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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
In the citing paper
“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
In the citing paper
“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
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“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
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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).”
BackgroundInfluentialComplement factor H variant increases the risk of age-related macular degeneration
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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).”
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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).”
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“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).”
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Mueller‐Buehl, A. M., Safaei, A., & Tsai, T. (2022) · Der Ophthalmologe · doi:10.1007/s00347-021-01565-4
“In vielen Laboren, wie auch in unserem, wird daher an der Interaktion aus Neuroretina und RPE- Zellen geforscht [17, 18].”
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Primary Culture of Porcine Retinal Pigment Epithelial Cells
Wen, F., Wang, Y., He, D., Liao, C., Ouyang, W., Liu, Z., Li, W. S., & Liao, Y. (2022) · Journal of Visualized Experiments · doi:10.3791/64244
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Schikora, J., Nickel, A., Bergert, J., Hähnel, R., Dort, A., Schayan-Araghi, S. Y., Banerjee, P., Wolf, H. N., May‐Simera, H., & Pauly, D. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.02.05.636432
The Importance of Differentiated RPE Cultures for studying Cell Biological Processes
Hazim, R. A., & Williams, D. S. (2025) · Advances in experimental medicine and biology · doi:10.1007/978-3-031-76550-6_58
Geiller, B., Cevallos, C., Tsybenko, I., Arnoux, L., Felder‐Schmittbuhl, M., & Pfaff, A. W. (2025) · Research Square · doi:10.21203/rs.3.rs-6171737/v1
Hansman, D. S., Lim, K. S., Thomas, D., Casson, R. J., & Peet, D. J. (2025) · Scientific Reports · doi:10.1038/s41598-025-93882-w
“ARPE-19 cells were differentiated in 10 mM nicotinamide using previously described methods 30 , with differentiation confirmed by qRT-PCR analysis of the RPE marker genes RLBP1 , RPE65, and BEST1 (Fig.”
Hulleman, J. D., Jeon, S., Bali, S., DiCesare, S. M., Abbas, A., Daniel, S., Ortega, A. J., Collier, G. E., Yang, J., Bhattacharyaa, A., et al. (2025) · ACS Chemical Biology · doi:10.1021/acschembio.4c00818
Wu, Y., Tong, Y., Byrnes, K. G., Zhou, Q., Dong, C., Benjamin, C., Parker, E., Bao, D., Ren, Z., Anderson, C. A., et al. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.03.03.709397
Circadian regulation of the retinal pigment epithelium–photoreceptor complex
Milićević, N. (2020) · theses.fr (ABES) · doi:10.70675/2a3b679cz473bz4c14zb33az700581cd794a
Zhao, Z., Ou, Q., Zhu, J., He, Z., Yang, Y., Jiang, J., Wang, Q., Zhou, Y., Liu, Y., Zhu, X., et al. (2026) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.67.4.30 · Cited by 1
Li, H., Yang, Y.-H., Dong, L., Zhang, R.-H., Zhou, W., Wu, H.-T., Dong, J., Yu, C., Wang, S., & Wei, W.-B. (2026) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.67.4.38
“34 These barrier defects may have direct consequences for ocular growth.”
Palakkan, A. A., Shankar, G., Vignesh, T. P., & Kim, R. (2026) · Biomedical Materials · doi:10.1088/1748-605x/ae6498
“For experiments, ARPE-19 cells were cultured in low serum medium to induce tight junctions [18].”
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“…medium (MEM alpha with GlutaMAX, 1% FBS, 1% Penicillin/Streptomycin, 1% N1 supplement, taurine (0.25 mg/mL), hydrocortisone (20 ng/mL), triiodothyronine (0.013 ng/mL), and 10 mM nicotinamide) with media changes three times per week for two weeks following a previously published protocol [26].”
InfluentialLu, Y. R., Shen, H., Kajderowicz, K. M., 艾家和, Chen-Neumann, Y. H., Hu, Y., Tyshkovskiy, A., Lou, Q., Dong, T., Grauvogel, L., et al. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.09.29.755187
“To mitigate the known limitations of undifferentiated ARPE-19 cells 39,40 , we used an optimized nicotinamide-based maturation protocol to differentiate them at confluence before stress challenges 41,42 , and also used mouse models to validate the top candidates.”
Matei, I., Grimes, D., Suwanmanee, G., Bagnall, J., Spiller, D. G., & Paraoan, L. (2026) · Cells · doi:10.3390/cells15201846
“…to create appropriate culture conditions for ARPE-19 cells to achieve a suitably differentiated, physiologically relevant phenotype in vitro [7,9,13–16], highlighting the importance of factors such as a high glucose-containing medium [7], pyruvate [7,9], nicotinamide supplementation [13–16],…”
Influential
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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