In Vitro Maturation of Retinal Pigment Epithelium Is Essential for Maintaining High Expression of Key Functional Genes

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialized world. AMD is associated with dysfunction and atrophy of the retinal pigment epithelium (RPE), which provides critical support for photoreceptor survival and function. RPE transplantation is a promising avenue towards a potentially curative treatment for early stage AMD patients, with encouraging reports from animal trials supporting recent progression toward clinical treatments. Mature RPE cells have been reported to be superior, but a detailed investigation of the specific changes in the expression pattern of key RPE genes during maturation is lacking. To understand the effect of maturity on RPE, we investigated transcript levels of 19 key RPE genes using ARPE-19 cell line and human embryonic stem cell-derived RPE cultures. Mature RPE cultures upregulated PEDF, IGF-1, CNTF and BDNF—genes that code for trophic factors known to enhance the survival and function of photoreceptors. Moreover, the mRNA levels of these genes are maximized after 42 days of maturation in culture and lost upon dissociation to single cells. Our findings will help to inform future animal and human RPE transplantation efforts.

Arap1 loss causes retinal pigment epithelium phagocytic dysfunction and subsequent photoreceptor death

“…To ensure that protein expression patterns were similar to those in in vivo RPE, cultures were maintained until they adopted physical characteristics of maturity as defined previously by pigmentation and ‘cobblestone’ hexagonal morphology ( Fig. 5 B-D) ( Adijanto and Philp, 2014 ; Al-Ani et al, 2020 ). Western blot analysis was performed on FRPE lysate, which detected a band at the same molecular mass ( Fig.…”

Scaffold-Free Retinal Pigment Epithelium Microtissues Exhibit Increased Release of PEDF

“…We next sought to characterize RPE-µTs’ signaling potential in more depth by comparisons with conventional monolayer cultures. A group of 21 RPE-specific genes was selected to evaluate the function of our microtissue, and the genes were categorized into secreted factors for choroid stability ( FASL, TIMP3, TGFB, VEGFA and PDGF-AA ), secreted factors for photoreceptor stability ( PEDF, IGF1, BDNF, GAS6 and FGF2 ) and general RPE functional markers ( RPE65, CFH, TRMP1, BEST1, FGF2R, MYRIP, CCL2, IL8, LHX2, LOXL and KDR ) [ 58 ] ( Figure 4 a). When comparing ARPE-19 microtissue to adherent culture, RT-qPCR data indicated upregulation of mRNA levels of PEDF and IGF-1 and downregulation of VEGF and TGF-β ( Figure 4 a).…”

“…As expected, our differentiated RPE cells showed dramatic increases in expression for these genes and a downregulation of the pluripotency gene OCT4 ( Figure 5 b). In addition to adopting a hexagonal morphology, our differentiated RPE had increased pigmentation [ 58 ], as RPE-µT compared to monolayer cultures ( Figure 5 c). Our differentiated RPE cells stained positively and specifically for key RPE markers, including RPE65, LRAT, CRALBP, Melanopsin, Sox9 and Best1, as well as ZO-1, thus suggesting the formation of tight junction ( Figure 5 d).…”

“…To estimate oxygen delivered to cultured cells under the aforementioned experimental conditions, we utilized our previously published method [ 58 , 96 ]. For the purpose of the calculation, we used 42 amol∙cell −1 ∙s −1 as the RPE oxygen consumption rate [ 97 ].…”

“…The resultant cDNA was then used to carry out real-time quantitative reverse transcription PCR (RT-qPCR) to compare gene-expression profiles between an RPE-µT and adherent culture ( Section 2.4 and Section 2.5 ). SYBR Green RT-qPCR was carried out with technical triplicates using 7500 Fast Real-Time PCR System (see Reference [ 58 ] for primer sequences), and analyzed with the 2 −ΔΔCT method [ 98 ], and with stable internal reference gene (CNTF).…”