Comparison of FRPE and human embryonic stem cell-derived RPE behavior on aged human Bruch's membrane.

TitleComparison of FRPE and human embryonic stem cell-derived RPE behavior on aged human Bruch's membrane.
Publication TypeJournal Article
Year of Publication2011
AuthorsSugino IK, Sun Q, Wang J, Nunes CF, Cheewatrakoolpong N, Rapista A, Johnson AC, Malcuit C, Klimanskaya I, Lanza R, Zarbin MA
JournalInvestigative ophthalmology & visual science
Date Published2011 Jul
KeywordsAged, Aged, 80 and over, Aging, Bruch Membrane, Carrier Proteins, Cells, Cultured, Embryonic Stem Cells, Female, Fetal Stem Cells, Fluorescent Antibody Technique, Indirect, Humans, In Situ Nick-End Labeling, Integrins, Keratins, Ki-67 Antigen, Macular Degeneration, Male, Microphthalmia-Associated Transcription Factor, Microscopy, Confocal, Microscopy, Electron, Scanning, Middle Aged, Organ Culture Techniques, Retinal Pigment Epithelium, Reverse Transcriptase Polymerase Chain Reaction, RNA, Messenger
AbstractPURPOSE: To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch’s membrane (BM) from aged and AMD donors. METHODS: hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. RESULTS: hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. CONCLUSIONS: Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.
Alternate JournalInvest. Ophthalmol. Vis. Sci.
PubMed ID21460262