{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,21]],"date-time":"2026-03-21T21:43:58Z","timestamp":1774129438044,"version":"3.50.1"},"reference-count":71,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2021,7,30]],"date-time":"2021-07-30T00:00:00Z","timestamp":1627603200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100006325","name":"ARVO Foundation for Eye Research","doi-asserted-by":"publisher","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}],"id":[{"id":"10.13039\/100006325","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The Bruch\u2019s membrane (BrM) is a five-layered extracellular matrix (ECM) that supports the retinal pigment epithelium (RPE). Normal age-related changes in the BrM may lead to RPE cell damage and ultimately to the onset and progression of age-related macular degeneration (AMD), which is the most common cause of visual loss among the elderly. A role for the complement system in AMD pathology has been established, but the disease mechanisms are poorly understood, which hampers the design of efficient therapies to treat millions of patients. In an effort to identify the mechanisms that lead from normal aging to pathology, we have developed a cell-based model using complement deficient human induced pluripotent stem cell (iPSC)-derived RPE cells cultured on an AMD-like ECM that mimics BrM. The data present evidence that changes in the ECM result in loss of differentiation and promote epithelial mesenchymal transition (EMT) of healthy RPE cells. This pathological process is mediated by complement activation and involves the formation of a randomly oriented collagen meshwork that drives the dedifferentiation of the RPE monolayer. Genetic ablation of complement component 3 has a protective effect against EMT but does not prevent the abnormal deposition of collagens. These findings offer new insights into the sequence of events that initiate AMD and may guide the design of efficient therapies to treat this disease with unmet medical needs.<\/jats:p>","DOI":"10.3390\/ijms22158183","type":"journal-article","created":{"date-parts":[[2021,7,30]],"date-time":"2021-07-30T12:59:24Z","timestamp":1627649964000},"page":"8183","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["AMD-Like Substrate Causes Epithelial Mesenchymal Transition in iPSC-Derived Retinal Pigment Epithelial Cells Wild Type but Not C3-Knockout"],"prefix":"10.3390","volume":"22","author":[{"given":"Blanca","family":"Chinchilla","sequence":"first","affiliation":[{"name":"The Ocular Genomics Institute at Mass Eye and Ear, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rosario","family":"Fernandez-Godino","sequence":"additional","affiliation":[{"name":"The Ocular Genomics Institute at Mass Eye and Ear, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,30]]},"reference":[{"key":"ref_1","first-page":"1615","article-title":"The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro","volume":"27","author":"Campochiaro","year":"1986","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_2","first-page":"465","article-title":"Structure, function, and pathology of bruch\u2019s membrane","volume":"Volume 1","author":"Curcio","year":"2012","journal-title":"Retina"},{"key":"ref_3","first-page":"499","article-title":"Analysis of retinal pigment epithelium integrin expression and adhesion to aged submacular human Bruch\u2019s membrane","volume":"101","author":"Zarbin","year":"2003","journal-title":"Trans. Am. Ophthalmol. Soc."},{"key":"ref_4","first-page":"1110","article-title":"Reattachment of cultured human retinal pigment epithelium to extracellular matrix and human Bruch\u2019s membrane","volume":"38","author":"Ho","year":"1997","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_5","first-page":"775","article-title":"Increase in the advanced glycation end product pentosidine in Bruch\u2019s membrane with age","volume":"40","author":"Handa","year":"1999","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1016\/j.jbiomech.2005.02.013","article-title":"Glycation increases human annulus fibrosus stiffness in both experimental measurements and theoretical predictions","volume":"39","author":"Wagnera","year":"2006","journal-title":"J. Biomech."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5370","DOI":"10.1167\/iovs.14-14126","article-title":"Age-dependent changes in heparan sulfate in human Bruch\u2019s membrane: Implications for age-related macular degeneration","volume":"55","author":"Keenan","year":"2014","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1146\/annurev-genom-090413-025610","article-title":"Age-related macular degeneration: Genetics and biology coming together","volume":"15","author":"Fritsche","year":"2014","journal-title":"Annu. Rev. Genom. Hum. Genet."},{"key":"ref_9","first-page":"2970","article-title":"The effect of age on the macromolecular permeability of human Bruch\u2019s membrane","volume":"42","author":"Moore","year":"2001","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1055\/s-0037-1598183","article-title":"Do advanced glycation end products and its receptor play a role in pathophysiology of hypertension?","volume":"26","author":"Mishra","year":"2017","journal-title":"Int. J. Angiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.devcel.2011.06.026","article-title":"Shaping cells and organs in drosophila by opposing roles of fat body-secreted collagen IV and perlecan","volume":"21","author":"Xu","year":"2011","journal-title":"Dev. Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/S0002-9440(10)62248-1","article-title":"Decreased thickness and integrity of the macular elastic layer of Bruch\u2019s membrane correspond to the distribution of lesions associated with age-related macular degeneration","volume":"166","author":"Chong","year":"2005","journal-title":"Am. J. Pathol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.exer.2004.09.006","article-title":"Impaired RPE survival on aged submacular human Bruch\u2019s membrane","volume":"80","author":"Gullapalli","year":"2005","journal-title":"Exp. Eye Res."},{"key":"ref_14","first-page":"123","article-title":"Retinal pigment epithelium resurfacing of aged submacular human Bruch\u2019s membrane","volume":"102","author":"Gullapalli","year":"2004","journal-title":"Trans. Am. Ophthalmol. Soc."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5555","DOI":"10.1093\/hmg\/ddv287","article-title":"A local complement response by RPE causes early-stage macular degeneration","volume":"24","author":"Garland","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1093\/hmg\/ddx392","article-title":"Changes in extracellular matrix cause RPE cells to make basal deposits and activate the alternative complement pathway","volume":"27","author":"Godino","year":"2017","journal-title":"Hum. Mol. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"9679","DOI":"10.1038\/s41598-018-28143-0","article-title":"C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway","volume":"8","author":"Pierce","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"18277","DOI":"10.1073\/pnas.1109703108","article-title":"Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration","volume":"108","author":"Johnson","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1167\/iovs.16-21060","article-title":"Subretinal pigment epithelial deposition of drusen components including hydroxyapatite in a primary cell culture model","volume":"58","author":"Pilgrim","year":"2017","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.preteyeres.2009.11.003","article-title":"The pivotal role of the complement system in aging and age-related macular degeneration: Hypothesis re-visited","volume":"29","author":"Anderson","year":"2010","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1016\/S1350-9462(01)00010-6","article-title":"An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch\u2019s membrane interface in aging and age-related macular degeneration","volume":"20","author":"Hageman","year":"2001","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1106","DOI":"10.1016\/j.imbio.2012.07.008","article-title":"The tick-over theory revisited: Is C3 a contact-activated protein?","volume":"217","author":"Nilsson","year":"2012","journal-title":"Immunobiology"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Chinchilla, B., Foltopoulou, P., and Fernandez-Godino, R. (2021). Tick-over mediated complement activation is sufficient to cause basal deposit formation in cell-based models of macular degeneration. J. Pathol.","DOI":"10.1002\/path.5747"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1172\/JCI31487","article-title":"Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases","volume":"117","author":"Wynn","year":"2007","journal-title":"J. Clin. Investig."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1002\/path.2277","article-title":"Cellular and molecular mechanisms of fibrosis","volume":"214","author":"Wynn","year":"2008","journal-title":"J. Pathol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"419","DOI":"10.14336\/AD.2018.0601","article-title":"The role of pathological aging in cardiac and pulmonary fibrosis","volume":"10","author":"Murtha","year":"2019","journal-title":"Aging Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4979","DOI":"10.1167\/iovs.10-5386","article-title":"Comparison of FRPE and human embryonic stem cell\u2013derived RPE behavior on aged human Bruch\u2019s membrane","volume":"52","author":"Sugino","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_28","first-page":"467","article-title":"Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch\u2019s membrane","volume":"40","author":"Tezel","year":"1999","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1186\/s13073-015-0183-x","article-title":"Restoration of mesenchymal retinal pigmented epithelial cells by TGF\u03b2 pathway inhibitors: Implications for age-related macular degeneration","volume":"7","author":"Radeke","year":"2015","journal-title":"Genome Med."},{"key":"ref_30","first-page":"267","article-title":"Phenotypic variation of retinal pigment epithelium in age-related macular degeneration","volume":"43","author":"Guidry","year":"2002","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1438","DOI":"10.1172\/JCI38019","article-title":"Epithelial-mesenchymal transitions: The importance of changing cell state in development and disease","volume":"119","author":"Acloque","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1420","DOI":"10.1172\/JCI39104","article-title":"The basics of epithelial-mesenchymal transition","volume":"119","author":"Kalluri","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1681\/ASN.2008040434","article-title":"C3a mediates epithelial-to-mesenchymal transition in proteinuric nephropathy","volume":"20","author":"Tang","year":"2009","journal-title":"J. Am. Soc. Nephrol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1412","DOI":"10.4049\/jimmunol.1501886","article-title":"Complement component 3 is regulated by TWIST1 and mediates epithelial\u2013mesenchymal transition","volume":"196","author":"Cho","year":"2016","journal-title":"J. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1093\/hmg\/ddt395","article-title":"Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD\/ML, an inherited macular degeneration","volume":"23","author":"Garland","year":"2013","journal-title":"Hum. Mol. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2281","DOI":"10.1038\/nprot.2013.143","article-title":"Genome engineering using the CRISPR-Cas9 system","volume":"8","author":"Ran","year":"2013","journal-title":"Nat. Protoc."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1016\/j.omtm.2019.10.014","article-title":"AAV-mediated gene augmentation therapy restores critical functions in mutant PRPF31+\/\u2212 iPSC-derived RPE cells","volume":"15","author":"Brydon","year":"2019","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_38","first-page":"e56274","article-title":"Rapid, directed differentiation of retinal pigment epithelial cells from human embryonic or induced pluripotent stem cells","volume":"128","author":"Foltz","year":"2017","journal-title":"J. Vis. Exp."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1089\/jop.2016.0022","article-title":"Induced pluripotent stem cell-derived retinal pigmented epithelium: A comparative study between cell lines and differentiation methods","volume":"32","author":"Leach","year":"2016","journal-title":"J. Ocul. Pharmacol. Ther."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1038\/9722","article-title":"A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy","volume":"22","author":"Stone","year":"1999","journal-title":"Nat. Genet."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"703","DOI":"10.3389\/fimmu.2019.00703","article-title":"Development and optimization of an ELISA to quantitate C3(H2O) as a marker of human disease","volume":"10","author":"Elvington","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"E8214","DOI":"10.1073\/pnas.1710430114","article-title":"Drusen in patient-derived hiPSC-RPE models of macular dystrophies","volume":"114","author":"Galloway","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"501","DOI":"10.3389\/fcell.2020.00501","article-title":"Role of epithelial-mesenchymal transition in retinal pigment epithelium dysfunction","volume":"8","author":"Zhou","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Shu, D.Y., Butcher, E., and Saint-Geniez, M. (2020). EMT and EndMT: Emerging roles in age-related macular degeneration. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21124271"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1429","DOI":"10.1172\/JCI36183","article-title":"Biomarkers for epithelial-mesenchymal transitions","volume":"119","author":"Zeisberg","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Georgiadis, A., Tschernutter, M., Bainbridge, J.W.B., Balaggan, K.S., Mowat, F., West, E., Munro, P.M.G., Thrasher, A.J., Matter, K., and Balda, M.S. (2010). The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0015730"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2610","DOI":"10.1016\/j.bbadis.2018.04.016","article-title":"Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers","volume":"1864","author":"Shah","year":"2018","journal-title":"Biochim. Biophys. Acta (BBA) Mol. Basis Dis."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Wang, A.L., Lukas, T.J., Yuan, M., Du, N., Tso, M.O., and Neufeld, A.H. (2009). Autophagy and exomoses in the aged retinal pigment epithelium: Possible relevance to drusen formation and age-related macular degeneration. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0004160"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1776","DOI":"10.1172\/JCI200320530","article-title":"Epithelial-mesenchymal transition and its implications for fibrosis","volume":"112","author":"Kalluri","year":"2003","journal-title":"J. Clin. Invest."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1006\/jsbi.2002.4449","article-title":"Analysis of the collagen VI assemblies associated with Sorsby\u2019s fundus dystrophy","volume":"137","author":"Knupp","year":"2002","journal-title":"J. Struct. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1038\/JID.2015.352","article-title":"Type VI collagen regulates dermal matrix assembly and fibroblast motility","volume":"136","author":"Theocharidis","year":"2016","journal-title":"J. Investig. Dermatol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1019","DOI":"10.1002\/jemt.20829","article-title":"3D reconstruction of histological sections: Application to mammary gland tissue","volume":"73","year":"2010","journal-title":"Microsc. Res. Tech."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.12659\/MSM.889887","article-title":"Age-related macular degeneration and changes in the extracellular matrix","volume":"20","author":"Romaniuk","year":"2014","journal-title":"Med. Sci. Monit."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1007\/978-3-319-17121-0_8","article-title":"Extracellular matrix alterations and deposit formation in AMD","volume":"Volume 854","author":"Pierce","year":"2016","journal-title":"Advances in Experimental Medicine and Biology"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S0161-6420(90)32619-2","article-title":"Aging changes in Bruch\u2019s membrane. A histochemical and morphologic study","volume":"97","author":"Pauleikhoff","year":"1990","journal-title":"Ophthalmology"},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Fields, M.A., Bowrey, H.E., Gong, J., Moreira, E.F., Cai, H., and Del Priore, L.V. (2017). Extracellular matrix nitration alters growth factor release and activates bioactive complement in human retinal pigment epithelial cells. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0177763"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"5479","DOI":"10.1167\/iovs.16-19816","article-title":"Associations between retinal pigment epithelium and drusen volume changes during the lifecycle of large drusenoid pigment epithelial detachments","volume":"57","author":"Balaratnasingam","year":"2016","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"AMD104","DOI":"10.1167\/iovs.18-24132","article-title":"A role for \u03b2A3\/A1-crystallin in type 2 EMT of RPE cells occurring in dry age-related macular degeneration","volume":"59","author":"Ghosh","year":"2018","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1038\/eye.1988.106","article-title":"Evolution of geographic atrophy of the retinal pigment epithelium","volume":"2","author":"Sarks","year":"1988","journal-title":"Eye"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1002\/jcp.21129","article-title":"Complement 3 is involved in changing the phenotype of human glomerular mesangial cells","volume":"213","author":"Wan","year":"2007","journal-title":"J. Cell. Physiol."},{"key":"ref_61","first-page":"8312","article-title":"Integrin \u03b11\u03b21 and \u03b12\u03b21 are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment","volume":"63","author":"Yang","year":"2003","journal-title":"Cancer Res."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1368","DOI":"10.1016\/S0161-6420(92)31792-0","article-title":"Ulrastructural immunocytochemistry of subretinal neovascular membranes in age-related macular degeneration","volume":"99","author":"Das","year":"1992","journal-title":"Ophthalmology"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"18602","DOI":"10.1038\/srep18602","article-title":"Col4a1 mutations cause progressive retinal neovascular defects and retinopathy","volume":"6","author":"Alavi","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"944","DOI":"10.1136\/bjo.79.10.944","article-title":"Preparation of Bruch\u2019s membrane and analysis of the age-related changes in the structural collagens","volume":"79","author":"Karwatowski","year":"1995","journal-title":"Br. J. Ophthalmol."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.exer.2015.03.009","article-title":"Molecular mechanisms of subretinal fibrosis in age-related macular degeneration","volume":"142","author":"Ishikawa","year":"2016","journal-title":"Exp. Eye Res."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"1917","DOI":"10.1126\/science.1151526","article-title":"Induced pluripotent stem cell lines derived from human somatic cells","volume":"318","author":"Yu","year":"2007","journal-title":"Science"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1038\/nmeth.1593","article-title":"Chemically defined conditions for human iPSC derivation and culture","volume":"8","author":"Chen","year":"2011","journal-title":"Nat. Methods"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"e109","DOI":"10.1182\/blood-2010-07-298331","article-title":"Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells","volume":"117","author":"Hu","year":"2011","journal-title":"Blood"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"3612","DOI":"10.1167\/iovs.05-1622","article-title":"Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue","volume":"47","author":"Maminishkis","year":"2006","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"1206","DOI":"10.1038\/nprot.2016.065","article-title":"Isolation, culture and characterization of primary mouse RPE cells","volume":"11","author":"Godino","year":"2016","journal-title":"Nat. Protoc."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/15\/8183\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:37:12Z","timestamp":1760164632000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/15\/8183"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,30]]},"references-count":71,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["ijms22158183"],"URL":"https:\/\/doi.org\/10.3390\/ijms22158183","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,30]]}}}