In vitro models of Retinal Pigment Epithelium: a brief literature review
The Retinal Pigment Epithelium (RPE) is a monolayer of pigmented cells directly abutting the photoreceptors of the retina. It plays many important roles in vision and in maintaining the health and integrity of the retina (Strauss, 2005). Different in vitro models of human RPE have been developped over the past decades : fetal RPE (Song and Lui, 1990), immortalized RPE cell lines such as ARPE19 (Dunn et al., 1996), and more recently RPE differentiated from human induced pluripotent stem cells (hiPSC) (Buchholz et al., 2009; Osakada etal., 2009).This brief review details how hiPSC-RPE and ARPE19 compare to fetal RPE (fRPE)in terms of morphology, marker expression and functions.
Morphology of Retinal Pigment Epithelium
Within a few weeks of reaching confluence, hiPSC-RPE exhibit a characteristic polygonal and pigmented morphology, similar to fRPE (Buchholz et al., 2009; Gamm et al.,2008). Under standard culture conditions, ARPE19 cells typically show a less regular morphology and absence of pigmentation (for passages commercially available),while the use of specific media and long term culture -2-6 months- can lead to a more polygonal and pigmented morphology (Ahmado et al., 2011; Luo et al.,2006).
Ultrastructurally, hiPSC-RPE and fRPE display numerous apical microvilli and melanin containing melanosomes (Carr et al., 2009; Maminishkis etal., 2006). Apical microvilli are also observed in ARPE19 cells, while melanin granules aremuch less prevalent (Dunn et al., 1996).
RNA and protein expression profile of Retinal Pigment Epithelium
Many markers specific of RPE key functions are similarly expressed in fRPE and hiPSC-RPE, while differentially expressed in ARPE19. Among 154 native RPE signature genes (Strunnikova et al., 2010),43 of them were down-regulated in ARPE19 against 3 to 5 of them in several independent hiPSC-RPE lines (Kamao et al., 2014). Genes expressed at reduced levels in ARPE19 include key RPE markers such as Tyrosinase, PMEL17, RPE65, RLBP1 and BEST1 (Ablonczy et al., 2011; Kamao et al.,2014; Klimanskaya et al., 2004). Further transcriptomic studies show grouping of hiPSC-RPE together with fRPE, while ARPE19 are on a different cluster (Kamao et al., 2014; Smith et al.,2019). In addition, the metabolome of fRPE and hiPSC-RPE are closely related (Krohne et al., 2012)
Function of Retinal Pigment Epithelium
Tight junctions betwen RPE cells allow for the formation of an epithelial barrier, whose transepithelial resistance (TER) can be measured. While the TER of ARPE19 is comprised between 50 and 90 Ω/cm2 after 4 to 6 weeks in culture (Dunn et al., 1996), fRPE monolayers usually reach about 250-500 Ω/cm2(Ablonczy et al., 2011; Maminishkiset al., 2006), similar to hiPSC-RPE (Brandl et al., 2014; Kamao et al.,2014).
Daily clearance of shed photoreceptorouter segments (POS) is a key task performed by RPE in vivo (Strauss, 2005). All three RPE in vitro models are able to phagocytose POS (Buchholz et al., 2009; Lin andClegg, 1998; Mukherjee et al., 2007). However, hiPSC-RPE and fRPE display more similar dynamics of POS binding and internalization compared to ARPE19 (Westenskow et al., 2012).
Growth Factor secretion
In vivo, RPE cells secrete two main growth factors : PEDF, to the apical side, and VEGF to the basal side. This polarized pattern of secretion is observed in vitro in fRPE (Maminishkis et al., 2006) and hiPSC-RPE (Kokkinaki et al., 2011; Maruotti etal., 2013). In contrast, in ARPE19, VEGF secretion in standard culture conditions is mostly directed toward the apical side (Ablonczy et al., 2011; Ahmado etal., 2011), while PEDF secretion is correctly polarized.
Photoreceptors lack cis-transisomerase function, and are therefore unable to regenerate all-trans-retinal into 11-cis-retinal, after biological conversion of a photon into an electric signal (Baehr et al., 2003). The reisomerization of all-trans-retinal into11-cis-retinal is performed by the RPE. Because ARPE19 have low or absent expression of key proteins involved in retinoid recycling, such as RPE65 and RLBP1, they are unable to correctly convert all-trans-retinal into 11-cis-retinal (Ablonczy et al., 2011; Maeda et al.,2013). On the contrary, fRPE and hiPSC-RPE have a functional visual cycle andreisomerize all-trans-retinal into 11-cis-retinal (Ablonczy et al., 2011; Maeda et al.,2013; Muniz et al., 2014).
hiPSC-RPE cells display a response similar to fRPE when treated with cytotoxic agent such as recombinant tissue plasminogen activator (rtPA), while ARPE19 cell line shows a tolerance to higher concentration of rtPA, making it less predictive of cytotoxic effects (Kamao et al., 2019).
The availability and ease of culture of immortalized RPE cell lines, such as ARPE19, have made them useful tools to modelize some aspects of RPE biology. However, immortalized RPE lines differ significantly from cultured fRPE when considering morphology, marker expression and, more importantly, key RPE functions. With the recent advances in stem cell technology, a new model of human RPE has become available : hiPSC-RPE not only closely ressemble fRPE in terms of morphology, marker expression and functionality, they also represent a cost-effective alternative available in virtually unlimited quantity.
Genetic disease study
Comparison table of RPE models: Fetal RPE, ARPE19 & hiPCS-RPE
Carr,A.J., Vugler, A.A., Hikita, S.T., Lawrence, J.M., Gias, C., Chen, L.L.,Buchholz, D.E., Ahmado, A., Semo, M., Smart, M.J., Hasan, S., da Cruz, L.,Johnson, L.V., Clegg, D.O., Coffey, P.J., 2009. Protective effects of humaniPS-derived retinal pigment epithelium cell transplantation in the retinaldystrophic rat. PLoS One 4, e8152.
Dunn,K.C., Aotaki-Keen, A.E., Putkey, F.R., Hjelmeland, L.M., 1996. ARPE-19, a humanretinal pigment epithelial cell line with differentiated properties. Exp EyeRes 62, 155-169.
Gamm,D.M., Melvan, J.N., Shearer, R.L., Pinilla, I., Sabat, G., Svendsen, C.N.,Wright, L.S., 2008. A novel serum-free method for culturing human prenatalretinal pigment epithelial cells. Invest Ophthalmol Vis Sci 49, 788-799.
Kamao,H., Mandai, M., Okamoto, S., Sakai, N., Suga, A., Sugita, S., Kiryu, J.,Takahashi, M., 2014. Characterization of human induced pluripotent stemcell-derived retinal pigment epithelium cell sheets aiming for clinicalapplication. Stem Cell Reports 2, 205-218.
Kamao,H., Miki, A., Kiryu, J., 2019. Evaluation of Retinal Pigment Epithelial CellCytotoxicity of Recombinant Tissue Plasminogen Activator Using Human-InducedPluripotent Stem Cells. J Ophthalmol 2019, 7189241.
Klimanskaya,I., Hipp, J., Rezai, K.A., West, M., Atala, A., Lanza, R., 2004. Derivation andcomparative assessment of retinal pigment epithelium from human embryonic stemcells using transcriptomics. Cloning Stem Cells 6, 217-245.
Kokkinaki,M., Sahibzada, N., Golestaneh, N., 2011. Human iPS-Derived Retinal PigmentEpithelium (RPE) Cells Exhibit Ion Transport, Membrane Potential, PolarizedVEGF Secretion and Gene Expression Pattern Similar to Native RPE. Stem Cells29, 825-835.
Krohne,T.U., Westenskow, P.D., Kurihara, T., Friedlander, D.F., Lehmann, M., Dorsey,A.L., Li, W., Zhu, S., Schultz, A., Wang, J., Siuzdak, G., Ding, S.,Friedlander, M., 2012. Generation of retinal pigment epithelial cells fromsmall molecules and OCT4 reprogrammed human induced pluripotent stem cells.Stem Cells Transl Med 1, 96-109.
Lin, H.,Clegg, D.O., 1998. Integrin alphavbeta5 participates in the binding ofphotoreceptor rod outer segments during phagocytosis by cultured human retinalpigment epithelium. Invest Ophthalmol Vis Sci 39, 1703-1712.
Luo,Y., Fukuhara, M., Weitzman, M., Rizzolo, L.J., 2006. Expression of JAM-A, AF-6,PAR-3 and PAR-6 during the assembly and remodeling of RPE tight junctions.Brain Res 1110, 55-63.
Maeda,T., Lee, M.J., Palczewska, G., Marsili, S., Tesar, P.J., Palczewski, K.,Takahashi, M., Maeda, A., 2013. Retinal pigmented epithelial cells obtainedfrom human induced pluripotent stem cells possess functional visual cycleenzymes in vitro and in vivo. J Biol Chem 288, 34484-34493.
Maminishkis,A., Chen, S., Jalickee, S., Banzon, T., Shi, G., Wang, F.E., Ehalt, T., Hammer,J.A., Miller, S.S., 2006. Confluent monolayers of cultured human fetal retinalpigment epithelium exhibit morphology and physiology of native tissue. InvestOphthalmol Vis Sci 47, 3612-3624.
Maruotti,J., Wahlin, K., Gorrell, D., Bhutto, I., Lutty, G., Zack, D.J., 2013. A simpleand scalable process for the differentiation of retinal pigment epithelium fromhuman pluripotent stem cells. Stem Cells Transl Med 2, 341-354.
Mukherjee,P.K., Marcheselli, V.L., de Rivero Vaccari, J.C., Gordon, W.C., Jackson, F.E.,Bazan, N.G., 2007. Photoreceptor outer segment phagocytosis attenuatesoxidative stress-induced apoptosis with concomitant neuroprotectin D1synthesis. Proc Natl Acad Sci U S A 104, 13158-13163.
Muniz,A., Greene, W.A., Plamper, M.L., Choi, J.H., Johnson, A.J., Tsin, A.T., Wang,H.C., 2014. Retinoid uptake, processing, and secretion in human iPS-RPE supportthe visual cycle. Invest Ophthalmol Vis Sci 55, 198-209.
Osakada,F., Jin, Z.B., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y.,Takahashi, M., 2009. In vitro differentiation of retinal cells from humanpluripotent stem cells by small-molecule induction. J Cell Sci 122, 3169-3179.
Smith,E.N., D'Antonio-Chronowska, A., Greenwald, W.W., Borja, V., Aguiar, L.R.,Pogue, R., Matsui, H., Benaglio, P., Borooah, S., D'Antonio, M., Ayyagari, R.,Frazer, K.A., 2019. Human iPSC-Derived Retinal Pigment Epithelium: A ModelSystem for Prioritizing and Functionally Characterizing Causal Variants at AMDRisk Loci. Stem Cell Reports 12, 1342-1353.
Song,M.K., Lui, G.M., 1990. Propagation of fetal human RPE cells: preservation oforiginal culture morphology after serial passage. J Cell Physiol 143, 196-203.
Strauss,O., 2005. The retinal pigment epithelium in visual function. Physiol Rev 85,845-881.
Strunnikova,N.V., Maminishkis, A., Barb, J.J., Wang, F., Zhi, C., Sergeev, Y., Chen, W.,Edwards, A.O., Stambolian, D., Abecasis, G., Swaroop, A., Munson, P.J., Miller,S.S., 2010. Transcriptome analysis and molecular signature of human retinalpigment epithelium. Hum Mol Genet 19, 2468-2486.
Westenskow, P.D., Moreno, S.K.,Krohne, T.U., Kurihara, T., Zhu, S., Zhang, Z.N., Zhao, T., Xu, Y., Ding, S.,Friedlander, M., 2012. Using Flow Cytometry to Compare the Dynamics ofPhotoreceptor Outer Segment Phagocytosis in iPS-Derived RPE Cells. InvestOphthalmol Vis Sci 53, 6282-6290.