MERTK-Dependent Ensheathment of Photoreceptor Outer Segments by Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

Almedawar, Seba; Vafia, Katerina; Schreiter, Sven; Neumann, Katrin; Khattak, Shahryar; Kurth, Thomas; Ader, Marius; Karl, Mike O.; Tsang, Stephen H.; Tanaka, Elly M.

doi:10.1016/j.stemcr.2020.02.004

Cited by 21 publications

(31 citation statements)

References 29 publications

(39 reference statements)

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“…binding of POS and subsequent phagocytosis through CD36 and MERTK [3,[6][7][8][9]. Our results also fit with gene expression profiles that have been published for ARPE19 cells [101], fetal and adult human RPE cells [102,103] as well as stem cell-derived RPE cells [104,105].…”

Section: Discussionsupporting

confidence: 89%

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

Heller

Heintz

Kwok

et al. 2020

Preprint

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Retinal pigment epithelial (RPE) cells have been used in disease modelling and transplantation studies in retinal diseases. Several types of RPE cells have been trialed, ranging from primary cells and immortalized cell lines to stem cell-derived RPE cells. During aging and in disease, the extracellular environment of the RPE cells changes, interfering with RPE cell adhesion. We hypothesize that this could be a key problem in transplantation studies that have reported lack of adhesion and survival of the transplanted RPE cells. Integrins are essential for the proper function of the RPE, mediating adhesion to Bruch's membrane, and the binding and subsequent phagocytosis of photoreceptor outer segments. Variability that has been found in clinical trials might be due to the variability of cell types used and their expression profiles of surface molecules. Here, we set out to analyze integrin expression in primary rat RPE cells and in the human cell line ARPE-19 using immunochemistry. We found that both cell types express integrins to varying degrees. After long-term culturing, ARPE-19 cells resemble mature RPE cells, and increase integrin expression. We hence argue that it is important to test the properties of these cells prior to transplantation to avoid failure of adhesion and to facilitate correct function.

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Section: Discussionsupporting

confidence: 89%

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

Heller

Heintz

Kwok

et al. 2020

Preprint

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“…We developed a miniaturized POS phagocytosis assay suitable for high-throughput screening, based on RPE cells derived from human embryonic stem cells (hESCs) cultured in 384-well plates, and we checked their quality and relevant functionality. We observed that RPE cells express mature RPE markers in a polarized fashion (Figure 1A) similar to what we previously observed in the more physiological transwell culture plate format (Almedawar et al, 2020). We further sonicated the POS, as previously described (Almedawar et al, 2020), to obtain a more homogeneous POS size, which facilitates image analysis ( Figures 1B and 1C).…”

Section: Human Rpe In a Miniaturized 384-well Plate Assay Are Functiosupporting

confidence: 79%

“…We observed that RPE cells express mature RPE markers in a polarized fashion (Figure 1A) similar to what we previously observed in the more physiological transwell culture plate format (Almedawar et al, 2020). We further sonicated the POS, as previously described (Almedawar et al, 2020), to obtain a more homogeneous POS size, which facilitates image analysis ( Figures 1B and 1C). Then, we titrated the POS by seeding them on RPE cells for 3 h based on our findings in Almedawar et al (2020).…”

Section: Human Rpe In a Miniaturized 384-well Plate Assay Are Functiosupporting

confidence: 79%

“…Thus, any dysfunction in POS phagocytosis leads to vision loss (Strauss, 2005). Indeed, dysfunctional phagocytosis has been described in both acquired age-related macular degeneration (AMD) (Golestaneh et al, 2017;Inana et al, 2018) and genetically inherited retinal degenerative diseases, including retinitis pigmentosa (RP) caused by mutations in Mer receptor tyrosine kinase (MERTK) (Almedawar et al, 2020;Niemann et al, 2000) and bestrophinopathies (Zorych et al, 2017). Thus, enhancing POS phagocytosis in the RPE might be of therapeutic relevance to many retinal degenerative diseases, which can be applied as a standalone therapy or in combination with gene-and cell-based therapies to enhance their outcomes.…”

Section: Introductionmentioning

confidence: 99%

“…During the initial steps of phagocytosis, POS expose phosphatidylserines (PSs) on their outer membranes, similar to apoptotic cells (Finnemann and Silverstein, 2001). RPE api-cal membrane, containing actin-rich ensheathing membranes, recognizes the so-called ''eat-me'' signals presented on the membrane of POS through bridging opsonins such as milk fat globule-EGF8 (MFGE8) and growth arrest-specific protein 6 (GAS6), which bind to their respective RPE membrane receptors aVb5 integrin (Nandrot et al, 2007) and MERTK (Hall et al, 2001;Law et al, 2015), initiating POS fragmentation and internalization (Almedawar et al, 2020). Following internalization, POS undergo lysosomal degradation followed by recycling of some of the components such as fatty acids stemming from docosahexaenoic acid (DHA) and retinoic acid back to the photoreceptors (Strauss, 2005).…”

Section: Introductionmentioning

confidence: 99%

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A Human Retinal Pigment Epithelium-Based Screening Platform Reveals Inducers of Photoreceptor Outer Segments Phagocytosis

et al. 2020

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Phagocytosis is a key function in various cells throughout the body. A deficiency in photoreceptor outer segment (POS) phagocytosis by the retinal pigment epithelium (RPE) causes vision loss in inherited retinal diseases and possibly age-related macular degeneration. To date, there are no effective therapies available aiming at recovering the lost phagocytosis function. Here, we developed a high-throughput screening assay based on RPE derived from human embryonic stem cells (hRPE) to reveal enhancers of POS phagocytosis. One of the hits, ramoplanin (RM), reproducibly enhanced POS phagocytosis and ensheathment in hRPE, and enhanced the expression of proteins known to regulate membrane dynamics and ensheathment in other cell systems. Additionally, RM rescued POS internalization defect in Mer receptor tyrosine kinase (MERTK) mutant hRPE, derived from retinitis pigmentosa patient induced pluripotent stem cells. Our platform, including a primary phenotypic screening phagocytosis assay together with orthogonal assays, establishes a basis for RPE-based therapy discovery aiming at a broad patient spectrum.

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Evaluation of CRISPR/Cas9 exon‐skipping vector for choroideremia using human induced pluripotent stem cell‐derived RPE

Iwagawa

Masumoto²,

Tabuchi

et al. 2022

The Journal of Gene Medicine

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Background Exon‐skipping is a powerful genetic tool, especially when delivering genes using an AAV‐mediated full‐length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9‐based exon‐skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X‐linked inherited retinal disease caused by mutation of the CHM gene. Methods We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon‐skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6‐skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. Results Artificial intelligence‐based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6‐skipping of CHM. Conclusions CHM exon 6‐skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

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MERTK-Dependent Ensheathment of Photoreceptor Outer Segments by Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

Cited by 21 publications

References 29 publications

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

A Human Retinal Pigment Epithelium-Based Screening Platform Reveals Inducers of Photoreceptor Outer Segments Phagocytosis

Evaluation of CRISPR/Cas9 exon‐skipping vector for choroideremia using human induced pluripotent stem cell‐derived RPE

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