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Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells.

ABSTRACT: Transplantation of retinal pigment epithelial (RPE) sheets derived from human induced pluripotent cells (hiPSC) is a promising cell therapy for RPE degeneration, such as in age-related macular degeneration. Current RPE replacement therapies, however, face major challenges. They require a tedious manual process of selecting differentiated RPE from hiPSC-derived cells, and despite wide variation in quality of RPE sheets, there exists no efficient process for distinguishing functional RPE sheets from those unsuitable for transplantation. To overcome these issues, we developed methods for the generation of RPE sheets from hiPSC, and image-based evaluation. We found that stepwise treatment with six signaling pathway inhibitors along with nicotinamide increased RPE differentiation efficiency (RPE6iN), enabling the RPE sheet generation at high purity without manual selection. Machine learning models were developed based on cellular morphological features of F-actin-labeled RPE images for predicting transepithelial electrical resistance values, an indicator of RPE sheet function. Our model was effective at identifying low-quality RPE sheets for elimination, even when using label-free images. The RPE6iN-based RPE sheet generation combined with the non-destructive image-based prediction offers a comprehensive new solution for the large-scale production of pure RPE sheets with lot-to-lot variations and should facilitate the further development of RPE replacement therapies.

SUBMITTER: Ye K

PROVIDER: S-EPMC7462996 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells.

Ye Ke K Takemoto Yuto Y Ito Arisa A Onda Masanari M Morimoto Nao N Mandai Michiko M Takahashi Masayo M Kato Ryuji R Osakada Fumitaka F

Scientific reports 20200901 1

Transplantation of retinal pigment epithelial (RPE) sheets derived from human induced pluripotent cells (hiPSC) is a promising cell therapy for RPE degeneration, such as in age-related macular degeneration. Current RPE replacement therapies, however, face major challenges. They require a tedious manual process of selecting differentiated RPE from hiPSC-derived cells, and despite wide variation in quality of RPE sheets, there exists no efficient process for distinguishing functional RPE sheets fr ...[more]

PMID: 32873827

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Project description:Background Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated. Methods A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes. Results In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix. Conclusions This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02651-9.

Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Project description:: Induced pluripotent stem cells (iPSCs) can be efficiently differentiated into retinal pigment epithelium (RPE), offering the possibility of autologous cell replacement therapy for retinal degeneration stemming from RPE loss. The generation and maintenance of epithelial apical-basolateral polarity is fundamental for iPSC-derived RPE (iPSC-RPE) to recapitulate native RPE structure and function. Presently, no criteria have been established to determine clonal or donor based heterogeneity in the polarization and maturation state of iPSC-RPE. We provide an unbiased structural, molecular, and physiological evaluation of 15 iPSC-RPE that have been derived from distinct tissues from several different donors. We assessed the intact RPE monolayer in terms of an ATP-dependent signaling pathway that drives critical aspects of RPE function, including calcium and electrophysiological responses, as well as steady-state fluid transport. These responses have key in vivo counterparts that together help determine the homeostasis of the distal retina. We characterized the donor and clonal variation and found that iPSC-RPE function was more significantly affected by the genetic differences between different donors than the epigenetic differences associated with different starting tissues. This study provides a reference dataset to authenticate genetically diverse iPSC-RPE derived for clinical applications. SIGNIFICANCE:The retinal pigment epithelium (RPE) is essential for maintaining visual function. RPE derived from human induced pluripotent stem cells (iPSC-RPE) offer a promising cell-based transplantation therapy for slowing or rescuing RPE-induced visual function loss. For effective treatment, iPSC-RPE must recapitulate the physiology of native human RPE. A set of physiologically relevant functional assays are provided that assess the polarized functional activity and maturation state of the intact RPE monolayer. The present data show that donor-to-donor variability exceeds the tissue-to-tissue variability for a given donor and provides, for the first time, criteria necessary to identify iPSC-RPE most suitable for clinical application.

Dataset Information

Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells.

Publications

Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells.

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