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RNA-Seq analysis of the retinal pigment epithelial cell line, ARPE19, during a time course of serum deprivation.

ABSTRACT: ARPE19 cells, derived from human retinal pigment epithelium (RPE), were cultured in serum supplemented media, then the serum was removed, and culture continued in serum free media. We use this culture system as a model for the early stages of Age-related macular degeneration. RNA-Seq was performed on RNAs from seven time points: day0 (serum supplemented), day1, 3, 4, 5, 6, and 9 serum deprived samples.

ORGANISM(S): Homo sapiens

PROVIDER: GSE129964 | GEO | 2020/05/13

REPOSITORIES: GEO

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Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Keywords: Tissue interactions, retina, retinal pigment epithelium, blood-retinal barrier, tight junctions

Project description:The therapeutic potential of pluripotent stem cells is great as they promise to Q7 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 Q8 this technology as it lends itself to in vivo monitoring and sophisticated 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 dozens of eye diseases. However, after transplantation, they 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 iPSC-RPE with adult primary RPE and the immortalized ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and adult human RPE for one month, evaluating morphology, RPE marker staining, and transepithelial electrical resistance as quality control indicators. We then isolated RNA for bulk RNAsequencing and DNA for genotyping. We genotyped adult RPE 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 more than ARPE-19. In addition, in iPSC-RPE, gene networks relating to epithelial to mesenchymal transition (EMT), stem cell maintenance, retina development, and muscle contraction were significantly elevated compared to those of adult RPE. We compared adult human RPE to iPSC-RPE in a model of 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.

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RNA-Seq analysis of the retinal pigment epithelial cell line, ARPE19, during a time course of serum deprivation.

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