Human iPSC-derived retinal pigment epithelium: a model system for identifying and functionally characterizing causal variants at AMD risk loci

Smith, Erin N.; D’Antonio‐Chronowska, Agnieszka; Greenwald, William W.; Borja, Victor; Aguiar, Lana Ribeiro; Pogue, Robert; Matsui, Hiroko; Borooah, Shyamanga; D’Antonio, Matteo; Ayyagari, Radha; Frazer, Kelly A.

doi:10.1101/440230

2018

DOI: 10.1101/440230

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Human iPSC-derived retinal pigment epithelium: a model system for identifying and functionally characterizing causal variants at AMD risk loci

Erin N. Smith

Agnieszka D’Antonio‐Chronowska

William W. Greenwald

et al.

Abstract: We evaluate whether human induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells can be used to prioritize and functionally characterize causal variants at age-related macular degeneration (AMD) risk loci. We generated iPSC-RPE from six subjects and show that they have morphological and molecular characteristics similar to native RPE. We generated RNA-seq, ATAC-seq, and H3K27acChIP-seq data and observe high similarity in gene expression and enriched transcription factor motif profile… Show more

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“…Corning ® Matrigel ® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230) 14. mTeSR TM 1 (Stem Cell Technologies, catalog number: 85850) 15. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330057) 16.…”

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confidence: 99%

“…DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330-057) 14. Accutase (Innovative Cell Technologies, Inc., catalog number: AT 104) 15. Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061) 16.…”

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confidence: 99%

“…50 ml conical tubes (Bio Pioneer, catalog number: CNT-50R) 14. Nalgene Cryogenic vials (Thermo Fisher Scientific, catalog number: 5000-1020) 15 e. Wipe off excess water from the vial, spray with 70% ethanol before placing in the hood. f. Remove thawed cells from the vial and add gently into 9 ml mTeSR containing 10 μM ROCK Inhibitor in a conical tube.…”

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In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE)

D’Antonio‐Chronowska¹,

D’Antonio²,

Frazer³

2019

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Induced Pluripotent Stem Cells (iPSCs) serve as an excellent model system for studying the molecular underpinnings of tissue development. Human iPSC-derived retinal pigment epithelium (iPSC-RPE) cells have fetal-like molecular profiles. Hence, biobanks like iPSCORE, which contain iPSCs generated from hundreds of individuals, are an invaluable resource for examining how common genetic variants exert their effects during RPE development resulting in individuals having different propensities to develop Age-related Macular Degeneration (AMD) as adults. Here, we present an optimized, costeffective and highly reproducible protocol for derivation of human iPSC-RPE cells using small molecules under serum-free condition and for their quality control using flow cytometry and immunofluorescence.While most previous protocols have required laborious manual selection to enrich for iPSC-RPE cells, our protocol uses whole culture passaging and yields a large number of iPSC-RPE cells with high purity (88-98.1% ZO-1 and MiTF double positive cells). The simplicity and robustness of this protocol would enable its adaption for high-throughput applications involving the generation of iPSC-RPE samples from hundreds of individuals. Keywords: Human induced pluripotent stem (hiPSC), Retinal pigment epithelium (RPE), Human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE), Age-related macular degeneration (AMD), Differentiation, Genetic studies, Small molecules, Genetic variant [Background] Age-related macular degeneration (AMD) is a leading cause of vision loss in developed countries affecting 11 million individuals in the United States and about 170 million worldwide (Pennington and DeAngelis, 2016). Moreover, considering age as a main factor, in our current aging society, the incidence of AMD is estimated to increase to 198 million in 2020 and 288 million by 2040 and to 22 million in the United States alone by 2050 (Wong et al., 2014, Pennington andDeAngelis, 2016). Current therapeutic strategies, although effective, are expensive and limited to delaying the speed of disease progression, and AMD still eventually leads to a complete loss of vision (Al-Zamil and Yassin, 2017;Mitchell et al., 2018). At the time of preparation of this article, there are seven active clinical trials aimed to evaluate the effectiveness and optimize the conditions of the transplantation of the human iPSC-RPEs or human embryonic stem cell-derived retinal pigment epithelium (ESC-RPE) cells (NIH-ClinicalTrials.gov). Thus, the development of a robust and cost-effective method for generating large amounts of high quality iPSC-RPEs is imperative for the advancement of future therapeutic treatments of AMD and potentially other eye diseases in humans as well as domestic animals (Sparrow et al., 2010).

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In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE)

D’Antonio‐Chronowska¹,

D’Antonio²,

Frazer³

2019

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Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting

et al. 2020

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Background: Despite increasing demand, current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time, labor, and cost intensive. Additionally, absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. Methods: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained, cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy, polarized VEGF expression, establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes, RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. Results: Using our 2D cytokine scarce protocol, hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless, at this stage, most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured, DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. Conclusions: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable, and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications.

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Human iPSC-derived retinal pigment epithelium: a model system for identifying and functionally characterizing causal variants at AMD risk loci

Cited by 2 publications

References 44 publications

In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE)

In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE)

Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting

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