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. 2019 Feb;179:18-24.
doi: 10.1016/j.exer.2018.10.009. Epub 2018 Oct 15.

Rapid Differentiation of the Human RPE Cell Line, ARPE-19, Induced by Nicotinamide

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Rapid Differentiation of the Human RPE Cell Line, ARPE-19, Induced by Nicotinamide

Roni A Hazim et al. Exp Eye Res. .
Free PMC article

Abstract

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.

Figures

Figure 1:
Figure 1:. Cellular morphology of differentiated ARPE-19 cells.
(A) Brightfield micrographs of ARPE-19 cells differentiated on plastic surfaces for one week in standard media (DMEM/F12 or DMEM-Pyruvate) or in MEM-Nic medium. (B) Immunofluorescence micrographs of ZO-1 labeling in ARPE-19 cells differentiated on Transwell inserts for one week in the same media as for A. Only cells differentiated in MEM-Nic medium show a cobblestone morphology after this one-week interval. (C) Nicotinamide is a necessary component in MEM-Nic for the differentiation of ARPE-19 cells. Brightfield micrographs of ARPE-19 cells plated at the same density and cultured on plastic tissue culture plates for two weeks in MEM-Nic medium with all supplements, except the nicotinamide, with 1 mM or 10 mM NR instead of the nicotinamide, or with 10 mM nicotinamide (i.e. complete MEM-Nic). Cobblestone morphology can only be observed in medium containing 10 mM Nic or 10 mM NR. The adjacent immunofluorescence images show ZO-1 labeling, after culture on Transwell inserts in MEM plus 10 mM Nic or 10 mM NR for two weeks. Note that different imaging systems were used for the brightfield images in A and C. NR, nicotinamide riboside. (D-G) ARPE-19 cells differentiated on Transwell inserts for 23 weeks in MEM-Nic medium. (D) TEM micrograph showing numerous microvilli emanating from the apical surface of ARPE-19 cells. (E) Phalloidin labeling showing actin filaments in the apical microvilli (lower) and in a circumferential ring at the level of the cell-cell junctions (upper). (F and G) Single plane confocal microscopy of α-tubulin immunolabeling, revealing horizontal microtubules in the apical part of the cell body (F) and vertical microtubules in the basal region (G) of polarized ARPE-19 cells. Scale bars, 100 μm (A and C), 20 μm (B, E, and G), or 0.5 μm (D). All images are representative from at least 3 experiments.
Figure 2:
Figure 2:. Expression profile and functional assessment of differentiated ARPE-19 cells.
(A) RTPCR shows expression of RPE-specific and RPE-related genes. N.T.C, no template control. (B) Immunolabeled western blot revealing expression of RPE65 in ARPE-19 cells differentiated in MEM-Nic for 4 weeks; the protein is undetectable in HEK cells and ARPE-19 cells differentiated in MEM without nicotinamide (MEM). See Supplementary Fig. 2A for images of the complete western blot after, first, Ponceau S-staining and, then, immunolabeling. HEK, human embryonic kidney cells. (C and D) Fluorescence micrographs of ARPE-19 cells differentiated on Transwell inserts for 3 weeks showing expression and polarized localization of OCLN, at the level of the junctional complexes (C), and BEST1, along the basolateral surface (D). The nuclei are counterstained with DAPI in the z-planes (taken at the yellow lines), which are shown beneath each panel. Although reconstructed z-axis resolution is considerably inferior to x- and y-axis resolution, creating significant vertical “blur”, it is evident that the OCLN labeling is more apical than the BEST1 labeling, indicating that the BEST1 labeling is below the junctional complex and in the lateral membrane that is part of the basolateral domain. (E) Immunolabeled western blot revealing the presence of Factor H in serum-free medium conditioned by differentiated ARPE-19 cells for 48 h. Quantification of band intensity showed significantly more Factor H present in the apical medium relative to the basal medium. Samples from three different cultures (n = 3) are shown; for each pair, the volume of basal medium loaded on to the gel was 6 times larger than the volume of apical medium in order to account for the difference in medium volume placed in the apical and basal chambers of the Transwell insert. See Supplementary Fig. 2B for image of complete western blot. (F) Z-projection of fluorescence micrograph of differentiated ARPE-19 cells showing expression and apical localization of PEDF, a protein secreted from the apical surface of polarized RPE cells. Scale bars, 20 μm (C, D, F). PEDF, pigment epithelium-derived factor. All images are representative of ≥ 3 experiments. Error bars in (E) represent ± S.E.M. *** P = 0.0002.
Figure 3:
Figure 3:. Phagocytic function in differentiated ARPE-19 cells.
(A) Micrographs of opsin-labeled ARPE-19 cells (before and after cell permeabilization) cultured on Transwell inserts in MEM or MEM-Nic, following exposure to porcine POSs for 2 h and fixed immediately (pulse), or fixed after a further 1-h chase period in the absence of POSs. Surface-bound POSs appear in yellow whereas internalized POSs appear in red. (B) Bar graph showing quantification of POSs. Data were normalized relative to the total number of POSs (bound and ingested) after the pulse (n = 7 fields of view, aggregated from separate experiments). The relative number of ingested POSs (i.e. POS phagosomes) after the pulse (n = 4) and the total number of POSs after the chase (n = 7) are also shown. Scale bar, 10 μm. Error bars in (B) represent ± S.E.M. Each field of view (n) contained ≥ 100 cells.

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