Dataset Information

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods.

ABSTRACT:

Purpose

The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE.

Methods

This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function.

Results

We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE.

Conclusions

The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.

SUBMITTER: Leach LL

PROVIDER: S-EPMC5911695 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Publications

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods.

Leach Lyndsay L LL Croze Roxanne H RH Hu Qirui Q Nadar Vignesh P VP Clevenger Tracy N TN Pennington Britney O BO Gamm David M DM Clegg Dennis O DO

Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics 20160516 5

<h4>Purpose</h4>The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE.<h4>Methods</h4>This study provides a comparison between 2 different methods for RPE diff ...[more]

PMID: 27182743

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Project description:The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE-derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting "immature" gene regulatory networks.

Dataset Information

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods.

Purpose

Methods

Results

Conclusions

Publications

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods.

Similar Datasets

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