The focus of our laboratory efforts is on cell replacement therapy as a treatment for age-related macular degeneration (AMD) and other retinal degenerations. AMD is currently the leading cause of blindness in the United States and Western Europe, yet current treatments are limited largely to slowing but not reversing the decline in vision associated with this disease. The rationale for cell transplantation in AMD is deceptively simple. RPE transplantation seeks to replace cells removed during submacular surgery for choroidal neovascularization in wet AMD, and to replace RPE that have undergone geographic atrophy in dry AMD. Our laboratory efforts are geared towards RPE transplantation in animal models of AMD, and to studying the role of immune suppression and the status of Bruch's membrane on graft survival.
1. Del Priore LV, Kaplan HJ, Hornbeck R, Jones Z, Swinn M. Retinal Pigment Epithelial Debridement as a Model for the Pathogenesis and Treatment of Macular Degeneration. Am J Ophthalmol 122:629-643, 1996.
2. Ho TC, Del Priore LV. Reattachment of Human Retinal Pigment Epithelium to Extracellular Matrix and Human Bruch's Membrane. Invest Ophthalmol Vis Sci 38:1110-1118,1997.
3. Del Priore LV, Tezel TH. Reattachment Rate of Human Retinal Pigment Epithelium to Layers of Human Bruch's Membrane. Arch Ophthalmol 116;335-341, 1998.
4. Tezel TH, Del Priore LV, Kaplan HJ. Fate of Human Retinal Pigment Epithelial Cells Seeded onto Layers of Human Bruch's Membrane. Invest Ophthalmol Vis Sci 1999;40:467-476.
5. Tezel TH, Del Priore LV. Repopulation of Different Layers of Host Human Bruch's Membrane by Retinal Pigment Epithelial Cell Grafts. Invest Ophthalmol Vis Sci 1999;40:767-774.
6. Del Priore LV, Kuo YH, Tezel TH. Age-Related Changes in Human RPE Cell Density and Apoptosis Proportion In Situ. Invest Ophthalmol Vis Sci 2002;43:3312-3318.
7. Del Priore LV, Geng L, Tezel TH, Kaplan HJ. Extracellular matrix ligands selectively promote RPE attachment to inner layers of Bruch's membrane. Current Eye Research 2002, in press.
8. Del Priore LV, Sheng Y, Johnson E, Edge A, Suzuki T, Geng L, Tezel TH, Kaplan HJ. Short-term Survival of Transplanted Fetal Pig Retinal Pigment Epithelium in a Xenograft Model. Current Eye Research, in press.
Our lab research is focused on retinal and RPE transplantation. Our laboratory efforts are geared towards RPE transplantation in animal models of AMD, and to studying the role of immune suppression and the status of Bruch's membrane on graft survival. We have developed an experimental animal model to determine the effects of RPE absence on the choriocapillaris and outer retina, in an animal model of RPE debridement in the porcine eye. Histologic examination reveals that Bruch's membrane is devoid of native RPE and the choriocapillaris is patent immediately after debridement. There is no proliferation of RPE 1 week after debridement, and choriocapillaris atrophy is displayed beneath areas of Bruch's membrane devoid of RPE. Four weeks after surgery, choriocapillaris atrophy persists in all debrided blebs, and outer retinal atrophy is present in areas of Bruch's membrane with no RPE and no choriocapillaris 4 weeks after surgery. This study proves that absence of the RPE leads to atrophy of the choriocapillaris within 1 week after surgery, and emphasizes the need for rapid RPE repopulation of Bruch's membrane if RPE is removed during submacular surgery.
At the current time no satisfactory animal model exists for exudative AMD. Despite this limitation, we can draw some inferences on the behavior of RPE transplants in humans from appropriate animal studies. We have determined the morphology after transplantation of organized primary porcine RPE sheets into the subretinal space of the pig eye. Primary female RPE sheets were harvested from freshly enucleated porcine eyes by removing the sclera and incubating the eyecup with 25 U/ml of Dispase for 30 minutes. Four days after surgery the subretinal space contains a multilayer of heavily pigmented RPE that are predominantly Barr body positive. One month after transplantation, there is marked shortening of the outer segments in the transplant bed but the external limiting membrane appears normal. The transplant bed contains a pigmented monolayer in some regions, whereas in other regions the graft is folded into multilayers with degeneration of the inner layers of transplanted cells despite the presence of transplanted basement membrane. The choroidal vessels and choriocapillaris remain patent in the transplant bed. Barr body positive cells can still be identified 3 months after surgery, although there are many Barr body negative pigment-laden cells that may represent host RPE or macrophages in the subretinal space. There is no infiltration of the graft site with inflammatory cells. This demonstrates that transplanted RPE sheets survive in the subretinal space up to three months after surgery and the choriocapillaris remains patent in the transplant bed, although there are many heavily pigmented cells within the transplant bed that are Barr body negative by 3 months.
We also have determined the effect of triple drug immune suppression on graft survival in a xenograft model of RPE transplantation. Primary fetal pig RPE micro aggregates (approximately 40,000 RPE) were injected into the subretinal space of 24 albino rabbits, with half the rabbits maintained on triple systemic immune suppression. RPE survival was estimated using a DNA probe (PRE) against a porcine-specific repetitive chromosomal marker or a RAM-11 antibody against rabbit macrophages. Numerous pigmented cells were visible in the subretinal space at all time points but most pigment-containing cells > 4 weeks after surgery were RAM-11 positive and PRE-negative. The number of PRE-positive cells in the immune suppressed group was greater than in immune competent controls but the difference was only statistically significant at 4 weeks. The time-dependent decrease in PRE-positive cells was more pronounced in immune suppressed animals. Thus, systemic immune suppression increases the 4-week survival of porcine RPE xenografts in the albino rabbit subretinal space, but there is poor survival in immune suppressed and immune competent animals 12 weeks after surgery. Many pigment-containing cells seen > 4 weeks after surgery are PRE negative indicating they are of host origin.
The last facet of our work involves RPE repopulation of Bruch's membrane in vitro. We have determined the ultimate fate of RPE plated onto different layers of Bruch's membrane in vitro. Explants of peripheral Bruch's membrane are prepared from donor human cadaver eyes, and the RPE, RPE basal lamina and inner collagenous layer removed sequentially by mechanical or enzymatic techniques. Explants are then stabilized on 4% agarose in 96-well plates. Synchronized first passage RPE from 83, 85, and 63 year-old donors are trypsinized and plated (15K viable/cells) onto each layer of Bruch's membrane. For each layer, plating efficiency and apoptosis rates at 24 h, proliferation rate and mitosis index 24 h after growth induction with 15% FBS and time required to reach confluence were determined. Our findings indicate that plating efficiency, proliferation rate, apoptosis rate, and ability of donor RPE to reach confluence depend on which layer of human Bruch's membrane is available for RPE reattachment. The poor survival of RPE plated onto deeper layers of Bruch's membrane may be due to the lack of extracellular matrix receptors for cell attachment in these deeper layers.
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