Publications (2)7.19 Total impact
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Article: Iris pigment epithelium attachment to aged submacular human Bruch's membrane.
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ABSTRACT: To determine whether iris pigment epithelium (IPE) cells can attach to aged submacular human Bruch's membrane and to assess whether IPE cells express the integrin subunits that may be necessary to bind to the known extracellular matrix ligands present in Bruch's membrane. IPE cells were seeded onto the RPE basement membrane (RPEbm) or inner collagenous layer (ICL) of aged submacular Bruch's membrane as microaggregates or were expanded in culture until enough cells could be obtained for seeding. Cell morphology and the percentage of cell coverage were determined 1 or 7 days after seeding. Messenger RNA was extracted from cultured and uncultured IPE cells and analyzed by RT-PCR. The expression of integrin subunits alpha1 to alpha6 and beta1 mRNA was examined. Coverage by uncultured IPE was low on both surfaces at day-1 (RPEbm, 7.9% +/- 4.8%; ICL, 5.0% +/- 2.5%) with few intact cells present. Culturing IPE improved attachment with similar coverage on both surfaces and no significant difference between day-1 (RPEbm, 89.9% +/- 9.1%; ICL, 63.4% +/- 26.5%) and day-7 (RPEbm, 97.8% +/- 2.3%; ICL, 94.7% +/- 6.6%). By day-7, cell morphology and coverage on both surfaces was variable, ranging from few intact cells to a high degree of coverage by flattened cells. All integrin subunits studied were expressed in cultured cells, whereas alpha2, alpha3, and alpha4 showed less or no expression in uncultured cells. Upregulation of integrin mRNA expression may be one explanation for the difference in coverage by cultured versus uncultured IPE cells. The presence of dead, dying, or flattened cells at day 7 indicates that IPE may not survive or differentiate on aged submacular Bruch's membrane.Investigative Ophthalmology & Visual Science 01/2005; 45(12):4520-8. · 3.60 Impact Factor -
Article: Adeno-associated virus encoding green fluorescent protein as a label for retinal pigment epithelium.
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ABSTRACT: To determine whether transduction with adeno-associated virus encoding green fluorescent protein (AAV-GFP) is useful for labeling transplanted retinal pigment epithelial cells (RPE). Transduction was performed by infection of confluent or subconfluent cultured feline RPE or by subretinal injection. Cells transduced in vitro were analyzed to determine label stability over time and label conservation with cell division. RPE transduced in vivo were harvested at 5 weeks for transplantation or immunohistochemical detection. Two cats received subretinal injections of harvested cells and were killed at 3 or 7 days. In vitro transduction of confluent RPE resulted in stable GFP fluorescence for at least 3 months. There was a marked decline in fluorescence after cell division. Nonconfluent transduced cells conserved label after cell division but showed a marked decline in the number of cells, due to cell death. In vivo transduction resulted in a high level of labeling, allowing labeled cells to be harvested and transplanted. Transplanted cells were detected immunohistochemically. Photoreceptor labeling was detected over areas containing a high density of transplanted, labeled RPE derived from cells transduced in vivo. Possible light toxicity to transduced RPE was observed. AAV-GFP-labeling of confluent cultured RPE and RPE in situ can be used to identify transplanted RPE, with some reservations. Cell division may cause dilution of the label, and release of cell contents into the subretinal space may cause label transfer to photoreceptors. Exposure to light of transduced cells should be limited.Investigative Ophthalmology & Visual Science 03/2003; 44(2):772-80. · 3.60 Impact Factor
