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

• Published in: Investigative ophthalmology & visual science Vol. 52; no. 8; pp. 4979 - 4997
• Main Authors: Sugino, Ilene K, Sun, Qian, Wang, Jianqiu, Nunes, Celia F, Cheewatrakoolpong, Noounanong, Rapista, Aprille, Johnson, Adam C, Malcuit, Christopher, Klimanskaya, Irina, Lanza, Robert, Zarbin, Marco A
• Format: Journal Article
• Language: English
• Published: United States Association for Research in Vision and Ophthalmology, Inc 01.07.2011
• Subjects: Aged; Aged, 80 and over; Aging - physiology; Bruch Membrane - cytology; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cells, Cultured; Embryonic Stem Cells - cytology; Female; Fetal Stem Cells - cytology; Fluorescent Antibody Technique, Indirect; Humans; In Situ Nick-End Labeling; Integrins - genetics; Keratins - metabolism; Ki-67 Antigen - genetics; Ki-67 Antigen - metabolism; Macular Degeneration - pathology; Male; Microphthalmia-Associated Transcription Factor - genetics; Microphthalmia-Associated Transcription Factor - metabolism; Microscopy, Confocal; Microscopy, Electron, Scanning; Middle Aged; Organ Culture Techniques; Retinal Pigment Epithelium - cytology; Retinal Pigment Epithelium - physiology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism
• ISSN: 1552-5783; 0146-0404; 1552-5783
• DOI: 10.1167/iovs.10-5386

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. 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. 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. 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.