Protective role of decellularized human amniotic membrane from

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Protective role of decellularized human amniotic membrane from oxidative stress induced damage on retinal pigment epithelial cells. Lekshmi Krishna, Kamesh Dhamodaran, Murali Subramani, Murugeswari Ponnulagu, Nallathambi Jeyabalan, Sai Rama Krishna Meka, Chaitra Jayadev, Rohit Shetty, Kaushik Chatterjee, Samanta Khora, and DEBASHISH DAS ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00769 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 26, 2018

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ACS Biomaterials Science & Engineering

Protective role of decellularized human amniotic membrane from oxidative stress induced damage on retinal pigment epithelial cells. Lekshmi Krishna1,2, Kamesh Dhamodaran1#†, Murali Subramani1#, Murugeswari Ponnulagu1, Nallathambi Jeyabalan3, Sai Rama Krishna Meka4 , Chaitra Jayadev5, Rohit Shetty6, Kaushik Chatterjee4, Samanta Sekhar Khora2, Debashish Das1* 1. Stem Cell Research Lab, GROW Laboratories, Narayana Nethralaya Foundation, 258/A, Bommasandra Industrial Area, Bangalore, Karnataka, India. 2. School of Bioscience and Technology, VIT University, Vellore, Tamil Nadu, India. 3. Grow Laboratories, Narayana Nethralaya Foundation, 258/A, Bommasandra Industrial Area, Bangalore, Karnataka, India. 4. Department of Materials Engineering, Indian Institute of Science, Bangalore, Karnataka, India. 5. Department of Vitreo-retinal Services, Narayana Nethralaya Eye Institute, 258/A, Bommasandra Industrial Area, Bangalore, Karnataka, India. 6. Department of Cornea and Refractive Surgery, Narayana Nethralaya Eye Institute, 258/A, Bommasandra Industrial Area, Bangalore, Karnataka, India # Authors equally contributed to the work for this manuscript. †

Present address: Department of Basic Sciences, The Ocular Surface Institute, College

of Optometry, University of Houston, Houston-77204, TX-USA. *Corresponding address: Dr. Debashish Das Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Narayana Nethralaya Eye Institute, 1 ACS Paragon Plus Environment

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Narayana Health City, 258/A, Bommasandra Industrial Area, Bangalore- 560 099, Karnataka, India Fax No: +91 80 6666 0650 Tel No: +91 80 6666 0722 Email: [email protected]; [email protected]

Abstract Oxidative stress is an important cause for several retinal aging diseases. Cell therapy using a decellularized human amniotic membrane (dHAM) as a tissue scaffold for retinal pigment epithelial cells has a potential therapeutic role in such pathological conditions. This is attributed by dHAM’s anti-inflammatory, anti-microbial, low immunogenicity aspects apart from harboring a drug reservoir potential. The underlying mechanisms for maintaining the physiological properties of transplanted cells and their survival in a diseased milieu using dHAM has remained unexplored/unanswered. Hence, we investigated the potential role of dHAM in preserving the cellular functions of retinal pigment epithelium in an oxidative stress environment. Adult human retinal pigment epithelial (ARPE-19) cells were cultured on dHAM or tissue culture dishes under hyperoxia.

Gene

expression,

immunofluorescence

staining,

enzyme-linked

immunosorbent assay (ELISA) and scanning electron microscopy (SEM) were performed to assess the levels of reactive oxygen species, proliferation, apoptosis, epithelial-mesenchymal transition, phagocytosis and secretion of vascular endothelial factors. These results indicate that reduced epithelial-mesenchymal transition, generation of reactive oxygen species (p≤0.0001), and apoptosis (p≤0.05) in cells 2 ACS Paragon Plus Environment

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cultured on dHAM compared to those on tissue culture dishes in oxidative stress conditions. Concomitantly, the secretion of the vascular endothelial growth factor was significantly reduced (p≤ 0.01) on dHAM. Phagocytic activity was significantly higher (p ≤0.001) in cells cultured on dHAM and were comparable to those cells cultured on tissue culture dishes. SEM images showed a clustered growth pattern on dHAM compared to an elongated morphology when cultured on tissue culture dishes under oxidative stress conditions. These findings demonstrate the utility of dHAM as a scaffold for growing retinal epithelial cells and to maintain their physiological properties in an oxidative stress condition with a potential to develop regenerative medicine strategies to treat degenerative eye diseases. Keywords: Decellularized human amniotic membrane, retinal pigment epithelium, oxidative stress.

Introduction Reactive oxygen species (ROS) released by oxidative stress on retinal pigment epithelial (RPE) cells has been associated with a number of retinal diseases such as age-related macular degeneration (AMD), glaucoma, diabetic retinopathy and retinal vein occlusion (RVO).1-2 Repopulating damaged cells with healthy RPE has potential to be a long term treatment strategy.3 In this aspect, an efficient method is the usage of natural and synthetic scaffolds for RPE to maintain the polarity and culture trait prior to transplantation. Amongst the several strategies utilized, RPE cells transplanted on decellularized human amniotic membrane (dHAM) has shown some promise.4

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The human amniotic membrane (HAM) is a thin and elastic tissue that forms the inner layer of the amniotic sac consisting of an inner layer of epithelial cells on a basement membrane that is composed of primarily Type IV COLLAGEN, LAMININ and HEPARAN SULPHATE. It has been widely used for transplantation of skin, reconstruction of oral cavity, bladder and ocular surface reconstruction (severe pterygium, chemical burns and Stevens–Johnson syndrome).5 The RPE is a major source of vascular endothelial growth factor (VEGF) in the eye and the retinal and choroidal vasculature is majorly driven by the VEGF levels in the eye.6 Apart from its role in vasculature, VEGF enables the survival of endothelial cells, protects the choroid, Müller cells, photoreceptors, and the retinal neurons.7 VEGF levels remain critical in defining the ocular physiology and patho-physiology. Increased levels of VEGF is one of the major drivers of vasoproliferative diseases, such as proliferative diabetic retinopathy (PDR), RVO, and wet-age related macular degeneration (wAMD).6 The secreted VEGF acts by binding to VEGF R1 and R2 receptors and activating the downstream cascade. Epithelial- mesenchymal transition (EMT) of RPE cells is a crucial step in the progression of PDR leading to contractile fibrotic membrane formation and tractional detachment of the retina.8-9 Recently, we have shown that VEGF inhibition of EMT can be reversed by the presence of bevacizumab in RPE cell cultures.10 Human RPE from cadaveric sources have been shown to maintain the RPE physiology with the expression of specific markers and morphologically when cultured on dHAM.11 Subretinal transplantation of HAM supports RPE growth and functionality and has been used as a replacement for the Bruch’s membrane. 4 ACS Paragon Plus Environment

4, 12-13

Most of the

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RPE transplantation studies have focused on transplanting the cell sheet in a nondiseased model to study the immunomodulatory effects. Though there are very few studies showing transplantation of RPE cells on dHAM in a disease model.13-15 RPE cell sheets alone have been transplanted in Royal College of London rats to restore the RPE cell functionality and vitality albeit with a short follow-up.16-19 However, further progress in the clinical applications of dHAM as a cell carrier can be greatly facilitated by developing a detailed understanding of the mechanisms of how dHAM can restore the RPE functionality in a diseased milieu. Towards that goal, in this study, cells cultured on dHAM and tissue culture dish were analyzed for cellular properties such as, proliferation, apoptosis, phagocytosis, EMT and secreted VEGF levels.

Experimental Materials and methods Preparation of human amniotic membrane: Samples were collected after obtaining approvals from the Institutional Ethical Committee and Institutional Review Board and the study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. To explain the procedure briefly, with the patients’ consent, the placenta was collected post caesarean section, washed with a 2X antibiotic solution (200U/ml penicillin, 200µg/ml streptomycin and 10µg/ml amphotericin B; Himedia, Mumbai, India) until all residual blood was removed. The amnion was then manually separated from the chorion. The orientation of the membrane (epithelial and endothelial surfaces) was determined based on the attachment of the umbilical cord to 5 ACS Paragon Plus Environment


the placenta. The amnion was spread out with its epithelial side facing downwards in a sterile pan containing 2X antibiotic solution. The mucus layer was cleaned with cotton swabs and sterilized nitrocellulose membrane was placed taking care to avoid entrapment of air bubbles on the amniotic membrane. Membranes of desirable sizes were then excised and stored in vials containing 15ml of 1:1 DMEM: glycerol medium at -80°C (Supplementary Figure 1A-F). The sterility of the processed membrane was further ensured by incubating it in fluid thioglycollate medium at 37°C and soybean casein digest medium (HiMedia, Mumbai, India) at 25°C for 7 days.20 Preparation of decellularized human amniotic membrane: Stored HAM at -80°C were thawed to room temperature and further decellularized as per the reported protocol.20 The HAM was placed carefully with the epithelial surface facing upward on a 60 mm petri dish containing a microscopic slide of size 2.5 x 2.5 cm2. The nitrocellulose paper was removed carefully rinsed in 1X phosphate buffered saline (PBS) and incubated with 0.25% trypsin-EDTA (Gibco, Life Technologies, Carlsbad, USA) for 15-20 minutes at 37°C. The dissociated epithelial layer from the membrane was scrapped off with sterile glass slides and rinsed with 1X PBS and observed under a phase contrast microscope to ensure the completeness of the decellularization procedure (Supplementary Figure G-J). This dHAM was then tucked onto a 2.5x2.5 cm2 glass slide and further used for culturing ARPE-19 cells.

Hematoxylin and eosin staining of HAM and dHAM: The membranes were fixed in 4% paraformaldehyde (Sigma Aldrich, MO, USA) and incubated for 10 min in hematoxylin solution, followed by decolorization in 1% acid 6 ACS Paragon Plus Environment

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alcohol, 2% sodium bicarbonate (Sigma Aldrich, MO, USA) and counter stained with eosin for 1 minute (Thermo Fisher Scientific, Mumbai, India). The stained membranes were dehydrated with a series of alcohol and xylene washes (Merck, Mumbai, India) and mounted with Distrene Dibutyl Phtalate Xylene (DPX; Merck, Darmstadt, Germany). The stained membranes were documented under a light microscope, using the NIS ELEMENTS imaging software (Nikon E200, Japan) (Supplementary Figure 1K-L; Scale bar = 5m).

Cell culture: ARPE-19 cells cultured on both, a tissue culture plate (TC) (Nunclon, Thermo Fisher Scientific, MA, USA) and dHAM, under normoxia and hyperoxia were analyzed during the study. The ARPE-19 cells were seeded at a density of 1X 105 per well in a 12 well plate (Nunclon, Thermo Fisher Scientific, MA, USA) and a 1X1 cm2 area on dHAM. Dulbecco’s Modified Eagle Medium: F12 (DMEM:F12) (Invitrogen, California, USA), containing 10% fetal bovine serum (Invitrogen, California, USA), and antibiotics (100U/ml of penicillin, 100g/ml of streptomycin, 5µg/ml amphotericin B) was used for culture and maintenance of cells under normoxia and hyperoxia for 5 days to mimic chronic oxidative stress conditions. It has been previously demonstrated that five days of mild oxidative stress leads to changes those are consistent with some of the pathological changes associated with AMD.21 ARPE-19 of passage 12-16 was used for this study.

Induction of oxidative stress:



The experiment commenced as cultures placed in a normoxic condition of 20% oxygen and 5% CO2 at 37ºC until reaching confluency and then transferred to a humidified hyperoxia chamber, a multi gas incubator (SMA-80DS/165, Astec, Japan) maintained at 40% oxygen and 5% CO2 at 37ºC with control plates under normoxic conditions.

Scanning electron microscopy: The surface characteristics of HAM and dHAM as well as the ARPE-19 cells grown on TC and dHAM under normoxia and hyperoxia were imaged using SEM (FEI Quanta 200 EDAX) at an acceleration voltage of 20 kV. Samples of 1cm x 1cm were processed for SEM observation by fixing them in 2% paraformaldehyde solution (Sigma Aldrich, Missouri, USA), followed by dehydration through a series of graded ethanol solutions (50, 75, 90, 95 and 100%) with a 10 min rinse each. Dehydrated samples were stored in desiccators and were sputter coated with gold (JEOL JFC-1200 fine coater) for 60 seconds and imaged by SEM.

Cell viability count - trypan blue: Cell viability of ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia was assessed using trypan blue 0.4% (w/v) (Mediatech, Cellgro, USA) staining. Viable and non-viable cells were counted with the aid of a hemocytometer, the percentage of viable cells determined, and represented graphically.

3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay:


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Metabolic activity of ARPE-19 cells cultured on TC and dHAM was measured colorimetrically by MTT - assay as per the manufacturer’s instructions (XpertTM, Himedia, Mumbai, India). The cells cultured on TC and dHAM were incubated with MTT solution (0.5 mg/ml, 37°C, 5% CO2) for 3 hours. After incubation the supernatant was removed and intense purple formazan crystals were dissolved in dimethyl sulfoxide (DMSO; MP Biochemicals, Mumbai, India). The optical absorbance of the solution at 570 nm wavelength was measured using a microplate reader 680 (Bio-Rad™, California, USA). The experiment was performed in triplicate.

RNA extraction and reverse transcription: Total RNA was extracted from the cells grown on TC and dHAM under normoxia and hyperoxia on Day 5 of incubation using a RNeasy Micro Kit, (Qiagen, Hilden, Germany) and quantified with a Nanodrop spectrophotometer 1000 (Thermoscientific, Wilmington, USA). Complementary DNA (cDNA)was generated using a high capacity cDNA Reverse Transcription Kit, (Life Technologies, CA, USA) and stored at -20ºC.

Quantitative real time polymerase chain reaction (RT-qPCR): RT-qPCR was performed according to the manufacturer’s instructions by using a KAPPA SYBER FAST qPCR kit Master Mix (Kapa Biosystems, Wilmington, MA, USA), and analyzed with CFX ConnectTM (Bio-Rad, Hercules, CA, USA) for the cDNA generated samples. All experiments were done in triplicate, and the data was normalized with the expression of the housekeeping gene gapdh. The delta cycle threshold (Ct) value was calculated and graphically represented as the difference



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between the Ct of the gene of interest (GOI) and Ct of the reference gene (RG). Delta Ct = CtGOI – CtRG. The list of gene specific primers along with the product size is mentioned in Supplementary Table 1. Immunofluorescence: Cells cultured on TC and dHAM under normoxia and hyperoxia were fixed using 2% paraformaldehyde on ice for 10 min, permeabilized with 0.1% Triton X 100 (Thermo Fischer Scientific, CA, USA) in 1X PBS for 15 min, blocked for 1 hour at room temperature with 1% bovine serum albumin (Himedia, Mumbai, India), and incubated overnight at 4ºC with the primary antibodies. After a brief wash in 1X PBST (0.02% Tween 20 (MP Biomedicals, CA, USA) in 1XPBS), cells were incubated with secondary antibodies for 1 hour. The slides were mounted using a VECTASHIELD® containing 2(4-amidinophenyl)-1H-indole-6-carboxamidine

(DAPI)

aqueous

mounting

medium

(Vector laboratories, CA, USA). Fluorescent images were documented using the ProgRes® Capture Pro 2.5 software on a fluorescent microscope (Olympus BX41, Tokyo, Japan). Fluorescence intensity was quantified using the Image J 1.48 version software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and represented graphically. At least 200 cells were counted from 12-17 images. The details for each graph with respect to numbers of cells counted and the number of images used are provided in the figure legends. The list of primary and secondary antibodies is furnished in Supplementary Table 2.

Cell density:


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Cell density of ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia were estimated as per the formula of density = mass/volume. The cells per area were calculated from the total number of particles fractioned by the total area of interest. The threshold DAPI fluorescent images were utilized for analysis using NIH ImageJ 1.48 version software (particle analyzer plugin).22

Cell Morphology: Cellular shape of ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia was estimated as per the specific morphology descriptors like the cell shape factor. The cell shape factor was calculated from the total projected actin area (A) and the total perimeter (P) of the cell from the fluorescent images using NIH ImageJ 1.48 version software. The cell shape is inferred from the two arbitrary values that lie between zero and one. If the shape is round or circular, the shape factor approaches one. As the shape becomes less round, elongated and irregular shaped, the shape factor approaches zero.23-24

ZO-1 Stability: The tight junction protein stability on the cell membrane was analyzed from the ARPE19 cells cultured on TC and dHAM under normoxia and hyperoxia. From the fluorescent images, a ratio was calculated between the fully maintained intact ZO-1 membrane and the total numbers of DAPI positive nuclei, and subsequently the percentage was estimated.



HAM protein extraction and western blotting: Both HAM and dHAM in 1.5 ml micro centrifuge tubes (Eppendorf, CA, USA) were immersed in liquid nitrogen and pulverized using a pestle. The crushed membranes were further lysed with the Radioimmunoprecipitation assay lysis buffer (25 mM Tris, 150 mM Sodium Chloride, 1% NP-40, 1% Sodium Deoxycholate, 0.1% Sodium dodecyl sulfate (SDS); G- Bioscience, St. Louis, MO, USA) and a protease inhibitor cocktail (Roche, CA,USA). ARPE-19 cells cultured on the dHAM/TC were trypsinized, collected in 1.5 ml centrifuge tubes and further lysed with the RIPA lysis buffer. After a series of freeze-thaw cycles, the protein lysate was collected post centrifugation. The lysates were mixed with 5X SDS buffer, separated on a 12% SDS-Polyacrylamide gel electrophoresis (PAGE) and wet transfer performed on a polyvinylidene difluoride (PVDF) membrane (Rugby WAR, UK). The membrane was blocked with 5% skimmed milk powder in 1X PBS with 0.1% Tween 20 (PBST) and incubated overnight at 4°C with the primary antibodies. This was followed by rinsing the membrane with 1XPBST and incubation for 1 hour with HRP conjugated secondary antibodies at room temperature. The respective protein bands were visualized using a chemiluminescence detection kit (Pierce® ECL Plus, Thermo scientific, Rockford, IL, USA) and documented with an Image Quant™ LAS 500 gel documentation/chemiluminescence detector (GE Healthcare Life Science, Uppsala, Sweden).6 The list of primary and secondary antibodies used for western blot is mentioned in Supplementary Table 2.

Phagocytosis assay:


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ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia were incubated with FITC labeled latex beads (1:50) (Cayman Chemicals, Michigan, USA) for 48 hours at 37°C. For immunofluorescence, the ARPE-19 cells on a cover slip and dHAM were mounted with a VECTASHIELD® mounting medium with DAPI and observed under a fluorescent microscope. Fluorescent images were documented using the ProgRes® Capture Pro 2.5 software.6, 10 In order to avoid counting beads stuck to the membrane and not internalized, post phagocytosis assay cells were incubated for 2 mins with trypan blue (quenching solution) followed by 3X wash with the assay buffer. The number of DAPI positive cells with opsonized latex beads were counted manually and represented graphically.

Caspase activity: Active caspase staining was performed using a FAM Caspase activity kit (Abgenex, San Diego, CA, USA). Cells were stained as per the manufacturer’s instructions. The stained cells were observed and documented using the ProgRes® Capture Pro 2.5 software on a fluorescent microscope. Fluorescence intensity was quantified using the Image J 1.48 version software and represented graphically as a percentage.

Reactive oxygen species: To depict the intracellular ROS changes induced by oxidative stress a fluorescent probe, 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen, Molecular Probes, USA), was used. Cultured cells were stained with DCFH-DA as per the manufacturer’s instructions. The stained cells were observed under an Olympus BX41



fluorescent microscope and fluorescent images were documented using the ProgRes® Capture Pro 2.5 software.. Fluorescence intensity was quantified using the Image J 1.48 version software and represented graphically.

Measurement of VEGF secretion: ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia as well as the dHAM controls were analyzed for secreted VEGF levels in the cell supernatant by using a sandwich Human VEGF DuoSet® ELISA (R&D Systems, MN, USA). Samples and standards were run in triplicate. The reaction product was quantified with a model 680 microplate reader (Bio-Rad, CA, USA) at a wavelength of 450 nm with the reference filter at 570 nm.

Statistical analysis: All experiments were performed at least in triplicate and the results of independent experiments were used for statistical analysis. All the data are represented graphically with the mean ± standard deviation (SD). Results were analyzed using the nonparametric Mann Whitney U test. Significance value denoted was p* < 0.05, **< 0.01, ***< 0.001. Statistical analysis was done by comparing cells cultured on dHAM and TC under normoxia, and dHAM and TC under hyperoxia. All the data including the mean, SD, n and p value are listed in Supplementary Table 3.

Results Characterization of HAM and decellularized HAM:


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The decellularization was assessed by studying cell morphology in HAM and dHAM (Supplementary Figure 1). Phase contrast images showed sheets of epithelial cells across the HAM that were not visible on the dHAM (Supplementary Figure 1I-J). Epithelial cells were tightly packed on the HAM as a single sheet as shown by hematoxylin and eosin staining, whereas the dHAM was devoid of epithelial cells. (Supplementary Figure 1K-L). SEM images of the HAM showed regions of intercellular as well as cellular contacts with the matrix. Such points were not visible on the dHAM (Supplementary Figure 1M, N; Scale bar = 10m, Insert- Scale bar = 3m). Furthermore, at higher magnification, interspersed COLLAGEN fibers of around 50nm diameter were visible on the dHAM. These randomly aligned fibers are shown in the inset (Supplementary Figure 1P, Scale bar = 1m, Insert- Scale bar = 100nm). We also characterized the profile of extracellular protein markers such as FIBRONECTIN, COLLAGEN I, and FGF-2. Quantification of the Western blot reveals a similar expression of FIBRONECTIN on both HAM as well as dHAM. There is a loss of COLLAGEN I and FGF-2 on dHAM compared to HAM (Supplementary Figure 1 O (i-ii)).

Morphological and ROS characterization of cultured ARPE-19 cells: Phase contrast images depicted the morphology of the cells cultured on dHAM and TC in normoxia and hyperoxia. A clustered pattern of growth was observed on dHAM and a well spread out fibroblastic growth pattern on TC (Figure 1A (i-iv)). SEM images further reconfirmed the colligated nature of cells cultured on dHAM and a well dispersed fibroblastic morphology of cells cultured on TC (Figure 1A (v - viii)).



Reactive oxygen species are molecules and free radicals derived from molecular oxygen that can damage the deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and oxidize proteins and lipids. Free radicals, such as superoxide radicals (O2) hydroxyl radicals (OH), hydrogen peroxide (H2O2), and singlet oxygen (1O2) predominantly lead to cellular damage.25 Oxidative species are produced under pathological situation, adverse conditions and during cellular metabolism. Low levels of ROS are necessary for protein phosphorylation, transcription factor activation, cell differentiation, apoptosis and cell immunity, while elevated levels are detrimental to cell survival. The enzymatic-non enzymatic antioxidant mechanisms protect the cells by converting ROS to free radicals and their metabolites. Superoxide dismutase (SOD), catalase and glutathione peroxidase are the most common antagonists of superoxide radicals. Vitamin C, E and beta-carotene are some of the non-enzymatic scavengers of ROS.26 A balance between the levels of ROS and antioxidants is essential for cellular health, while an increase in ROS levels gives rise to oxidative stress conditions.

For the detection of ROS, DCFH-DA is the most widely used fluorogenic dye. It measures the hydroxyl and peroxyl activities in the cells.27 Human amniotic membrane, amniotic fluid, and amniotic membrane epithelial cells are known to harbor anti-oxidant properties. The amniotic membrane’s anti-oxidant property, combined with hyaluronic acid as an anti-inflammatory agent, aid its ability to scavenge ROS.28 DCFH-DA staining for ROS levels showed similar levels of positivity in cells cultured on TC and dHAM under normoxia. Two distinct patterns were seen, puncta and diffuse with more puncta staining in cells cultured on dHAM and diffuse pattern on TC (Figure 1B (i-iii)). The


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puncta stained particles are intracellular oxidized material packaged in vesicles that eventually undergo degradation in cells grown on dHAM. The presence of diffuse staining implicates presence of ROS in the intracellular space. A higher intensity of diffuse DCFH-DA staining therefore suggests higher ROS levels in the cells.29 There was no difference in the calculated mean fluorescent intensity of cells cultured under normoxia (Figure 1C). However, there was a notable reduction in the DCFH-DA staining in cells cultured on dHAM compared to those cultured on TC under hyperoxia (Figure 1B (ii-iv)). Image quantification revealed a significant increase in DCFH-DA positivity in cells cultured on TC compared to dHAM under hyperoxia (Figure 1C). The slightly lower cell density on TC under hyperoxia conditions can be attributed to a higher percentage of cell death (Supplementary Figure 2A). The results show that culturing cells on dHAM reduces the generation of ROS as depicted by DCFH-DA positivity compared to those cultured on TC under hyperoxia.

Proliferative status of cultured ARPE-19 cells: Proliferation of RPE cells is inhibited in hyperoxia with increasing grades of oxygen tension.30 It is assumed that proliferation of RPE cells in a pathologic environment gives rise to aberrant cells thereby causing disease. A harmony between the cyclins and cyclin dependent kinases (CDKs) is important for cell cycle regulation. CDKs bind to cyclin and thereby phosphorylate other cell cycle regulators to initiate cell cycle. Cyclin A shuttles between nucleus and cytoplasm and binds to CDK2 and CDK1 regulating both S phase and G2/M transition phase of the cell cycle respectively. Cyclin B is a mitotic cyclin, required for the cells to progress into and



out of the M phase. This cyclin binds to CDK1 and its activity elevates through the cell cycle until M phase, where it abruptly falls down. Cyclin D1 is a cell cycle protein that shuttles between the nucleus and cytoplasm. It is a key regulator of proliferation with elevated levels during the G1 phase that lowers as the cell proceeds for DNA synthesis in S phase. Cyclin D1 binds to CDK4 and CDK6 for its functional activation. Cyclin E acts as a check gate for cells to enter to S phase by binding to the regulator subunit of CDK2.23 Ki67 is present in all the active phases of cell cycle- G1, S, G2 and M phase, but absent in the resting cells. Hence, it is widely used as a marker to determine the number of proliferating cells in a group of cells. Positivity of Ki67 staining implies that the cells are undergoing cell cycle.31

Hence, ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia were analyzed for the cellular proliferation rate. We found there was a significant decrease in the mRNA levels of cyclin A, cyclin B, cyclin D1, cyclin E, cdk1, cdk2, cdk4 and cdk6 in cells cultured on dHAM compared to those cultured on TC under normoxia (Figure 2AB). Cells cultured under hyperoxia revealed a significant decrease in the expression of cyclin A, cyclin B, cyclin D1, and cyclin E mRNA on dHAM compared to those on TC (Figure 2A). There was a significant increase in the mRNA level of kinases such as cdk1, cdk2, cdk4 and cdk6 (Figure 2B). Immunofluorescence staining of Ki67 showed a higher number of Ki67 positivity in cells cultured on TC compared to dHAM under normoxia (Figure 2C (i-iii)). Graphical representation of the quantified Ki67 positive cells revealed significantly lower Ki67 positivity in cells cultured on dHAM compared to those


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on TC under normoxia. However, there was no significant difference between cells cultured on TC and dHAM under hyperoxia (Figure 2C (ii-iv) and 2D).

Apoptotic status of the cultured ARPE-19 cells: Several studies have shown the anti-apoptotic property of dHAM on epithelial cells.32-33 Hence, we investigated the apoptosis status of the cells grown on dHAM and TC under normoxia and hyperoxia conditions using the vital stain, trypan blue. The percentage of viable cells was similar in cells cultured on TC and dHAM under normoxia. On the contrary, there was a significant decrease in the percentage of viable cells when cultured on TC when compared to those on dHAM under hyperoxia (Figure 3A). MTT is the measure of cell proliferation rate, cell size, metabolic rate and cell survival. MTT directly measures the cell metabolic rate based on the ability of the metabolically active cell to reduce the yellow Tetrazolium salt by forming the Formazan crystals that is measured at 570nm. This indirectly states that higher the metabolically active cells (viable cells) higher would be Formazan crystal formation and hence, higher the absorbance value. MTT assay showed that cells cultured on dHAM showed higher cell viability compared to TC, both under normoxia and hyperoxia. Cells grown on dHAM were metabolically active compared to TC (Figure 3B). To further validate the apoptosis levels we checked for the mRNA expression of bax, bcl2, caspase 3 and caspase 9 levels. BAX is a BCL2 family of pro-apoptotic proteins, present in higher eukaryotes that is able to pierce the mitochondrial outer membrane to release cytochrome C thereby inducing cell apoptosis.34 BCL2 is also a member of BCL2 family but belonging to the anti-apoptotic proteins.35 BCL2 prevents the action of



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BAX by limiting its number of insertion sites on the mitochondrial membrane.36 Caspases are protease enzymes mediating the programmed cell death machinery of cells. Caspase 3 is an executioner caspase that binds to caspase 8 and caspase 9 to initiate the signals for causing cell death. Caspase 9, on the other hand, is one of the initiator caspase besides caspase 2 and caspase 8 that signals the caspase 3 to execute the apoptosis signals.37

Our results revealed mRNA levels of bax, bcl2, caspase 3 and caspase 9 a nonsignificant differences though there was a trend of cells cultured on dHAM to show lower pro-apoptotic mRNA levels compared to those cultured on TC under normoxia. There was a significant decrease in the mRNA levels of bax, caspase 3 and caspase 9 in cells cultured on dHAM compared to those on TC under hyperoxia. The mRNA levels of bcl2 were similar in cells cultured on TC and dHAM under hyperoxia (Figure 3C). Immunofluorescence staining with anti-BCL2 antibody corroborated our findings by revealing a significantly higher percentage of positivity in cells cultured on dHAM compared to those on TC under normoxia and hyperoxia (Figure 3D, G). There was a significantly higher percentage of BAX positivity in cells cultured on TC compared to dHAM under normoxia and hyperoxia (Figure 3E, H).

Fluorochrome inhibitor of

caspases (FLICA) staining is done to determine the status of active caspase. The FLICA is a cell-permeable and non-cytotoxic reagent containing a caspase inhibitor sequence VAD that is linked to a green fluorescent probe (carboxy fluorescein, FAM). FLICA passes through an intact plasma membrane and covalently binds to the active caspase, thereby detecting only active caspase. Apoptosis is a multistep process and


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hence detection of an active caspase ensures execution of cell death induced by apoptosis.37 FLICA staining revealed a significantly elevated percentage of positivity in cells cultured in TC compared to those cultured on dHAM under normoxia

and

hyperoxia (Figure 3F, I). The results indicate the possibility that cells cultured on dHAM stay protected from hyperoxia induced apoptosis.

Status of EMT in cultured ARPE-19 cells: Studies have shown that amniotic membrane prevents EMT, thus maintaining the epithelial origin of the cells.38 TGF-beta signaling cascades are one of the primary modulators of EMT, which is inhibited by amniotic membrane. Apart from proliferation, EMT of RPE cells is regarded as a hallmark of proliferative vitreo-retinopathy and is regulated by TGF-β/Smad2/3 signaling.38

The filamentous actin (F-actin) form is present abundantly in the cells, primarily involved in cell motility and stability. Myosin binds to the F-actin filaments introducing the contractile movements in the cells. It has been shown that with reduced EMT, there is reduction in levels of F-actin.39 To determine the EMT status, cells were stained with Factin. A distinct staining pattern was observed with F-actin staining of cells cultured on TC and dHAM. Thick actin filaments traversed across the cells when cultured on TC. Moreover there was an increase in the staining intensity and the thickness of actin filaments when the cells were cultured on the TC under hyperoxic conditions. Under normoxia, the cells revealed a more elongated shape and fibroblastic nature when cultured on TC. On the contrary, cells cultured on dHAM showed a more spherical



morphology with the F-actin staining restricted to the cellular periphery (Figure 4A (iii & iv) independent of the culture conditions. However, the F-actin staining was restricted to the periphery when cells were cultured on dHAM under hyperoxic/normoxic conditions (Figure 4A). In addition, the size of the cells cultured on TC under hyperoxia condition were much bigger with much higher stress fibers and a more fibroblast like morphology compared to those cultured on dHAM in similar conditions (Figure 4A (ii-iv)). Quantification of the circularity assay of cells cultured on TC and dHAM revealed an arbitrary unit close to 0.5 and 1 respectively (Supplementary Figure 2C). This suggests that cells cultured on dHAM appeared more circular compared to those grown on TC independent of the culture conditions. Zona occludens 1 (ZO-1) the tight junction protein is present in the cell-cell adhesion junctions. The dissolution of tight junction proteins is one of indicators of EMT, resulting in decreased/diffused ZO-1 expression.40 Immunofluorescence staining with ZO-1, a tight junction protein, was visible in cells grown on TC and dHAM under normoxia (Figure 4B (i-iii)). An intact membrane staining was observed in cells grown on dHAM under hyperoxia compared to cells cultured on TC under hyperoxia (Figure 4B (ii & iv); Supplementary Figure 2B). Connexin-43 is a gap junction alpha 1protein, primarily functioning in cell-cell communication. Its levels are reduced during induction of EMT.41 Furthermore, CONNEXIN-43 (CXN-43) immunofluorescence staining revealed staining of cells cultured on TC and dHAM under normoxia without any notable difference in the staining intensity (Figure 4C (i-iii)). The staining intensity of CXN-43 was weak in cells cultured on TC compared to those on dHAM under hyperoxia (Figure 4C (ii-iv)). Epithelial cadherin (E-CADHERIN) is a calcium dependent cell adhesion molecule that forms adherens junctions for binding the


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cells. VIMENTIN is an intermediate filament protein expressed in mesenchymal cells and helps maintain cellular integrity and resistance to stress. Loss of E-CADHERIN and gain of VIMENTIN in a cell are indicators of ongoing EMT.42 Western blot results show a decrease in the E-CADHERIN levels in cells cultured on TC compared to cells cultured on dHAM under hyperoxia. There was no difference in the E-CADHERIN levels in cells cultured on TC and dHAM under normoxia. Contrarily, western blot for VIMENTIN show a strong expression in cells cultured on TC compared to those on dHAM under hyperoxia. The levels of VIMENTIN were similar in cells cultured on TC and dHAM under normoxia (Figure 4D (i-ii)). The results revealed lower EMT transition in cells cultured on dHAM compared to cells cultured on TC under hyperoxia.

Status of VEGF expression levels in cultured ARPE-19 cells: Vascular endothelial growth factor-A (VEGF A) is signal protein primarily playing a role in the formation and maintenance of blood vessels. Secreted VEGF executes its vascular functions by binding to VEGF R2 receptor.6 Secretion of VEGF is a major determinant of RPE functionality.7 It has been shown that expression of VEGF is enhanced on culturing cells on amniotic membrane in comparison to those grown on tissue culture dishes.11, 43 Hence, to assess the functional aspect of the RPE cells, we estimated the VEGF levels, gene expression of vegf A, and its receptors vegf R1 and vegf R2. Under normoxia, cells cultured on dHAM showed significantly higher mRNA levels of vegf A compared to those cultured on TC. Significantly higher expression of vegf A mRNA level was observed in cells cultured on dHAM compared to TC under hyperoxia (Figure 5A). There was no significant difference in the mRNA levels of vegf



R1 in cells cultured on TC and dHAM under normoxia and hyperoxia. Cells cultured on TC showed significantly lower mRNA levels of vegf R2 compared to those on dHAM under normoxia and hyperoxia (Figure 5A). VEGF A staining was higher in cells cultured on dHAM in comparison to those cultured on TC under normoxia (Figure 5B, C), though not significant. On the contrary VEGF A staining was significantly decreased in cells cultured on TC compared to those on dHAM under hyperoxia (Figure 5B, C). The ELISA result for secreted VEGF estimation showed a significantly higher level in cells cultured on dHAM compared to those cultured on TC under normoxia. Moreover, the levels of secreted VEGF in cells grown on TC compared to those cultured on dHAM under hyperoxia also showed a significant difference. Cells cultured on TC under hyperoxia had higher secreted VEGF levels compared to those grown on dHAM under hyperoxia (Figure 5D).

Phagocytosis levels in cultured ARPE-19 cells: Phagocytosis is a vital property of RPE and any alterations in its capacity results in pathological manifestations. Coated vesicles (CV) are the intracellular organelles responsible for the phagocytosis in RPE cells.44 Hyperoxia also affects the phagocytic property of RPE cells.45 Functional assay for phagocytosis was performed using FITC labelled latex beads. Since each of the human CV is of approximately 50nm, intracellular clustering of CVs as well as a higher number phagocytosed beads in each of the CV can results in the appearance of a green blob in the phagocytosed cell. On the other hand when the CVs are not clustered or the number of beads phagocytosed by them is less, they provide a intracellular scattered pattern.44 The phagocytosis


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opsonized bead assay was significantly higher on dHAM compared to TC under normoxia (Figure 6A (i & iii)). The phagocytosis bead assay showed a significantly lower number of opsonized latex beads in cells cultured on TC compared to dHAM under hyperoxia (Figure 6A (ii & iv)). Overall, based on the green fluorescent tag, phagocytosis was enhanced in cells cultured on dHAM compared to those cultured on TC (Figure 6A (i-iv)). The percentage of cells with opsonized beads in cells cultured on dHAM was significantly higher when compared to TC under hyperoxia (Figure 6B), thereby denoting lower phagocytosis levels in cells cultured on TC.

Discussion HAM remains the oldest natural scaffold (1910) that is still widely used in tissue engineering and regenerative medicine.46 Translational aspect of amniotic membrane has been extensively used in the branch of ophthalmology compared to other branches of medicine. Unlike other scaffolds such as blood vessel, diaphragm, esophagus and intestine, apart from cell adhesion properties amniotic membrane have antiinflammatory, anti-microbial, low immunogenicity aspects making them suitable for transplantation studies.47 Studies showed dHAM and poly(lactic-co-glycolic acid) (PLGA) scaffolds possessing better mechanical properties compared to hybrid hydrogels. Though, Young’s modulus and stiffness of PLGA was greater than dHAM, dHAM had a better modulus of toughness compared to PLGA.

Whereas, Young’s

modulus, stiffness and modulus of toughness was higher in both PLGA and dHAM compared to hybrid gels. The tensile strength was lower in hybrid hydrogels compared to that of PLGA and dHAM, which were comparable.48 One of the most significant



advantage of natural scaffolds is their extra-cellular matrix-like composition that promotes tissue repair and regeneration.49 Additionally, the amniotic membrane has a drug reservoir capacity that can be exploited in a cell therapy module for combinatorial treatment of cells along with the drugs of interest.50

Unlike most epithelial cells, RPE cells have diverse roles from a barrier function to maintaining normal vision and ocular vasculature with its secretome.7 With increasing age, the RPE gets more vulnerable to oxidative stress. To overcome this, there have been attempts to explore the possibility of cell therapy using exogenously differentiated RPE cells. The HAM is a suitable scaffold to carry RPE cells to the transplant site.43, 51 As a natural biomaterial, the use of HAM was first reported in 2005.52 Decellularized HAM has been used for surface reconstruction based on its capacity to transport ocular limbal epithelial cells.20 It has been shown that dHAM is a natural reservoir of many growth factors like Epidermal growth factor, Keratinocyte growth factor, Hepatocyte growth factors, basic Fibroblast growth factor as well as with anti-inflammatory and antiangiogenic factors.53 Studies have proven that HAM (excluding the native epithelial cells) expresses mRNAs for a number of growth factors as well as growth factor proteins enabling epithelialization post transplantation. Biological properties such as anti-inflammatory, anti-microbial, anti-fibrosis, anti-scarring and its mechanical strength make HAM an efficient scaffold in clinical ophthalmology.54 Simultaneously, HAM exerts its antioxidant and free radical scavenging activity to remove ROS from its environment.28, 55 Akrami et al. demonstrated higher expression of RPE genes, RPE65, Cellular retinaldehyde binding protein, VEGF, and Cluster of differentiation factor 68 on


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dHAM cultured RPE cells wherein the membrane restricts the dedifferentiation of cultured RPE cells.11 Amniotic membrane cultured RPE cells show increase in transepithelial resistance as well as secreted protein production, namely VEGF, thrombospodin-1, BESTROPHIN and Pigment epithelium derived factor, compared to TCs.43 Though dHAM is capable of supporting and restoring RPE functions, there is a lack of studies investigating its role as a carrier for RPE cells in a disease state. Hence, we have attempted to investigate the potential of dHAM in restoring the physiological properties of RPE in oxidative stress conditions.

ARPE-19 cells were cultured on TC or on dHAM in hyperoxic conditions for 5 days to mimic a short mild oxidative stress.45 We used 40% hyperoxia oxygen concentration and 20% normoxia oxygen concentration for RPE cells in in vitro conditions. RPE in vivo is exposed to high oxygen flux hence the experiments were conducted at 40% oxygen concentration for hyperoxia.56-57 Moreover, it is known that pathological conditions in AMD can be mimicked in vitro by culturing the cells in a mild oxidative stress for five days. We also investigated the role of Polycaprolactone (PCL) of 900nm fiber diameter scaffold in modulating the hyperoxia effects on ARPE-19 cells. The results revealed that culturing the cells on PCL nanofiber under hyperoxia conditions revealed ROS levels similar to the cells cultured on TC under hyperoxia conditions. In an attempt to depict the cell morphology, ZO-1 immunostaining was performed in cells cultured on PCL nanofiber and TC under hyperoxia conditions. The results revealed loss of gap junction protein (ZO-1) in the cells cultured on TC or PCL nanofiber when cultured under hyperoxia.(Supplementary Figure 4; Intact cell membrane is shown by yellow colored



arrows and destabilized membrane is shown by white colored arrows for ZO-1 staining; Scale bar=5m)

Cultured ARPE-19 cells showed a clustered growth pattern when grown on dHAM in hyperoxic conditions, while those on TC under hyperoxia had a more elongated morphology (Figure 1). The clustered growth pattern observed on dHAM could be due to the uneven surface of dHAM, creating an island of conductive milieu for cell growth. The close proximity of cells may be more conducive to a healthy cellular homeostasis.58

DCFH-DA staining for ROS revealed small sharp green puncta as well as diffused staining. There were more puncta staining observed in cells cultured on dHAM compared to TC under hyperoxia conditions. Moreover, we have shown that concurrent to the increased oxidized material there is no increase in the apoptotic levels in cells cultured on dHAM under hyperoxia compared to those cultured on TC. Hence, it is likely these oxidized material in the cells cultured on dHAM are undergoing degradation and further studies are needed to explore the mechanism by which this occurs.29 Diffused staining pattern represents ROS staining of the cells. In our studies we observe that cells cultured on TC show higher intensity of diffused staining compared to those cultured on dHAM in hyperoxic conditions. These findings suggest that the vesicles aid in eliminating the oxidized material, thereby protecting the cells from the adverse effects of oxidative stress, though further studies are needed to corroborate the same. In our study we determined the induction of ROS in cells cultured on TC and dHAM in normoxic/hyperoxic conditions. Similarly, our experiments also confirm the free radical


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scavenging property of dHAM. This was revealed by a lower ROS production depicted by DCFH-DA immunofluorescence intensity in cells cultured on dHAM and incubated in a hyperoxic environment compared to those cultured on TC (Figure 1). The ROS level is directly dependent on the cell density. The ROS levels are low in confluent cultures compared to sub-confluent cultures.59 Though cell density affects ROS, our results of ROS are comparable as the seeding cell density in our cultures were similar. There was slight decrease in the cell density in the cells cultured on TC under hyperoxia conditions on day 5. Higher percentage of apoptosis would be the most probable cause in lowering the cell density in cells cultured on TC under hyperoxia conditions (Supplementary Figure 2A).

Cells cultured in normoxic conditions on TC showed a significantly higher expression of cell proliferation markers than those cultured on dHAM. However, there was not much difference in cells cultured on TC or dHAM in a hyperoxic environment (Figure 2). Proliferation is reduced in ARPE-19 cells cultured on dHAM in normoxic conditions,4 which is important, as RPE cell proliferation is touted to be one of the major causes for proliferative vitreoretinopathy and AMD.60 It has been shown that the anti-proliferative ability of dHAM is regulated by heavy chain hylauronan/ pentraxin 3 present in the amniotic membrane.38 FIBRONECTIN, a 11.48KD protein, is a major component of dHAM has cell adhesion domain enabling cells to adhere and then promote epithelial phenotype rather than mesenchymal migratory behavior. Cell adhesion domain of FIBRONECTIN is found to be containing 108 residues of aminoacids without any cysteines and isoleucine at amino and methionine at the carboxyl terminals.61 It has



been shown that while the levels of FIBRONECTIN do not get affected in HAM and dHAM, COLLAGEN I is absent in dHAM.62 Integrin α5 has a specific affinity for FIBRONECTIN and it has been shown that RPE cells have a highest adhesive nature when cultured on FIBRONECTIN compared to COLLAGEN 1 (though not significant) or COLLAGEN 4.63 It has been documented that endothelial cells adhesion on substrate bound FGF-2 is primarily using αvβ3 integrins.64 McAvoy showed using lens epithelial cells that in the presence of FGF2, cells maintained the cell basic properties of proliferation, migration and differentiation.65 The direct role of substrate bound FGF2 on cultured RPE cell is unknown. It can very well be envisaged that FGF2 might be enabling a better retinal pigment epithelia cell phenotype in culture. We have previously shown that Notch signaling, which is a known regulator of RPE proliferative potential, is downregulated when cells are cultured on dHAM compared to TC.66 Gene expression of Cyclins-CDKs revealed that expression of all the Cyclins and CDKs were significantly lower in all the cultures compared to those grown in TC under normoxia conditions. Cyclins undergo phosphorylation and get activated on binding with CDKs. Furthermore, since Ki67 protein is present and regulates all the stages of cell cycle we investigated the percentage of Ki67 positivity in the cells cultured on TC as well as dHAM under normoxia and hyperoxia conditions. The results revealed that there was a significant decrease in the percentage of Ki67 positivity in cell grown on dHAM compared to those cultured on TC under normoxia environment. However, no difference in the percentage of Ki67 positive cells was obtained in cultures grown under hyperoxia conditions. Hence, it can be assumed that the inhibition of pro-proliferative capacity prevents


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accumulation of defective RPE cells in an oxidative stress milieu, thereby preventing disease progression (Figure 2).

Cell counting using vital stain revealed a significant increase in the percentage of live cells in cultures grown on dHAM compared to those on TC, under hyperoxia conditions. MTT assay revealed higher percentage of metabolically active cell viability in cells cultured on dHAM compared those on TC both in normoxia and hyperoxia. mRNA expression levels of pro-apoptotic markers such as bax, caspase 3 and caspase 9 were significantly reduced in cells cultured on dHAM compared to TC under hyperoxia conditions. Additionally, cells cultured on dHAM in hyperoxia conditions showed significantly higher percentage of anti-apoptotic protein BCL2 positivity but lower BAX as well as FLICA (active caspase) positivity compared to those grown on TC in hyperoxia conditions. Our results show that even in normoxic conditions, cells grown on TC showed significantly higher levels of apoptosis compared to those cultured on dHAM, which was confirmed by active caspase staining (Figure 3). Lower level of apoptosis in cells cultured on dHAM in a hyperoxic environment is a strong indicator of the protective mechanism of dHAM on ARPE-19 cells under oxidative stress. This suggests that cells transplanted with dHAM as a carrier in an oxidative stress milieu can survive longer and increase the chances for cell integration. Cells cultured on dHAM in hyperoxic conditions showed lower apoptotic levels compared to those cultured on TC and comparable in both under normoxia (Figure 3).



In an attempt to determine the RPE physiological properties, we ascertained the EMT, VEGF levels and phagocytosis activity. We observed there was loss of ZO-1 from gap junctions along with gain in levels of VIMENTIN in cells cultured on TC compared to those on dHAM under hyperoxia conditions. E-CADHERIN levels were increased in cultures grown on dHAM compared to those on TC under hyperoxia conditions. Likewise, F-actin staining was restricted to the membrane in cells grown on dHAM (under both normoxia and hyperoxia), in comparison to those on TC suggesting optimal biophysical microenvironment provided by the dHAM in contrast to the extremely rigid TC surface and thus aberrant mechanotransduction. The immunofluorescence staining of CXN-43 is significantly elevated in cells grown on dHAM under hyperoxia compared to those on TC. Apart from its role in EMT, CXN-43 plays a definitive role in the differentiation of RPE (Figure 4).67 It has also been shown that CXN-43 is vital for playing a protective role for cells under oxidative stress.68 There is also a significant downregulation of E-CADHERIN and concurrent increase of VIMENTIN was observed by western blot in cells cultured on TC compared to those on dHAM in hyperoxic conditions (Figure 4).

At gene expression levels, it can be seen that there is a significant increase in the mRNA levels in cells cultured on dHAM compared to those on TC under both normoxia and hyperoxia. While the VEGFA positivity in cells cultured on dHAM and TC under normoxia was comparable, a significantly lower percentage of VEGFA positivity was noted on cells cultured on TC in comparison to those on dHAM under hyperoxia (Figure 5). Similarly, there might be an increase in cap-dependent translation in ARPE-19 cells


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cultured on TC in normoxic conditions resulting in increased VEGFA expression levels with lower levels of vegf A mRNA. There are several reports suggesting that there may not be any concurrence between mRNA levels and protein levels.69 Expression of vegf A was significantly low in cells grown on TC compared to cells grown on dHAM in hyperoxic conditions. The concentration of the secreted VEGF was significantly lower in cells cultured on dHAM compared to those on TC under hyperoxia, but significantly higher in dHAM under normoxia (Figure 5). It has been shown that VEGF can induce ROS in cells and it is speculated that this increased ROS results in more severe damage to tissues. Hence, VEGF and ROS have a two-way cross talk with each other, and it has been shown that ROS induces VEGF secretion in ARPE-19 cells.70 We have seen that ROS levels in cells cultured on TC and dHAM under normoxia are similar. Hence, it can be assumed that under normoxia (low ROS levels), secreted VEGF levels are higher in cells grown on dHAM compared to those on TC. This could be attributed to a higher metabolic activity of the cells grown on dHAM. This is one of the major limitations in cell therapy based treatment in AMD patients, where the local ocular environment is under oxidative stress. In similar lines, our results also show that secreted VEGF levels are higher in cells cultured on TC compared to those cultured on dHAM under hyperoxia. This difference could be attributed to the higher levels of ROS in cells grown on TC compared to those on dHAM . It is therefore possible that higher ROS enhances the secretion of VEGF in cells cultured on TC, and contrarily lower ROS regulates the secreted VEGF levels in cell grown on dHAM. Furthermore, we show that though vegf A mRNA could be detected using RT-qPCR, the secreted VEGFA levels remained undetected from dHAM (Supplementary Figure 3A, B).



Phagocytosis was significantly reduced in cells cultured on TC in hyperoxic conditions compared to cells cultured on dHAM in similar conditions (Figure 6). Phagocytosis is a vital property of RPE and any alterations in its capacity results in pathological manifestations.71 Hyperoxia also affects the phagocytic property of RPE cells.45 Our findings show that cells cultured on TC in hyperoxic conditions show reduced phagocytic activity compared to cells cultured on TC in normoxic conditions. It has also been shown that RPE cells with higher F-actin stress fibers and low lateral circumferential staining show lower phagocytic property compared to those with high circumferential F-actin staining.72 Cells cultured on dHAM in normoxia as well as hyperoxia conditions showed circumferential F-actin staining whereas those cultured on TC showed abnormal F-actin staining pattern with higher stress fibers. Whereas, cells grown on dHAM in hyperoxic conditions showed significantly higher number of opsonized latex beads compared to those cultured on TC under hyperoxia (Figure 6).

One of the critical aspects of a successful cell therapy is that the transplanted cells need to survive in the diseased milieu while still maintaining their physiological properties. Hence, in an attempt to mimic the diseased milieu of mild hyperoxic stress, RPE cells were cultured in hyperoxic conditions in the presence/absence of dHAM. Our results suggest that dHAM has the potential to scavenge the ROS generated in a hyperoxic environment and thereby help cells to retain their critical properties. The findings of this work have potentially significant impact for cell therapy transplantation studies in a stressed/ diseased milieu. In an attempt to widen the proposed outcome, the amniotic


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membrane could be loaded with liposomes, nanoparticles, microspheres or capsules carrying the desired growth factors or signaling inducer molecules. This capacity of dHAM has been exploited in loading drugs such as 5-fluorouracil PG J2

74,

green silver nanoparticles

75,

levofloxacin

76.

73,

15-deoxy-Δ12,14-

Drug release as well as a barrier

function in drug penetration, facilitates the usage of dHAM in a planned clinical management of disease.77 The usage of amniotic membrane for personalized medicine could be enhanced by loading them with signaling molecules in the form of nanoparticles. It is well established that the signaling crosstalk in health and disease doesn’t follow an “on-off” pattern, but rather in a gradient. In-corporation of a temporally regulated gradient would make amniotic membrane the most attractive scaffold for cell therapy.

Hence, dHAM has the potential to be a carrier to transplant human embryonic stem cells or induced pluripotent stem cells derived RPE cells for cell therapy in conditions like AMD. Mechanotransduction pathway studies would provide better insight into the integration of the transplanted cells with a temporal outcome. Additionally, these findings pave way for expanding the application of dHAM as a carrier for cell therapy in a broader clinical application.

Conclusion Oxidative stress is a major cause of various retinal degenerative diseases. Transplantation of healthy RPE is a promising treatment option. It is imperative to understand the underlying mechanisms that define the success of dHAM in the



transplantation of RPE cells as it provides a suitable carrier. Our results showed that RPE cells grown under oxidative stress condition on dHAM not only survive better but also maintain their physiological properties such as VEGF secretion, EMT, and phagocytosis. Based on the findings of our study the potential of dHAM as a carrier can be expanded for other pathologic conditions driven by oxidative stress.

Supporting Information Available The following file is available free of charge: Supplementary: Figure S1: Preparation, decellularization and characterization of HAM (Figure S1). Figure S2: Quantification of cell density and morphology (Figure S2). Figure S3: Levels of vegf A mRNA and secretion from dHAM (Figure S3). Figure S4: Immunofluorescence staining of ROS and ZO-1 (Figure S4) Supplementary Table 1. Primers used in RT-qPCR (Table S1). Supplementary Table 2: List of primary and secondary antibodies with catalogue details (Table S2). Supplementary Table 3: Mean value± SD, n and p value of the assays (Table S3) Acknowledgements The authors would like to thank Narayana Nethralaya Foundation and Department of Science and Technology, Govt. of India [SR/SO/HS-228/2012] for providing financial support to conduct this work. The authors would also like to thank Dr. P. Narendra and Dr. Arkasubhra Ghosh for their administrative support. The authors express their gratitude to Dr. K Bhujang Shetty for providing all the logistics needed for this work.

References:


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Figure Legends: Figure 1. Morphological characterization and reactive oxygen species (ROS) in cultured ARPE-19 cells. (A) Representative phase contrast microscopic images of ARPE-19 cells cultured on TC and dHAM under normoxic (i & ii) and hyperoxic conditions (iii & iv), Scale bar= 10m. SEM images of cultured TC and dHAM in normoxic (v & vi) and hyperoxic conditions (vii & viii), Scale bar= 50m . (B) Representative ROS immunofluorescence staining using DCFH-DA on ARPE-19 cells cultured on TC under normoxia (i) and hyperoxia conditions (ii). DCFH-DA staining of cells cultured on dHAM in normoxic (iii) and hyperoxic conditions (iv), Scale bar= 5m. (C) Graphical representation of the quantified mean fluorescent intensity of the ROS images using Image J software. For immunofluorescence quantification, a total of 300 cells were counted from 17 images. Statistical calculation was performed using Mann-Whitney U test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005).

Figure 2. Proliferative status of ARPE-19 cells cultured on TC and dHAM in hyperoxic and normoxic conditions. (A) Graphical representation of quantitative real-time PCR gene expression results of proliferation markers (cyclins A, B, D1, E) in ARPE-19 cells cultured on TC and dHAM under normoxic and hyperoxic conditions. (B) mRNA expression levels of proliferation markers (cdks1,2,4,6) in ARPE-19 cells cultured on TC and dHAM in normoxic and hyperoxic conditions. mRNA expression level of gapdh gene was the internal control for the experiments. (C) Representative microscopic images of immunofluorescence



staining of Ki67 protein in cells cultured on TC and dHAM under normoxic (i & iii) and hyperoxic (ii & iv) conditions, Scale bar= 5m. (D) Graphical representation of the percentage of Ki67 positivity in ARPE-19 cells cultured on TC and dHAM in normoxic and hyperoxic environment. For immunofluorescence quantification, a total of 300 cells were counted from 15 images. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005).

Figure 3. Apoptosis status of ARPE-19 cultured on TC and dHAM in both normoxia and hyperoxia environment. (A) Bar graph representing the percentage of viability using Trypan Blue staining in ARPE-19 cells cultured on TC and dHAM under normoxic and hyperoxic conditions. (B) Graphical representation of the relative % of viability of cultured ARPE-19 cells on TC and dHAM under normoxic and hyperoxic conditions using MTT Assay. (C) Graphical representation of the mRNA expression levels estimated by real-time quantitative PCR of apoptotic markers (bax, bcl2, caspase 3 and caspase 9) in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia. Expression levels of gapdh gene were the internal control for the experiments. Representative immunofluorescence stained images of BCl2 (D), BAX (E) proteins and FLICA caspase activity (F) in ARPE-19 cells cultured on TC and dHAM under normoxic (i & ii) and hyperoxic (iii & iv) conditions, Scale bar= 5m. Graphical representation of the percentage of positivity of BCl2 (G), BAX (H) and FLICA (I) has been depicted. For immunofluorescence quantification, a total of 300 cells were counted from 15 images. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005).


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Figure 4. Status of EMT in ARPE-19 cells cultured on TC and dHAM under oxidative stress conditions. Representative immunofluorescence staining F-Actin (A), ZO-1 (B) and CXN-43 (C) protein in cells cultured on TC and dHAM under normoxia (i & iii) and hyperoxic (ii & iv) conditions. Yellow colored arrows show the F-actin staining pattern (A). Intact cell membrane is shown by yellow colored arrows and destabilized membrane is shown by white colored arrows for ZO-1 staining (B). Yellow colored arrows show CXN-43 staining status (C). Scale bar= 5m. (D) Western blot results showing the expression of E-CADHERIN, VIMENTIN and GAPDH in ARPE-19 cells cultured on TC and dHAM in normoxic and hyperoxic conditions (i). Graphical representation of the quantified Western blot images (ii).

Figure 5. Expression of VEGF in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia. (A) Quantitative real-time PCR results of expression of vegf A and its receptors (vegf R1, and vegf R2) in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia depicted as mRNA expression levels. Expression level of gapdh gene was the internal control for the experiments. (B) Representative immunofluorescence stained VEGF A protein in cells cultured on TC and dHAM under normoxia (i & iii) and hyperoxia (ii & iv), Scale bar= 5m. (C) Graphical representation of the percentage of positivity of VEGF A in cells. For immunofluorescence quantification, a total of 200 cells were counted from 20 images. (D) Concentration of secreted VEGF A as estimated by



sandwich ELISA has been depicted graphically for cultured ARPE-19 cells. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005).

Figure 6. Phagocytosis in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia. (A) Representative immunofluorescence staining of latex beads opsonized by cells cultured on TC and dHAM under normoxic (i & iii) and hyperoxic (ii & iv) conditions, Scale bar= 5m. (B) Graphical representation of the percentage of cells with opsonised beads in cultured ARPE-19 cells. For immunofluorescence quantification of the engulfed beads, a total of 100 cells were counted from 15 images. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005).


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Figure 1. Morphological characterization and reactive oxygen species (ROS) in cultured ARPE-19 cells. (A) Representative phase contrast microscopic images of ARPE-19 cells cultured on TC and dHAM under normoxic (i & ii) and hyperoxic conditions (iii & iv), Scale bar= 10μm. SEM images of cultured TC and dHAM in normoxic (v & vi) and hyperoxic conditions (vii & viii), Scale bar= 50μm . (B) Representative ROS immunofluorescence staining using DCFH-DA on ARPE-19 cells cultured on TC under normoxia (i) and hyperoxia conditions (ii). DCFH-DA staining of cells cultured on dHAM in normoxic (iii) and hyperoxic conditions (iv), Scale bar= 5μm. (C) Graphical representation of the quantified mean fluorescent intensity of the ROS images using Image J software. For immunofluorescence quantification, a total of 300 cells were counted from 17 images. Statistical calculation was performed using Mann-Whitney U test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005). 260x331mm (300 x 300 DPI)

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Figure 2. Proliferative status of ARPE-19 cells cultured on TC and dHAM in hyperoxic and normoxic conditions. (A) Graphical representation of quantitative real-time PCR gene expression results of proliferation markers (cyclins A, B, D1, E) in ARPE-19 cells cultured on TC and dHAM under normoxic and hyperoxic conditions. (B) mRNA expression levels of proliferation markers (cdks1,2,4,6) in ARPE-19 cells cultured on TC and dHAM in normoxic and hyperoxic conditions. mRNA expression level of gapdh gene was the internal control for the experiments. (C) Representative microscopic images of immunofluorescence staining of Ki67 protein in cells cultured on TC and dHAM under normoxic (i & iii) and hyperoxic (ii & iv) conditions, Scale bar= 5μm. (D) Graphical representation of the percentage of Ki67 positivity in ARPE-19 cells cultured on TC and dHAM in normoxic and hyperoxic environment. For immunofluorescence quantification, a total of 300 cells were counted from 15 images. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005).



245x300mm (300 x 300 DPI)


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Figure 3. Apoptosis status of ARPE-19 cultured on TC and dHAM in both normoxia and hyperoxia environment. (A) Bar graph representing the percentage of viability using Trypan Blue staining in ARPE-19 cells cultured on TC and dHAM under normoxic and hyperoxic conditions. (B) Graphical representation of the relative % of viability of cultured ARPE-19 cells on TC and dHAM under normoxic and hyperoxic conditions using MTT Assay. (C) Graphical representation of the mRNA expression levels estimated by real-time quantitative PCR of apoptotic markers (bax, bcl2, caspase 3 and caspase 9) in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia. Expression levels of gapdh gene were the internal control for the experiments. Representative immunofluorescence stained images of BCl2 (D), BAX (E) proteins and FLICA caspase activity (F) in ARPE-19 cells cultured on TC and dHAM under normoxic (i & ii) and hyperoxic (iii & iv) conditions, Scale bar= 5μm. Graphical representation of the percentage of positivity of BCl2 (G), BAX (H) and FLICA (I) has been depicted. For immunofluorescence quantification, a total of 300 cells were counted from 15 images. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005). 237x274mm (300 x 300 DPI)




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Figure 4. Status of EMT in ARPE-19 cells cultured on TC and dHAM under oxidative stress conditions. Representative immunofluorescence staining F-Actin (A), ZO-1 (B) and CXN-43 (C) protein in cells cultured on TC and dHAM under normoxia (i & iii) and hyperoxic (ii & iv) conditions. Yellow colored arrows show the F-actin staining pattern (A). Intact cell membrane is shown by yellow colored arrows and destabilized membrane is shown by white colored arrows for ZO-1 staining (B). Yellow colored arrows show CXN-43 staining status (C). Scale bar= 5μm. (D) Western blot results showing the expression of E-CADHERIN, VIMENTIN and GAPDH in ARPE-19 cells cultured on TC and dHAM in normoxic and hyperoxic conditions (i). Graphical representation of the quantified Western blot images (ii). 280x430mm (300 x 300 DPI)



Figure 5. Expression of VEGF in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia. (A) Quantitative real-time PCR results of expression of vegf A and its receptors (vegf R1, and vegf R2) in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia depicted as mRNA expression levels. Expression level of gapdh gene was the internal control for the experiments. (B) Representative immunofluorescence stained VEGF A protein in cells cultured on TC and dHAM under normoxia (i & iii) and hyperoxia (ii & iv), Scale bar= 5μm. (C) Graphical representation of the percentage of positivity of VEGF A in cells. For immunofluorescence quantification, a total of 200 cells were counted from 20 images. (D) Concentration of secreted VEGF A as estimated by sandwich ELISA has been depicted graphically for cultured ARPE-19 cells. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005). 255x325mm (300 x 300 DPI)


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Figure 6. Phagocytosis in ARPE-19 cells cultured on TC and dHAM under normoxia and hyperoxia. (A) Representative immunofluorescence staining of latex beads opsonized by cells cultured on TC and dHAM under normoxic (i & iii) and hyperoxic (ii & iv) conditions, Scale bar= 5μm. (B) Graphical representation of the percentage of cells with opsonised beads in cultured ARPE-19 cells. For immunofluorescence quantification of the engulfed beads, a total of 100 cells were counted from 15 images. Statistical calculation was performed using Mann Whitney U-test (*≤ 0.05, ** ≤ 0.01, ***≤ 0.005). 199x193mm (300 x 300 DPI)


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Protective role of decellularized human amniotic membrane from oxidative stress induced damage on retinal pigment epithelial cells. Lekshmi Krishna, Kamesh Dhamodaran, Murali Subramani, Murugeswari Ponnulagu, Nallathambi Jeyabalan, Sai Rama Krishna Meka, Chaitra Jayadev, Rohit Shetty, Kaushik Chatterjee, Samanta Sekhar Khora, Debashish Das.


Protective role of decellularized human amniotic membrane from

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