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Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide
Open accessAbstract
Human RPE cell lines, especially the ARPE-19 cell line, are widely-used in eye research, as well as general epithelial cell studies. In comparison with primary RPE cells, they offer relative convenience and consistency, but cultures derived from these lines are typically not well differentiated. We describe a simple, rapid method to establish cultures from ARPE-19 cells, with significantly improved epithelial cell morphology and cytoskeletal organization, and RPE-related functions. We identify the presence of nicotinamide, a member of the vitamin B family, as an essential factor in promoting the observed differentiation, indicating the importance of metabolism in RPE cell differentiation.
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“However, these cell lines have genetic abnormalities, with triploidy in the D407 line (Davis et al., 1995), and an abnormal karyotype (most notably, a loss of one copy of chromosome 15, and an unbalanced translocation between the chromosome 15q and 19q) in the ARPE-19 line (Fasler-Kan et al., 2018; Hazim et al., 2019).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Hazim et al., 2019
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“An important part of the protocol, reported by Hazim et al. for ARPE-19 cells, is a series of washes with trypsin that selectively removes the more proliferative cells, which tend to be more loosely adhered to the substrate, leaving behind firmly attached cells that more readily form a culture with a cobblestone appearance (Hazim et al., 2019).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Hazim et al., 2019
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“These protocols required 3–4 months for RPE differentiation, whereas, a more recent protocol was able to achieve a similar level of differentiation within 4 weeks through the addition of nicotinamide instead of pyruvate (Hazim et al., 2019) (Fig. 5).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Hazim et al., 2019
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Cites
The Retinal Pigment Epithelium in Visual Function
Strauss, O. (2005) · Physiological Reviews · doi:10.1152/physrev.00021.2004 · Cited by 2,828 · cited 1×
“It performs specific functions in support of the photoreceptor cells, including the reisomerization of visual chromophore, and phagocytosis of photoreceptor outer segment (POS) membrane (Strauss, 2005).”
BackgroundComplement factor H polymorphism and age-related macular degeneration
Edwards, A. O., Ritter, R., Abel, K. J., Manning, A., Panhuysen, C., & Farrer, L. A. (2005) · Science · doi:10.1126/science.1110189 · Cited by 2,449 · cited 5×
“The importance of the RPE for proper vision is underscored by several retinal degenerative diseases that involve RPE pathology, including age-related macular degeneration (AMD) (Bhutto and Lutty, 2012).”
BackgroundInfluentialComplement factor H variant increases the risk of age-related macular degeneration
Haines, J. L., Hauser, M. A., Schmidt, S., Scott, W. K., Olson, L. M., Gallins, P., Spencer, K. L., Kwan, S. Y., Noureddine, M., Gilbert, J. R., et al. (2005) · Science · doi:10.1126/science.1110359 · Cited by 2,424 · cited 5×
“The importance of the RPE for proper vision is underscored by several retinal degenerative diseases that involve RPE pathology, including age-related macular degeneration (AMD) (Bhutto and Lutty, 2012).”
BackgroundInfluentialGomes, A. P., Price, N. L., Ling, A. J. Y., Moslehi, J. J., Montgomery, M. K., Rajman, L., White, J. P., Teodoro, J. S., Wrann, C. D., Hubbard, B. P., et al. (2013) · Cell · doi:10.1016/j.cell.2013.11.037 · Cited by 1,482
ARPE-19, A Human Retinal Pigment Epithelial Cell Line with Differentiated Properties
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“When first reported, standard cultures of the ARPE-19 cell line readily exhibited many of the characteristics of well-differentiated RPE cells, including cobblestone morphology, apical-basal polarity, and expression of RPE-specific proteins (Dunn et al., 1996).”
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“Nicotinamide has been shown to protect against oxidative stress and maintain mitochondrial health (Gomes et al., 2013; Mitchell et al., 2018), while its related compound, NR, was demonstrated to promote oxidative metabolism by means of increasing the NAD+/ NADH ratio (Canto et al., 2012).”
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“The importance of the RPE for proper vision is underscored by several retinal degenerative diseases that involve RPE pathology, including age-related macular degeneration (AMD) (Bhutto and Lutty, 2012).”
BackgroundInfluentialOrganization and execution of the epithelial polarity programme
Rodriguez-Boulan, E. J., & Macara, I. G. (2014) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm3775 · Cited by 727 · cited 1×
“The cells typically exhibit incomplete execution of the epithelial polarity program (Rodriguez-Boulan and Macara, 2014), thus compromising their ability to represent in vivo biology.”
BackgroundStem cell metabolism in tissue development and aging
Shyh‐Chang, N., Daley, G. Q., & Cantley, L. C. (2013) · Development · doi:10.1242/dev.091777 · Cited by 602 · cited 1×
“Interestingly, many of the key enzymes and coenzymes in these metabolic pathways have been shown to become upregulated as cells differentiate into their terminal fate (Shyh-Chang et al., 2013; Hu et al., 2016).”
BackgroundRhyu, D. Y., Yang, Y., Ha, H., Lee, G. T., Song, J. S., Uh, S., & Lee, H. B. (2005) · Journal of the American Society of Nephrology · doi:10.1681/asn.2004050425 · Cited by 523 · cited 1×
“Interestingly, there is evidence to suggest that oxidative stress, and in particular reactive oxygen species, can promote epithelial-to-mesenchymal transition (Rhyu et al., 2005; Wang et al., 2010).”
BackgroundIdelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al. (2009) · Cell stem cell · doi:10.1016/j.stem.2009.07.002 · Cited by 486 · cited 2×
“Given the role that nicotinamide has been shown to play in the differentiation of RPE from pluripotent stem cells (Idelson et al., 2009; Maruotti et al., 2015; Saini et al., 2017), we tested the necessity of nicotinamide in the MEM-Nic medium for the observed morphological phenotype.”
BackgroundNicotinamide Improves Aspects of Healthspan, but Not Lifespan, in Mice
Mitchell, S. J., Bernier, M., Aon, M. A., Cortassa, S., Kim, E. Y., Fang, E. F., Palacios, H. H., Ali, A., Navas-Enamorado, I., Di Francesco, A., et al. (2018) · Cell Metabolism · doi:10.1016/j.cmet.2018.02.001 · Cited by 338 · cited 1×
“Nicotinamide has been shown to protect against oxidative stress and maintain mitochondrial health (Gomes et al., 2013; Mitchell et al., 2018), while its related compound, NR, was demonstrated to promote oxidative metabolism by means of increasing the NAD+/ NADH ratio (Canto et al.”
BackgroundMicrotubule Organization and Function in Epithelial Cells
Müsch, A. (2003) · Traffic · doi:10.1111/j.1600-0854.2003.00149.x · Cited by 268 · cited 1×
“In epithelial cells, the spatial organization of microtubules, in addition to their associated motors, have been shown to be critical for the polarized targeting of secretory vesicles, as well as the machinery of the endocytic pathway (Gilbert et al., 1991; Musch, 2004).”
BackgroundNicotinamide-induced Mitophagy
Jang, S., Kang, H. T., & Hwang, E. S. (2012) · Journal of Biological Chemistry · doi:10.1074/jbc.m112.363747 · Cited by 264 · cited 1×
“In particular, it serves as a precursor for the production of NAD +, NADP+, and other substrates that participate in metabolic pathways, including the TCA cycle, and the mitochondrial electron transport chain (Jang et al., 2012).”
BackgroundBuchholz, D. E., Pennington, B. O., Croze, R. H., Hinman, C. R., Coffey, P. J., & Clegg, D. O. (2013) · Stem Cells Translational Medicine · doi:10.5966/sctm.2012-0163 · Cited by 261
Gilbert, T., Le Bivic, A., Quaroni, A., & Rodriguez-Boulan, E. (1991) · The Journal of Cell Biology · doi:10.1083/jcb.113.2.275 · Cited by 234 · cited 1×
“In epithelial cells, the spatial organization of microtubules, in addition to their associated motors, have been shown to be critical for the polarized targeting of secretory vesicles, as well as the machinery of the endocytic pathway (Gilbert et al., 1991; Musch, 2004).”
BackgroundTombran‐Tink, J., Shivaram, S., Chader, G. J., Johnson, L., & Bok, D. (1995) · Journal of Neuroscience · doi:10.1523/jneurosci.15-07-04992.1995 · Cited by 218 · cited 1×
“2F), a protein known to be secreted from the apical surface of polarized RPE cells (Tombran-Tink et al., 1995).”
BackgroundSaini, J. S., Corneo, B., Miller, J. D., Kiehl, T. R., Wang, Q., Boles, N. C., Blenkinsop, T. A., Stern, J. H., & Temple, S. (2017) · Cell stem cell · doi:10.1016/j.stem.2016.12.015 · Cited by 189 · cited 1×
“Given the role that nicotinamide has been shown to play in the differentiation of RPE from pluripotent stem cells (Idelson et al., 2009; Maruotti et al., 2015; Saini et al., 2017), we tested the necessity of nicotinamide in the MEM-Nic medium for the observed morphological phenotype.”
BackgroundMaruotti, J., Sripathi, S. R., Bharti, K., Fuller, J., Wahlin, K. J., Jaskula-Ranga, V., Sluch, V. M., Berlinicke, C. A., Davis, J. A., Kim, C., et al. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1422818112 · Cited by 144 · cited 1×
“Given the role that nicotinamide has been shown to play in the differentiation of RPE from pluripotent stem cells (Idelson et al., 2009; Maruotti et al., 2015; Saini et al., 2017), we tested the necessity of nicotinamide in the MEM-Nic medium for the observed morphological phenotype.”
BackgroundEnergy Metabolism Plays a Critical Role in Stem Cell Maintenance and Differentiation
Hu, C., Fan, L., Cen, P., Chen, E., Jiang, Z., & Li, L. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17020253 · Cited by 143 · cited 1×
“Interestingly, many of the key enzymes and coenzymes in these metabolic pathways have been shown to become upregulated as cells differentiate into their terminal fate (Shyh-Chang et al., 2013; Hu et al., 2016).”
BackgroundFunctional Analysis of Serially Expanded Human iPS Cell-Derived RPE Cultures
Singh, R., Phillips, M. J., Kuai, D., Meyer, J., Martin, J. M., Smith, M. A., Perez, E. T., Shen, W., Wallace, K. A., Capowski, E. E., et al. (2013) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.13-11943 · Cited by 136 · cited 1×
“Furthermore, stem cell-derived RPE cells have been found to lose their polarized organization and RPE functions after only a few passages in culture (Singh et al., 2013).”
BackgroundWang, Z. P., Li, Y., & Sarkar, F. H. (2010) · Current Stem Cell Research & Therapy · doi:10.2174/157488810790442813 · Cited by 123
Retinal pigment epithelium polarity in health and blinding diseases
Caceres, P. S., & Rodriguez-Boulan, E. J. (2019) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2019.08.001 · Cited by 105
Ahmado, A., Carr, A.-J., Vugler, A. A., Semo, M., Gias, C., Lawrence, J. M., Chen, L. L., Chen, F. K., Turowski, P., da Cruz, L., et al. (2011) · Investigative Opthalmology & Visual Science · doi:10.1167/iovs.10-6374 · Cited by 100 · cited 1×
“Although the premelanosome marker, Pmel17, has been detected in ARPE-19 cells (Ahmado et al., 2011), images of normal RPE melanosomes have never been shown.”
BackgroundThe Retinal G Protein-coupled Receptor (RGR) Enhances Isomerohydrolase Activity Independent of Light
Wenzel, A., Oberhauser, V., Pugh, E. N., Lamb, T. D., Grimm, C., Samardzija, M., Fahl, E., Seeliger, M. W., Remé, C. E., & von Lintig, J. (2005) · Journal of Biological Chemistry · doi:10.1074/jbc.m503603200 · Cited by 92 · cited 1×
“For western blotting, the antibodies used included: rabbit anti-RPE65 (pin 5) (Wenzel et al., 2005) (gift from Andreas Wenzel) and goat anti-FH (Quidel A312).”
BackgroundMicrotubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis
Jiang, M., Esteve-Rudd, J., Lopes, V. S., Diemer, T., Lillo, C., Rump, A., & Williams, D. S. (2015) · Journal of Cell Biology · doi:10.1083/jcb.201410112 · Cited by 85
Phenotypic Heterogeneity of Retinal Pigment Epithelial Cells In Vitro and In Situ
Burke, J. M., Skumatz, C. M. B., Irving, P. E., & McKay, B. S. (1996) · Experimental Eye Research · doi:10.1006/exer.1996.0008 · Cited by 84 · cited 1×
“Moreover, these primary cultures exhibit significant heterogeneity among cultures from different donors (Burke et al., 1996; Kuznetsova et al., 2014).”
BackgroundBoles, N. C., Fernandes, M., Swigut, T., Srinivasan, R., Schiff, L., Rada-Iglesias, Á., Wang, Q., Saini, J. S., Kiehl, T. R., Stern, J. H., et al. (2020) · Stem Cell Reports · doi:10.1016/j.stemcr.2020.03.009 · Cited by 81
Regulated secretion of complement factor H by RPE and its role in RPE migration
Kim, Y. H., He, S., Kase, S., Kitamura, M., Ryan, S. J., & Hinton, D. R. (2009) · Graefe s Archive for Clinical and Experimental Ophthalmology · doi:10.1007/s00417-009-1049-y · Cited by 78 · cited 1×
“This observation is consistent with immunofluorescence data showing Factor H localization on the apical surface of human fetal RPE cultured on Transwell inserts (Kim et al., 2009).”
ResultCell Models to Study Regulation of Cell Transformation in Pathologies of Retinal Pigment Epithelium
Kuznetsova, A. V., Kurinov, A. M., & Aleksandrova, M. A. (2014) · Journal of Ophthalmology · doi:10.1155/2014/801787 · Cited by 77 · cited 1×
“Moreover, these primary cultures exhibit significant heterogeneity among cultures from different donors (Burke et al., 1996; Kuznetsova et al., 2014).”
BackgroundHazim, R. A., Karumbayaram, S., Jiang, M., Dimashkie, A., Lopes, V. S., Li, D., Burgess, B. L., Vijayaraj, P., Alva-Ornelas, J. A., Zack, J. A., et al. (2017) · Stem Cell Research & Therapy · doi:10.1186/s13287-017-0652-9 · Cited by 66 · cited 2×
“cells can be derived from embryonic and induced pluripotent stem cells (Idelson et al., 2009; Buchholz et al., 2013; Hazim et al., 2017).”
BackgroundMethodWavre-Shapton, S. T., Meschede, I. P., Seabra, M. C., & Futter, C. E. (2014) · Journal of Cell Science · doi:10.1242/jcs.154757 · Cited by 60 · cited 1×
“The ARPE-19 cells differentiated by our method establish a polarized epithelial cell model that can be used to investigate general epithelial processes, including secretory and endocytic vesicular transport, as well as RPE-specific processes such as the trafficking of phagosomes and the degradative organelles that are…”
BackgroundMiceli, M. V., Newsome, D. A., & Tate, D. J. (1997) · PubMed · Cited by 52
Selective Proteolysis of Arrestin by Calpain
Azarian, S. M., King, A. J., Hallett, M. A., & Williams, D. S. (1995) · Journal of Biological Chemistry · doi:10.1074/jbc.270.41.24375 · Cited by 47 · cited 1×
“POSs were isolated using a previously published protocol for isolation of bovine POSs (Azarian et al., 1995), with a few modifications.”
MethodEsteve-Rudd, J., Hazim, R. A., Diemer, T., Paniagua, A. E., Volland, S., Umapathy, A., & Williams, D. S. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1709211115 · Cited by 42 · cited 1×
“The ARPE-19 cells differentiated by our method establish a polarized epithelial cell model that can be used to investigate general epithelial processes, including secretory and endocytic vesicular transport, as well as RPE-specific processes such as the trafficking of phagosomes and the degradative organelles that are…”
BackgroundMilićević, N., Mazzaro, N., de Bruin, I., Wils, E., ten Brink, J. B., ten Asbroek, A. L. M. A., Mendoza, J., Bergen, A. A. B., & Felder‐Schmittbuhl, M. (2019) · Scientific Reports · doi:10.1038/s41598-019-48203-3 · Cited by 28
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Fasler-Kan, E., Aliu, N., Wunderlich, K., Ketterer, S., Ruggiero, S., Berger, S., & Meyer, P. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-7680-5_17 · Cited by 20 · cited 1×
“The immortalization of the ARPE-19 cell line occurred spontaneously, and in a recent study (Fasler-Kan et al., 2018), it was shown that different sources of the cell line exhibited abnormal karyotypes.”
BackgroundCell Culture Analysis of the Phagocytosis of Photoreceptor Outer Segments by Primary Mouse RPE Cells
Hazim, R. A., & Williams, D. S. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-7720-8_4 · Cited by 11 · cited 1×
“A previously described double immunofluorescence labeling strategy (Hazim and Williams, 2018) was used to distinguish between POSs bound to the surface of the ARPE-19 cells and POSs that have been internalized (Fig.”
MethodRole of FGF and Hyaluronan in Choroidal Neovascularization in Sorsby Fundus Dystrophy
Wolk, A., Hatipoğlu, D., Cutler, A., Ali, M., Bell, L., Qi, J. H., Singh, R., Batoki, J. C., Karle, L., Bonilha, V. L., et al. (2020) · Cells · doi:10.3390/cells9030608 · Cited by 8
Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis
Buyandelger, U., Walker, D. G., Yanagisawa, D., Morimura, T., & Tooyama, I. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21103635 · Cited by 8
Cited by
Sorted by citations
The cell biology of the retinal pigment epithelium
Lakkaraju, A., Umapathy, A., Tan, L. X., Daniele, L., Philp, N. J., Boesze-Battaglia, K., & Williams, D. S. (2020) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2020.100846 · Cited by 422
“However, the morphological features of these cells do not appear as RPE-like as those reported by others (Ahmado et al., 2011; Hazim et al., 2019).”
BackgroundInfluentialRole of Epithelial-Mesenchymal Transition in Retinal Pigment Epithelium Dysfunction
Zhou, M., Geathers, J. S., Grillo, S. L., Weber, S. R., Wang, W., Zhao, Y., & Sundstrom, J. M. (2020) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2020.00501 · Cited by 199
Retinal pigment epithelium polarity in health and blinding diseases
Caceres, P. S., & Rodriguez-Boulan, E. J. (2019) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2019.08.001 · Cited by 105
Bharti, K., den Hollander, A. I., Lakkaraju, A., Sinha, D., Williams, D. S., Finnemann, S. C., Rickman, C. B., Malek, G., & D'Amore, P. A. (2022) · Experimental Eye Research · doi:10.1016/j.exer.2022.109170 · Cited by 98
Boles, N. C., Fernandes, M., Swigut, T., Srinivasan, R., Schiff, L., Rada-Iglesias, Á., Wang, Q., Saini, J. S., Kiehl, T. R., Stern, J. H., et al. (2020) · Stem Cell Reports · doi:10.1016/j.stemcr.2020.03.009 · Cited by 81
“In addition, NAM improves cobblestone phenotype in immortalized ARPE19 cells (Hazim et al., 2019).”
Background
Metadata
- Authors
- Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., & Williams, D. S.
- Affiliations
- University of California, Los Angeles (US)
- Corresponding author
- Williams, David S.
- Venue
- Experimental Eye Research
- Publisher
- Elsevier BV
- Published
- 2019-02
- Volume
- 179
- Pages
- 18-24
- ISSN
- 0014-4835 (ISSN-L)
- PMC
- PMC6360117
- OpenAlex
- W2897486375
- Cited by
- 126
- References
- 41
- Citations by year
- 2026: 16 · 2025: 24 · 2024: 26 · 2023: 16 · 2022: 15 · 2021: 7 · 2020: 18 · 2019: 4
- FWCI
- 7.29
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Experimental Eye Research (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institutes of Health (P30EY0331, R01EY013408, R01EY027442, F31EY026805)
- Topics
- Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
- Health Sciences › Medicine › Ophthalmology › Retinal Development and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Connexins and lens biology
- Keywords
- retinal pigment epithelium, RPE differentiation, nicotinamide, epithelial morphology, cytoskeletal organization, vitamin B metabolism
- MeSH terms
- Bestrophins, Retinoid Isomerohydrolase, Cell Differentiation, Cell Line, Cytoskeleton, Gene Expression Regulation, Humans, Microvilli, Niacinamide, Vitamin B Complex, Biomarkers, Fluorescent Antibody Technique, Indirect, cis-trans-Isomerases, Integrin beta Chains, Retinal Pigment Epithelium, Real-Time Polymerase Chain Reaction, Occludin, Zonula Occludens-1 Protein
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