The study of disease onset and progression in human is limited by the fact that patients are often symptomatic only at the end stage disease and there is limited access to patient material. iPS cell technology provides a possibility to develop in vitro disease models using patient-specific iPS cells, study disease onset and progression, and identify potential therapeutic intervention. We have focused our attention on BEST disease, a monogenic RPE-specific disease caused by mutations in gene VMD2. Patients show characteristic phenotypes such as reduced light peak response and accumulation of sub-retinal deposits. Over time, the photoreceptor and RPE layers detach leading to the death of photoreceptors and vision loss in these patients. Although, it is clear that disease is caused by mutation in the gene VMD2 that encodes for an anion channel BESTROPHIN 1 expressed in the RPE, the exact mechanism leading to disease pathogenesis is not well understood. We have developed iPS cell lines from families (affected and unaffected siblings) with BEST disease and are now using RPE derived from those iPS cells to study disease pathogenesis in vitro. In addition, we have developed methods to culture iPS cell derived RPE cells in 96-well and 384-well microtiter plates to optimize high throughput screening assays. RPE cells were transferred to 384-well plates at committed and immature RPE stages. Our results show that these cells continue to differentiate and mature in these plates and attain RPE phenotype. We have also successfully optimized a multiplex gene expression assay that monitors the differentiation stage of iPS cell derived RPE cells. In this assay, antisense oligonucleotide probes labeled on fluorescent magnetic beads are used to pull down specific mRNAs. Using another set of antisense oligonucleotide, a detection label is attached to that mRNA. The bead, mRNA, label complex is detected by flow cytometry. In our assay, we simultaneously used probes against eight different genes (SOX2, PAX6, TYR, BEST1, RPE65, RDH5, CSPG5, and TRPM1). Expression of these eight genes was compared between primary human RPE and iPS cell derived RPE at two stages of differentiation. We were able to detect the expression of all these genes in iPS cell derived RPE grown in 384-well plates. As expected, as compared to the primary RPE, the expression of progenitor genes was higher in iPS cell derived RPE at both differentiation stages and the expression of late-stage RPE-specific genes was much lower. Furthermore, for all these genes we were also able to detect a difference in expression between these two stages of iPS cell to RPE differentiation. Our results suggest that this assay can be used to identify compounds that improve RPE maturation in vitro. In addition, this assay provides the possibility of identifying potential therapeutic drugs that work by modulating the endogenous expression of genes of interest.
