Abstract
Abstract: :
Purpose: To examine attachment ability of human iris pigment epithelium (IPE) to aged submacular Bruch’s membrane. Coverage of transplanted cells in organ culture and expression of integrin mRNA were examined. Methods: Human eyes were obtained from National Disease Research Interchange and the North Carolina Eye Bank. Donor ages ranged from 59 to 91 years. (mean ± sd; 75.8 ± 8.9) IPE cells were isolated using 1mg/ml collagenase and either cultured or used directly (uncultured). For attachment studies, cells (1.21x105 cells) were seeded onto submacular Bruch’s membrane debrided to expose either the native retinal pigment epithelium basement membrane (RPE bm) or the inner collagenous layer (ICL). Five specimens were prepared for each condition. Uncultured IPE specimens at 7 days were not prepared, because the coverage at 1 day was too low. Following 1 or 7 day incubation, specimens were analyzed by scanning electron microscopy. Messenger RNA of cultured and uncultured cells (N=4) was extracted and processed for reverse transcription polymerase chain reaction. Integrin subunits of α1–6, and ß1 were examined. Results: No significant difference was seen in the percent coverage on RPE bm vs ICL with cultured IPE cells at either 1 or 7 days. (1d; bm 89.9 ± 9.1%, ICL 63.4 ± 26.5%, 7d; bm 64.2 ± 48.4%, ICL 77.0 ± 38.6%) No significant difference was seen in the coverage on RPE bm vs ICL by uncultured IPE cells at 1 day (bm 7.9 ± 4.8%, ICL 5.0 ± 2.5%). Significant difference was seen in the coverage of cultured vs uncultured IPE on RPE bm and ICL at 1 day. (bm; Student’s t–test p=5.6x10–7 ICL; Welch’s t–test p=7.9x10–3) All the integrin subunits were expressed in cultured IPE cells, while α2–4 were less or not expressed in uncultured IPE cells. Conclusions: Culturing IPE improves attachment to submacular Bruch’s membrane. Upregulation of integrin mRNA expression may be one explanation for the difference in coverage by cultured vs uncultured IPE cells.
Keywords: age–related macular degeneration • transplantation • iris