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

Abstract

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby's fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid-and protein-rich "drusen-like" composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE-ECM interface.human induced pluripotent stem cells | retinal pigment epithelium | macular dystrophies | drusen | sub-RPE deposits M aculopathies are a major cause of blindness, with agerelated macular degeneration (AMD) being the leading cause of irreversible vision loss in adults in the United States. Histopathological and clinical studies have shown that AMD and a subset of inherited macular dystrophies (MDs) share extensive phenotypic and clinical similarities (1-4). Specifically, AMD and related MDs have a cumulative etiology with adult onset of signs and symptoms and similar disease presentation including drusen formation, extracellular matrix (ECM) protein accumulation, thickening of Bruch's membrane, development of choroidal neovascularization, retinal pigment epithelium (RPE) atrophy, and ultimately the loss of vision (1-5). Although, the major pathological manifestations in these maculopathies are localized to the RPE-ECM interface in the retina, the multifactorial nature of these diseases, including the involvement of multiple retinal cell layers (photoreceptors, RPE, and choriocapillaris) (3, 6-9) and environmental risk factors (e.g., cigarette smoke) (10), has complicated the pursuit of t...

Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interactions

“…The TER in AMD patients (187 ± 6, 194 ± 7, 189 ± 4, and 198 ± 4 Ω × cm 2 ) vs the TER in normal controls (202 ± 5, 191 ± 7, and 197 ± 3 Ω × cm 2 ) revealed no significant difference ( P > .05) (Figure M). These TER results are consistent with TER in other reported embryonic stem cell (ESC)‐derived and iPSC‐derived RPE cell lines . Given the TER, high expression of claudin‐3 and claudin‐19 (Supplemental Table S3) and circumferential bands of ZO‐1, these iPSC‐derived RPE cells from patient samples can establish barrier function in vitro.…”

“…Saini et al showed increased expression of mRNAs for apolipoprotein E (APOE), CFI, C1R, ICAM‐1, and of C3 protein in some AMD lines, whereas cells cultured under normal conditions in our study revealed a decrease in mRNA. Additional to C1R and C3, Galloway et al found increases in MCP, C1S, and C5. Furthermore, there were increases in drusen‐like deposits with increased expression of APOE, TIMP3, and collagen IV.…”

“…The current study joins two others that studied RPE‐related macular dystrophies by using patient‐derived iPSC and age‐matched controls. Saini et al studied AMD including samples from donors with the age‐related maculopathy susceptibility 2 (ARMS2)/HtrA serine peptidase 1 (HTRA1) homozygous genotype, and Galloway et al studied three slow retinal degenerations: Sorsby's fundus dystrophy, Doyne honeycomb retinal dystrophy/malattia leventinese, and autosomal dominant radial drusen. Although the number of patients used in each study was small, together they constituted an expanding database.…”

Sorsby fundus dystrophy: Insights from the past and looking to the future

“…This possibly recapitulates the lipofuscin accumulation in the RPE of BVMD patient eyes (Singh et al, ). Similarly, the formation of confluent‐drusen like deposits in hiPSC‐RPE monocultures derived from patients with SFD and another inherited macular dystrophies, Doyne honeycomb retinal dystrophy (DHRD) has been reported (Galloway et al, ). It is plausible that the ability of hiPSCs to differentiate into RPE cells that display several physical and functional characteristics akin to the human adult RPE in vivo contributes to the development of disease‐relevant pathology in culture (Buchholz et al, ; Kokkinaki et al, ; Singh et al, ).…”

“…Given that TIMP3 in the eye is primarily synthesized by RPE cells (Della, Campochiaro, & Zack, ), hiPSC‐derived RPE cells plausibly provide a suitable platform to complement the existing cell culture and animal model platforms for investigating the SFD disease mechanism(s). Furthermore, the fact that (a) hiPSC‐RPE in culture lay a basement membrane containing TIMP3 and secrete TIMP3 into the ECM (Galloway et al, ) (Figure ) with banding pattern on Western blotting consistent with known monomer (~24 kDa), glycosylated‐monomer (~27 KDa) and dimer (~55 kDa) forms of TIMP3 and (b) patient‐derived SFD hiPSC‐RPE develop confluent drusen‐like deposits containing known drusen‐resident proteins (e.g. APOE, TIMP3) (Galloway et al, ) (Figure ) highlights their utility for investigating the role of TIMP3 mutations in the development of specific disease‐relevant phenotypes of SFD (e.g.…”