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Steven J Fliesler, Elizabeth D. Au, Lara A. Skelton, Sriganesh Ramachandra Rao, Michael H Farkas; Comparative RNA-Seq analysis of iPSC-derived human RPE cells from normal and Smith-Lemli-Opitz Syndrome (SLOS) patients. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4586.
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We previously reported defective phagosome maturation in iPSC-derived RPE cells from SLOS patients, relative to controls (Ramachandra Rao et al., ARVO 2016). Here, we performed RNA-Seq analysis on iPSC-derived RPE cells from normal vs. SLOS human donors, to further validate these cells for in vitro studies.
iPSCs from “mildly” (CWI) and “severely” (A2) affected SLOS patients (F.D. Porter, NICHD/NIH) and a normal neonatal human donor (Dys0100; ATCC) were converted to RPE cells (Ferrer M et al., 2014). Cells were grown to confluence and maintained in culture for 2.5 mo. Total RNA was isolated (N=6 replicates per RPE line); RNA-Seq libraries were prepared and sequenced (Illumina HiSeq 2500 System). ≥70 million raw reads per sample were generated; these were aligned to the hg38 human genome build using STAR, and gene/isoform quantification was performed using RSubread and the Gencode v24 database. Differential expression was determined using DESeq (threshold: fold-change ≥ 2, adjusted p<0.01). Downstream data processing was performed using custom R or Perl scripts.
>60 million reads were aligned per sample. Isoform expression quantification revealed 28,688, 31,976, and 33,291 isoforms were expressed in the Dys0100, A2-SLOS, and CWI-SLOS RPE cells, respectively (~25% of all annotated transcripts). Intra-sample concordance: 0.96-0.99; inter-sample concordance: A2-SLOS vs. Dys0100 RPE (0.90); CWI-SLOS vs. Dys0100 RPE (0.78); A2-SLOS vs. CWI-SLOS RPE (0.65). Out of 86 RPE markers analyzed, A2-SLOS RPE expressed 82, while both CWI-SLOS and Dys0100 RPE expressed 85. Intra-sample RPE marker concordance: A2-SLOS RPE (0.98); CWI-SLOS RPE (0.99); Dys0100 RPE (0.72). Unexpectedly, inter-sample concordance for these RPE markers was high between the CWI-SLOS vs. Dys0100 RPE cells (0.92), but was extremely low between A2-SLOS vs. Dys0100 (0.13) and A2-SLOS vs. CWI-SLOS (0.05) cells. Differential expression analysis (# dysregulated isoforms): A2-SLOS vs. Dys0100 RPE (16,151); CWI-SLOS vs. Dys0100 RPE (10,443); A2SLOS vs. CWI-SLOS RPE (14,082).
All three iPSC-derived RPE lines exhibited gene expression patterns characteristic of RPE cells. CWI (mild) and A2 (severe) SLOS RPE lines exhibited dysregulated gene expression with respect to control (Dys0100) RPE cells and to each other, consistent with disease severity.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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