIBCO), 1% non-essential amino acids (GIBCO), 1mM L-glutamine with 0.1mM beta-mercaptoethanol, 30ug/ml gentamicin (GIBCO) and 4ng/ml bFGF or feeder free on hES-qualified Matrigel (BD) in mTesR1 (StemCell Technologies).  These were fed daily until superconfluent (approximately 2 weeks post-seeding) before changing to knockout DMEM media as above but without FGF.  Flasks were fed thrice weekly until pigmented RPE foci had appeared and were large enough to cut out.  The foci were then excised with a scalpel, washed with PBS (-MgCl2, - CaCl2) and incubated with Accutase (GIBCO) for 1-1.5h in a shaking water bath at 37oC.  Dissociated RPE were pelleted at 700xg for 5min, resuspended in warm knockout DMEM media without FGF as above, and then strained through a 70um cell strainer to remove any large aggregates. RPE were counted and seeded at a density of 100000 cells/cm2 on 96-well plates coated with the extracellular matrix  CellStart (Invitrogen).	Cells were cultured for between 0 and 56 days as specified by the TIME factor, feeding with media twice weekly with 0.2ml/well, before samples were collected for analysis.	Total RNA was extracted from RPE cells using the RNEasy Mini or Micro Kit (Qiagen) with on-column DNase digestion. 	miRNA was labelled with biotin and hybridized to Affymetrix GeneChip miRNA 2.0 by Aros Inc. according to the manufacturer's instructions.			Raw data was normalised using vsn.	Data was log2 transformed
Term Source Name	ArrayExpress
Term Source File	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MTAB-960.sdrf.txt