| Metadata |
| datasetIdentifier | PASS01012 |
| datasetType | Other |
| submitter | Antti Jylhä <antti.jylha@uta.fi> |
| submitter_organization | Department of Ophthalmology, Faculty of Medicin and Life Sciences, University of Tampere |
| lab_head_full_name | Hannu Uusitalo |
| lab_head_email | hannu.uusitalo@uta.fi |
| lab_head_organization | Department of Ophthalmology, Faculty of Medicin and Life Sciences, University of Tampere |
| lab_head_country | Finland |
| datasetTag | 08017080235600 |
| datasetTitle | Comparative proteomic analysis of human embryonic stem cell-derived and primary human retinal pigment epithelium |
| publicReleaseDate | 2017-07-10 00:00:00 |
| finalizedDate | 2017-07-10 01:39:31 |
| summary | Isobaric tags for relative quantitation (iTRAQ) based proteomic analysis was
used to compare the proteome of human embryonic stem cell-derived retinal
pigment epithelial cells (hESC-RPE) to primary human RPE (hRPE). Human ESC-RPE
were differentiated from two independent hESC lines and hRPE samples were
extracted from three different cadaveric human donor eyes. 1041 common proteins
were present in both hESC-RPE cells and native hRPE with majority of the
proteins similarly regulated. The hESC-RPE proteome reflected that of normal
hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and
transport proteins expressed. No signs of increased stress, apoptosis, immune
response, proliferation, or retinal degeneration related changes were noted in
hESC-RPE, while important RPE specific proteins involved in key RPE functions
such as visual cycle and phagocytosis, could be detected in the hESC-RPE.
Overall, the results indicated that the proteome of the hESC-RPE cells closely
resembled that of their native counterparts. |
| contributors | Heidi Hongisto
Antti Jylhä
Janika Nättinen
Jochen Rieck
Tanja Ilmarinen
Zoltan Vereb
Ulla Aapola
Roger W Beuerman
Goran Petrovski
Hannu Uusitalo
Heli Skottman |
| publication | [Paper #SREP-16-29237C]
Heidi Hongisto, Antti Jylhä, Janika Nättinen,
Jochen Rieck, Tanja Ilmarinen, Zoltan Vereb,
Ulla Aapola, Roger W Beuerman, Goran Petrovski,
Hannu Uusitalo, and Heli Skottman, Comparative
proteomic analysis of human embryonic stem
cell-derived and primary human retinal pigment
epithelium. Scientific Reports, submitted. |
| growth | The two hESC lines: Regea08/017 (46;XX) and Regea08/023 (46;XY) derived at
University of Tampere as previously described (Skottman. In Vitro Cell Dev
Biol Anim. 2010 Apr;46(3-4):206-9.) were used in the study.
The undifferentiated hESCs were cultured and differentiated to RPE as
previously described (Vaajasaari et al. Mol Vis. 2011 Feb 22;17:558-75).
Cells from three independent differentiation experiments (replicates 1-3)
were used for the study. Each biological replicate consisted of three cell
culture inserts. The hESC-RPE cells were collected with TrypLE Select, washed
twice with PBS, pelleted by centrifugation and stored as dry pellets at −80°C
until protein extraction.
Primary hRPE from three different donors with no macroscopical or microscopical
signs of retinal degeneration were used. For human retinal pigment epithelium
isolation, following enucleation, the eye bulb was washed with 5% povidone
iodine (Betadine), the anterior segment was cut approximately 3.5 mm posterior
to the limbus and the anterior segment, the vitreous and neural retina were
removed. The eyecup was then filled with RPE cell culture medium (DMEM/F12,
supplemented with 10% FCS, 1% antibiotics-antimycotics and 1% L-Glutamin) and
the cells collected by a special glass cell scraper to gently remove the RPE
from Bruch's membrane while continuously bathed in their own cell culture
medium. The RPE was collected as a pellet by centrifugation (1000 rpm, 10 min).
The RPE pellet was washed twice with PBS, followed by centrifugation after
each wash, and shipped for further analysis at -20°C. |
| treatment | No experimental treatments. |
| extraction | See published paper https://www.nature.com/articles/srep14684 |
| separation | See published paper https://www.nature.com/articles/srep14684 |
| digestion | See published paper https://www.nature.com/articles/srep14684 |
| acquisition | See published paper https://www.nature.com/articles/srep14684 |
| informatics | See published paper https://www.nature.com/articles/srep14684 |
| instruments | Ab Sciex TripleTOF 5600+ |
| species | Human |
| massModifications | none |
