Morphological, Biochemical, and Transcriptomic Characterization of Ipsc-Derived Human RPE Cells from Normal and Smith-Lemli- Opitz Syndrome Patients

Purpose: Smith-Lemli-Opitz syndrome (SLOS) is a human recessive disease that affects cellular cholesterol (Chol) synthesis due to mutations in the gene encoding 7-dehydrocholesterol-reductase (DHCR7), resulting in defective reduction of 7-dehydrocholesterol (7-DHC) to Chol. Genetic and pharmacological rodent models of SLOS reveal progressive retinal and retinal pigmented epithelium (RPE) pathology. In this study, we aimed to characterize in vitro models of human RPE SLOS pathology utilizing two SLOS patient-derived induced pluripotent stem cell-derived RPE (iPSC-RPE) cell lines- "mild " SLOS (CWI) and "severe " SLOS (A2)-versus a normal human iPSC-RPE cell line (DYS) and performed comparative transcriptomic, morphological, and biochemical analyses. Methods: iPSC-derived RPE cultures were differentiated and matured in parallel for 2.5 months and monitored by brightfield microscopy. DHCR7 mutations were verified by PCR amplification spanning the target region and sequencing. Total RNA was isolated from replicate cultures, and RNA-Seq libraries were prepared and sequenced. Whole cell lysates were prepared from parallel cultures and subjected to Western blot analysis to assess the expression levels of RPE-specific and differentiation markers. Each RPE cell line was analyzed by confocal microscopic immunohistochemistry to further assess morphology, cell border integrity (zona occludens/tight junction protein-1 (TJP-1) and occludin), and F-actin distribution and organization (fluor-conjugated phalloidin). Sterol profiling (by reverse-phase HPLC analysis) was performed on similarly matured cultures that were switched to delipidated (Chol-free) media for 9 days prior to analysis. Results: RNA-Se q libraries from the RPE cell lines showed a comparable number of total isoforms. Inter-sample concordance was high between A2-SLOS versus Dys0100 RPE (0.90) but was low when comparing CWI-SLOS versus Dys0100 RPE (0.78), or A2-SLOS versus CWI-SLOS RPE (0.65). Out of the 86 RPE markers analyzed, 82 were expressed in A2-SLOS RPE, whereas 85 were expressed in both CWI-SLOS and Dys0100 RPE. Inter-sample concordance for RPE markers was high for CWI-SLOS versus Dys0100 RPE cells (0.92) but low between A2-SLOS versus Dys0100 (0.13) and A2-SLOS versus CWI-SLOS (0.05). Upregulation of transcripts for epithelial-to-mesenchymal transition markers and downregulation of RPE-specific markers were observed in both SLOS iPSC-RPE cell lines, in agreement with Western blot results that revealed elevated expression of smooth muscle actin in SLOS-derived RPE cell lines. CWI and A2 RPE cells exhibited altered monolayer organization, marked stress fiber formation, and aberrant TJP1 and occludin labeling patterns. Sterol analysis demonstrated a significantly elevated 7DHC/Chol mol ratio in A2 RPE cells compared to CWI and DYS RPE cells. Both the A2 and CWI RPE cells were responsive to Chol depletion and exhibited lipid droplet accumulation. Conclusion: Our data demonstrate DHCR7 mutation -dependent accumulation of 7DHC and lipid droplets, as well as altered RPE marker expression in SLOS iPSC-derived RPE cell lines. The degree of the observed abnormalities correlated with disease severity. These cell lines represent a novel biological resource for studies aimed at further understanding the pathobiology of SLOS, particularly as it involves SLOS-associated visual system dysfunction, and for in vitro testing of candidate therapeutic drugs or gene therapy approaches to improve clinical management of SLOS patients10