Title: An iPSC patient specific model of CFH (Y402H) polymorphism displays characteristic features of AMD and indicates a beneficial role for UV light exposure

Age related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease, however these do not exist for the dry form. Complement factor H ( CFH ) polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, retinal pigment epithelium (RPE) damage and visual decline. We have derived and characterised induced pluripotent stem cell (iPSCs) lines from two patients without AMD and low risk genotype and two patients with advanced AMD and high risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H (FH), factor I (FI) and factor H like 1 (FHL-­1). The iPSC RPE cells derived from high risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy and deposition of “drüsen” like deposits. The low and high risk RPE cells respond differently to intermittent exposure to UV light which leads to an improvement in cellular and functional phenotype only in the high risk AMD-­RPE cells. Taken together our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. hormones RPE


Introduction
Age related macular degeneration (AMD) is the most common cause of blindness in the developed world, affecting one in three people by age 75 and is characterised by a loss of central vision, affecting the macular area of the retina. It accounts for 50% of blind and partially sighted registration with an estimated prevalence of ~600,000 significantly visually impaired people in the UK and over 8 million worldwide (1)(2)(3)(4). 70,000 new diagnoses are made every year in UK and 13% of people aged over 80 are affected by late stage AMD.
The number of AMD affected people in UK is expected to rise to 1.3 M by 2050 with healthcare costs rising to 16.4 billion during 2010-2020 (5). Visual loss associated with AMD is caused by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors.
AMD occurs in two forms: "dry" AMD where cellular debris, called drüsen accumulates between the RPE and Bruch's membrane (BrM), appearing as yellow specks on the retina.
"Wet" AMD is usually characterised by aberrant blood vessel growth and encroachment from the choroid underneath the retina although it can also originate from the inner retinal vasculature. Treatments are evolving for wet AMD including anti-VEGF treatments, photodynamic and laser therapy (6)(7)(8);; however there are no effective treatments to prevent progression of the underlying disease process and advanced dry AMD.
AMD is a multifactorial progressive disease with a complex interaction between environmental, metabolic, hereditary factors and chronic innate immune activation (9). A variety of alleles and haplotypes associated with early and late AMD have been identified from genome wide association studies (GWAS) (10)(11)(12)(13), but the precise roles of these genes and the mechanisms by which they increase disease risk are ill defined. One of the most significant genetic findings for AMD has been the complement factor H (CFH) polymorphisms. Complement Factor H (FH) protein functions by limiting the formation of C3 convertase of the complement system and by promoting the degradation of C3b to iC3b.
Failure to control the activity of C3 convertase results in overproduction of C3b and C3a causing a shift in the complement cascade to its terminal lytic pathway. A significantly deleterious consequence of this is the formation of the anaphylotoxin, C5a and the membrane attack complex both of which deliver potent inflammatory signals. The T>C substitution in exon 9 (Y402H) of the CFH gene is strongly associated with susceptibility to AMD and has led to recognition of the importance of complement activation in AMD pathogenesis (10). There is now evidence from large case-control association studies to confirm association with a variety of other complement cascade genes including CFHR1-3, CFI, CFB, C3, and C9 (10,11). The polymorphisms within the 10q26 gene loci containing the PlEKHA1/HTRA1/ARMS2 genes have also consistently demonstrated strong associations with AMD in GWAS (10,12). In addition to data gathered from large genetic cohorts, biochemical and molecular studies have provided substantial evidence to support an important role for complement activation in AMD. This is illustrated by the presence of activators and regulators of the complement system in drüsen (14) and the increased expression of membrane attack complex (MAC) proteins in choriocapillaris and BrM of aged individuals as well as those with the Y402H polymorphism (15)(16)(17).
The Y402H polymorphism can confer >5 fold increase risk of developing AMD and is present in approximately 30% of people of European descent. Although FH protein is synthesised by the choroid, it is not able to diffuse passively through BrM into the retina;; however its alternatively spliced, truncated form, named FH-like protein 1 (FHL-1), is able to do so (18).
FHL-1 retains all the necessary domains for complement regulation and binds to BrM through interactions with heparan sulphate (18)(19)(20). The Y402H polymorphism affects the ability of both FH and FHL-1 to bind to heparan sulphate (21). Furthermore, FH and lipoproteins compete for binding to heparan sulphate in BrM (22), thus it has been suggested that impaired binding of FH/FHL-1 to heparan sulphate in individuals with Y402H polymorphism results in fewer binding sites for FH/FHL-1, increased C3b depositions, lipoprotein accumulation and failure to regulate complement activation, leading to recruitment of mononuclear phagocytes, RPE damage, and visual function decline.
Recent advances in the field of induced pluripotency have permitted generation of patient specific induced pluripotent stem cells (iPSCs) which have the ability to differentiate into cells of any tissue type including photoreceptors and RPE (23). The ability to produce large quantities of functional patient-specific retinal cells from iPSCs offers an unparalleled chance to elucidate disease mechanisms and evaluate new therapeutic agents. Since the pathogenesis of AMD is largely unknown, creating a disease model using iPSC technology could be a valuable tool to address fundamental questions about disease biology as well as creating a biological tool to perform drug discovery and toxicity screening. The validity of this approach has been illustrated by two recent publications reporting derivation of iPSCs from AMD patients with ARMS2/HTRA1 high risk genotypes displaying reduced SOD2 defence, rendering RPE more susceptible to oxidative damage (24,25). We focused on derivation and characterisation of iPSC from individuals homozygous for the low and high risk CFH (Y402H) polymorphism. When compared to iPSC-RPE derived from age matched control low risk individuals, the high risk iPSC-RPE cells show a range of cellular, ultrastructural and functional deficiencies that mimic several key features of AMD including increased inflammation, hallmarks of cellular stress, accumulation of lipid droplets and deposition of "drüsen" like deposits. Exposure to intermittent ultra violet light (UV) elicited different responses from low and high risk RPE cells and in the latter revealed an improvement in the cellular and ultrastructural features associated with AMD. Together our data, suggest that the patient specific iPSC disease modelling provides a robust tool to assess potential therapeutic agents to treat AMD before long expensive trials.

Generation of iPSCs from high risk AMD donors and unaffected controls
To investigate how the Y402H polymorphism in CFH leads to the pathology associated with AMD, DNA was extracted from donor cell fibroblasts and sequenced to detect single 6 nucleotide polymorphisms in the CFH, HTRA1 and ARMS2 genes (Supplementary Figure   1, A). The two homozygous low risk donors were selected on the basis of low risk for all three SNP's rs11200638 (HTRA1), rs1061170 (CFH) and rs10490924 (ARMS2) and no clinical manifestation of AMD. The high risk donors were specifically selected as having advanced AMD with unilateral wet AMD and reticular pseudodrüsen (a known high risk feature for both types of advanced AMD) in their fellow eyes (Supplementary Figure  1, B-C') and high risk SNP for CFH and low risk HTRA1 and ARMS2. The high risk CFH in combination with low risk HTRA1 polymorphism has been consistently associated with central drüsen formation in the older age group (26).
iPSCs were generated from dermal fibroblasts using non-integrative Sendai viral vectors expressing Yamanaka reprogramming transgenes. Between twenty and thirty clones were generated from each donor. At least three clones from each individual were expanded, adapted to feeder free culture conditions and thoroughly characterised using well

Establishing iPSC-RPE from high risk AMD patients and unaffected controls
7 iPSCs were differentiated to RPE using a defined serum and feeder free protocol described in the methods section. The RPE patches were mechanically isolated and expanded on laminin coated trans-well inserts or tissue culture plates. Hexagonal cells with pigmentation both visible macro and microscopically (Figure  1,  A,  B), which expressed the putative RPE cell marker ZO-1, CRALBP and BEST1 (Figure  1,  C). Polarity in the RPE cells is important for their physiological function, we checked the presence of Na + K + -ATPase in both low and high risk iPSC-RPE cells and showed apical localisation in both (Figure 1, D). iPSC-RPE cells also secreted pigment epithelium-derived factor (PEDF) also known as serpin F1 (SERPINF1) in a physiologically similar fashion to adult RPE (27) (Figure  1, E) all cultures.
RPE cells form a tight barrier in the retina which can be measured by trans-epithelial resistance (TER). We observed no significant differences in TER between RPE derived from high or low risk AMD individuals (Figure 1, F). Phagocytosis assays also indicated that iPSC-RPE were able to phagocytose bovine rod outer segments with no differences observed between low and high risk AMD donors (data not shown).

RPE cells.
Expression of, FH/FHL-1 ( Figure  2 Interestingly C3 was down regulated in high-risk iPSC-RPE, while C5 showed no significant 8 difference (Figure  3, C, D). Together these results add to the evidence for local complement synthesis in the eye as previously documented in the literature (28,29).

Gene expression profiles of cytokines in AMD and control iPSC-RPE
High-risk iPSC-RPE had reduced gene expression of mitochondrial Superoxide dismutase 2, (SOD2) (Figure 3, F), which acts to transform superoxide, a toxic by-product of oxidative phosphorylation, into less harmful hydrogen peroxide and diatomic oxygen. It has also been reported that ARMS2/HTRA1 polymorphism leads to compromised superoxide dismutase 2 response (23) while knockout of SOD2 in mice is used as an early model of AMD (30).  (33). We noted a significant difference between genotypes with highrisk donors expressing IL1β at a higher level than low risk RPE (Figure 3, K). Elevated levels of IL-6 are found in the vitreal fluid of AMD patients and have been used as predictors of AMD progression (34), however we did not detect any difference in IL6 between genotypes suggesting that the release of this cytokine is likely from another source such as microglia (Figure 3, L). Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic RPE genes. There was a slight decrease (although not significant) in OTX2 in high-risk donors, perhaps linked to TNF-α expression as previously stated (Figure 3 When all genes with > 1.5 fold expression changes between the high and low risk iPSC RPE were analysed using Enrichr, (OMIM disease) (40), macular degeneration, diabetes mellitus type 2 and protein glycosylation disorder diseases were found to be overrepresented in the responsible for post-translational glycol modification of proteins, and this is considered a location specific modification as the enzymes required are normally compartmentalised.
Glycosylation status is suggested to be important for efficient transport/diffusion of FHL-1 though BrM, FHL-1 is normally non-glycosylated and passes easily (41), while glycosylated CFH does not. Interfering with this status could be detrimental to location and diffusion characteristics. Additionally, advanced glycosylation end products (AGE) are a classical indication of an aged RPE cell layer (42). Together these data suggests that the in vitro iPSC-RPE model we have created mimics the disease at the molecular level.

Many investigations have described the proteomic and lipid composition of drüsen (43).
APOE is ubiquitously associated with drüsen formation and shown to comprise 36% of all proteins found extracellularly (43). The terminal complement complex (TCC) (C5b-C6-C7-C8-C9 n , [C5b-9]) is comprised of five proteins, C5b, C6, C7, and C8, with the fifth, C9 forming a transmembrane ring structure. We found the presence of aggregates that either contained ApoE, C5b-9 or both proteins in low and high risk iPSC-RPE;; however the size of deposits containing both ApoE and C5b 9 was larger in the high risk RPE (Figure 4, A-D).
Significantly, larger lipid globules were also detected in high risk donors compared with low risk (Figure  4, E & F).

Ultrastructural changes to AMD iPSC-RPE
Transmission electron microscopy (TEM) showed that the length of microvilli was reduced in RPE derived from the high risk donors (Figure 5, A). The mitochondrial number also decreased ( Figure 5, C), however the area covered by them was slightly larger in high risk donors ( Figure  5, B) suggestive of fewer but larger mitochondria which could be the result of age related mitochondrial dysfunction or stress (44). Long range PCR assays indicated the absence of mtDNA deletions in the fibroblasts and RPE derived from both low and high risk individuals (data not shown). We also observed the formation of asymmetric vacuoles (marked with red stars) almost exclusively in RPE generated from high-risk donors ( Figure  5, These vacuoles, which are indicative of "adaptive survival" in response to environmental or oxidative stress, have also been observed in a SOD2 knockdown mouse model of early AMD (28). They have the potential to lead to vacuolation-mediated cell death, however our flow cytometric analysis did not indicate significant changes in apoptosis between low and high risk AMD iPSC-RPE (data not shown).

Autophagy is upregulated in high risk AMD iPSC-RPE cells
Due to the increased lipid build-up and ultrastructural changes we suspected that autophagy may have a role in AMD pathogenesis. It has also been documented previously that dysregulated autophagy may sensitise RPE cells to oxidative stress (45). Autophagy is associated with intra/inter cellular waste removal and is upregulated during nutrient starvation and general stress response. In donor fibroblasts no difference in expression of two key autophagy markers, LC3 puncta and p62 aggregates was observed between low and high risk donors (Figure 6 A along with the intensity of p62, which was also increased (Figure 6, J), which potentially suggests a block in autophagy in high risk RPE-iPSC.

The response of low and high risk AMD-RPE cells to UV exposure
UV can induce the generation of ROS derived from diatomic oxygen (O 2 ), superoxide anion (O 2 -), hydroxyl and peroxyl radicals, resulting in DNA damage. The retina is highly susceptible to photochemical damage due to continuous light and UV exposure. This photochemical induction is exacerbated by the retinal oxygen tension (70 mmHg), which is higher than many other tissues, thereby increasing the probability of ROS formation.
Although the relationship between UV light exposure and AMD is unclear, epidemiological evidence indicates an association between the severity of light exposure and the occurrence of AMD (46). Light in the visible UV spectrum (441 nm) is deleterious for RPE cells, being the most energetic radiation reaching the macula and causes photo-oxidation generating reactive photoproducts including N-retinylidene-N-retinylethanolamine (A2E), DNA oxidation, and cell apoptosis (47,48). Drüsen and outer segments are composed largely of lipids (polyunsaturated fatty acids) and are particularly vulnerable to photo-oxidation leading to a chain reaction mechanism of lipid peroxidation and peroxide organic free radical production (49).
To investigate whether UV exposure acts to exacerbate the gene expression, functional or structural defects observed in RPE cells derived from AMD patients, we exposed iPSC-RPE cells continuously to 0.0045 mW/cm 2 of 390-410nm light for 1 hour each day for 5 days which resulted in an increase in the concentration of intracellular reactive oxygen species and decreased mitochondrial membrane potential (data not shown). Pigmentation levels can affect the absorption of UV light, however pigmentation levels between low and high risk donors did not differ significantly (p=0.5; ; Supplementary figure 3, D). IL6 expression, a cytokine previously associated with AMD (50), was increased in response to UV in both low and high risk iPSC-RPE cells (Figure  7,  A). SOD2, VEGF, IL18, CFH and FHL-1 expression increased only in the high risk RPE cells (Figure 7, B, C, D, E) N). Mitochondrial area decreased in both low and high-risk iPSC-RPE when exposed to 390-410nm UV light (Figure 7, P), while the overall number of mitochondria remained similar (Figure 7, Q). While there was no significant increase in the number of vacuoles or drüsen-like deposits in low risk iPSC-RPE cells, in the high risk iPSC-RPE both parameters decreased significantly (below levels observed in the low risk iPSC-RPE cultures;; Additionally microvilli length in high-risk donor cells increased in response to UV (Figure 7, O). Together our data suggest that low and high risk iPSC-RPE cells respond differently to UV light exposure and set precedence for using iPSC-RPE disease modelling as a platform for testing existing and new therapeutic regimes.

Discussion
To date there are no effective treatments that target the underlying disease process in AMD.
Availability of patient specific models which can generate large numbers of RPE cells would provide a significant advance for a better understanding of AMD physiopathology, the contribution of environmental, lifestyle and dietary factors and drug testing. The advent of iPSC technology has made the in vitro modelling of many inherited diseases possible;; however to date this method has predominantly been viewed as useful to physiopathologies that manifest early during development or in childhood. Three recent studies have implemented premature aging approaches to model Parkinson's disease (52) and AMD (23) using iPSC, paving the way for modelling of complex age related disease.
In this manuscript, we investigated whether iPSC could be used to provide a disease modelling tool which mimics an AMD phenotype in the laboratory in the absence and presence of stress stimuli. Our rationale was to focus on patients phenotyped with a significant risk factor for AMD, such as Y402H polymorphism in the CFH gene. Using the iPSC derived RPE cells generated from low and high risk donors in the absence of any stress stimuli, we have been able to confirm several key cellular features of AMD as follows: (i) increased expression of inflammatory markers (for example IL1β);; (ii) lower expression of the protective oxidative stress markers (SOD2);; (iii) increased number of stress vacuoles (and their surface area);; (iv) increased accumulation of lipid droplets and (v) increased expression of LC3 vesicles and higher p62 expression/aggregate suggestive of impaired autophagy. Most importantly, we were able to identify the formation of deposits comprising of components including Apolipoprotein E and C5b-9, in keeping with drüsen formation. These deposits occupied a significantly higher volume in the RPE derived from high-risk lines. The presence of drüsen and its larger volume in RPE derived from the high risk iPSC lines, together with confirmation of key molecular features observed in previous AMD studies, suggest that this iPSC model closely mimics the disease phenotype observed in AMD patients.
The complement proteins associated with AMD (FH, FI, FHR1, FHR3, C2, C3) are plasma proteins predominantly produced in the liver;; however biosynthesis at extrahepatic sites is now well recognised (17,18). As with the blood-brain barrier, the blood-retinal barrier limits access to circulating plasma proteins and it has been suggested that local complement synthesis may be required for its effects in such areas. Indeed, it has been shown that choroid and RPE as well as cultured unstimulated RPE cells produce transcripts for most classical (CP) and alternative (AP) pathway complement genes (17,18). Data summarised in this manuscript indicate that iPSC derived RPE cells express the active complement proteins and are able to modulate their expression in response to stress stimuli without having to rely on secretion from the choroid and diffusion through the BrM as previously suggested (18).
In view of this local complement regulation by RPE cells themselves, we were interested to assess the response of low and high risk RPE responses to stress stimuli. Since a trend towards an association between severity of light exposure and AMD has been suggested by epidemiological studies, we exposed the iPSC-derived RPE to repeated doses of UV for five consecutive days. Pigmentation level between the two groups was not significantly different.

The low risk RPE cells responded by increasing the expression of inflammatory marker IL6
and complement factor I (CFI). More significant changes were observed in high risk RPE cells, which upregulated the expression of protective oxidative stress defence protein, SOD2 as well as complement factor H (CFH) and its truncated form, FHL-1, in addition to showing an improved ultrastructural (increased microvilli length, reduced number of stress vacuoles and lower mitochondrial area) and functional (lower volume of drüsen like deposits) properties. These results indicate that the low and high-risk AMD-RPE cells respond very differently to UV exposure and moreover this provides evidence for UV mediated functional and cellular improvement of AMD-associated cellular changes in high-risk AMD-RPE cells.
These intriguing results which we attribute to increased SOD2 expression need to be validated in a larger number of cell lines derived from additional high risk donors over longer intervals and with different UV doses. They do however highlight an important role for increased oxidative stress defence as a potential therapeutic strategy for AMD, corroborating recent data obtained with the HTRA1/ARMS2-iPSC model and exposure to nicotinamide (25). Nonetheless, human clinical trials are complex, expensive and require prolonged periods to assess the long-term effect of a therapy in large numbers of patients with specific phenotypes to provide a consistent end point. The assessment is further complicated by differing progression rates in patients and the uncertain choice of disease endpoints to assess progression (55). This is a significant problem and can lead to trials having negative but disputed conclusions (e.g. the COMPLETE study on Eculizimab for dry AMD) (56). A robust and well characterised in vitro model such as the one described herein provides an efficient tool to assess potential therapeutic agents to treat AMD (such as complement pathway modulation), to better understand disease physiopathology and to test/repurpose drugs.

Human donors
Written informed consent was obtained from each donor, all samples were obtained as part

iPSC characterisation
The pluripotency of iPSC lines was confirmed with immuno-fluorescence, flow cytometry and RT-PCR. Cells were fixed with 4% paraformaldehyde for 15 minutes and stained with primary antibodies OCT4 and SSEA4 (Abcam, UK). Primary antibodies were detected using Alexa Fluor secondary antibodies (Supplementary figure 4, B). Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI). Images were captured using a fluorescence microscope (Leica Axiovert, Germany). iPSC were assessed for their propensity to generate all three germ layers using primary antibodies against AFP, TUJ1 and SMA, primary

DNA extraction and sequencing
DNA was extracted using a column biased method (Qiagen, Germany), sequence tagged PCR was performed using 100ng of DNA. Sanger sequencing was performed (GATC biotech, Germany) and results were interpreted using finch TV (Geospiza, USA).

Quantitative RT-PCR
RNA was extracted from frozen cell pellets using ReliaPrep™ RNA Cell Miniprep System as per the manufactures instructions. RNA quantification was performed with a NanoDrop™2000 spectrophotometer (Thermo, USA). We ensured that the 260/280 ratio and concentration was between 1.7-2.1 and yields of >250ng/μl respectively. cDNA synthesis was performed using Promega GoScript™ Reverse Transcription System as per the manufactures instructions. All experiments were performed using a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, UK), using SYBR green reaction technology (Promega, UK). Cycle parameters are as follows: 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, finalising with a Melt Curve Stage. The Livak method (ΔΔC t ) was used (57), C t results of the target genes were normalised to the C t of the reference gene GAPDH finally the fold difference in expression was determined (2 -ΔΔC t ). A list of the primers used can be found in (Supplementary  figure  5,  B).

RNA Sequencing
RNA was extracted using RNeasy Micro Kit (Qiagen)) according to the manufactures instructions, from six cell culture inserts, three of each genotype. cDNA was generated whereby genes that lie three standard deviations from the mean (μ- 3σ) were selected (≥5 fold change). This gene list was queried against the PANTHER (protein annotation through evolutionary relationship) classification system to highlight disproportionally expressed pathways. RNA-seq data is deposited into GEO (accession number GSE91087). A list of the overrepresented glycogenesis genes can be found in Table  6.

Pigment Bleaching
Post fixation and prior to immunocytochemistry, RPE cells were bleached using a Melanin Bleach Kit (Polysciences, USA) to remove pigmentation, as melanosomes can cause excessive auto-fluorescence. Pre-treatment Solution A was added for 5 minutes at room temperature, the solution was removed and cells washed two times with PBS. Pre-treatment Solution B was then added for 1-3 minutes until pigmentation was removed. This solution was removed and cells washed again with PBS.

In vitro and in vivo 3 germ layer differentiation
In vitro: iPSCs were spontaneously differentiated to allow the emergence of cell types representative of the three embryonic germ layers. iPSCs were allowed to reach 80% confluency after which the media was switched to DMEM/F12 (Thermo Fisher Scientific), 20% Foetal bovine serum (FBS) (Thermo Fisher Scientific), 1% Penicillin-Streptomycin Solution (Thermo Fisher Scientific) and 1% MEM Non-essential Amino Acids Solution (Thermo Fisher Scientific  Figure  4,  B), for 1 hour, after which the cells were washed as previously stated and 1-2 drops/mL of NucBlue was added. Plates were stored at 4°C prior to imaging on a Zeiss Axioplan microscope. All incubations occurred at room temperature unless otherwise stated.

Teratoma formation in immuno-deficient mice
All procedures were approved and conformed to institutional guidelines. 1x10 6 iPSCs were injected subcutaneously into the right flank of adult NOD/SCID mice. All cells were cotransplanted in a 50µl Matrigel carrier, (BD Biosciences) to enhance teratomae formation.

Trans-epithelial resistance
Trans-epithelial resistance (TER) was measured with a Millicell ERS-2 Voltohmmeter (Merck Millipore). Firstly the electrical resistance of a blank cell culture insert with media in both apical and basal compartments was measured, after which inserts with cells were measured.

Transmission electron microscopy
Cells were fixed with 2% glutaraldehyde and kept at 4°C. TEM including all the cell processing was performed at Newcastle University Electron Microscopy Research Services, Ultrathin sections were stained with heavy metal salts (uranyl acetate and lead citrate) and imaged on a Philips CM100 TEM.

Statistical analysis
Shapiro-Wilk test was used to determine normality, for normally distributed data sets, one-       and APOE in high risk iPSC-RPE cells, * p=0.0002.