Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

Abstract

PurposeExtracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components.MethodsRetinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry.ResultsApparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks.ConclusionsThe data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Drusen in patient-derived hiPSC-RPE models of macular dystrophies

“…S3 and S4). In fact, similar to data published on porcine RPE in culture (14), our results demonstrate spontaneous development of APOE-positive sub-RPE deposits in aged (D90) Ctrl hiPSC-RPE cultures (Figs. 2 A, C, and D and 4 A and D and SI Appendix, Fig.…”

“…Given that drusen in macular degeneration patient eyes are lipoproteinaceous deposits underlying RPE cells, we next evaluated if sub-RPE deposits in SFD and DHRD hiPSC-RPE in culture were also rich in neutral lipids and the RPE-synthesized drusen proteins APOE, CRYAA/CRYAB, and TIMP3 (32)(33)(34)(35). Consistent with published studies on mammalian RPE (14,36), staining of hiPSC-RPE cultures with Nile red demonstrated the presence of neutral lipids in Ctrl, SFD, and DHRD hiPSC-RPE cultures (Fig. 3A).…”

“…Although RPE cells in culture do not lay a complex ECM like the in vivo Bruch's membrane, previous studies using primary RPE cells of human, mouse, and porcine origin (13)(14)(15) have demonstrated the formation of sub-RPE deposits in cell culture. Consistent with the aforementioned studies, we show that Ctrl hiPSC-RPE cells in culture are capable of forming sub-RPE deposits (Fig.…”

“…ECM thickening could then potentially inhibit the flux of metabolic waste products by RPE cells and lead to development of sub-RPE deposits (drusen) (12). In fact, recent studies using both mouse- (13) and porcine-derived (14) RPE have demonstrated that RPE cells in culture are capable of forming basal deposits in a cell-autonomous fashion. Furthermore, human fetal RPE (hfRPE) cultures, when exposed to exogenous stressors such as serum (15) or POS (16), have been shown to develop drusen-like basal deposits.…”

Nicotinamide, iRPE-in-a dish, and age-related macular degeneration therapy development

“…A main stream AMD hypothesis is that nonpathalogical agerelated retinal changes insidiously turn in into pathological (dry) AMD changes, while a more sudden twist on top of the ongoing pathology causes (wet) AMD (9,10). Thus, culturing RPE by itself may be a too simple model to use in AMD: more advanced in vitro models with appropriate controls taking a number of these extra-RPE aspects into account are under development (11), and could preferably be used in combination with the IPSCs-RPE approach.…”