Detection of Residual iPSCs Following Differentiation of iPSC-Derived Retinal Pigment Epithelial Cells

<b><i>Purpose:</i></b> The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. <b><i>Methods:</i></b> RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. <b><i>Results:</i></b> <i>ZSCAN10</i> and <i>LIN28A</i> were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs <i>ZSCAN10</i> and <i>LIN28A</i> were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of <i>ZSCAN10</i> and <i>LIN28A</i> in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, <i>ZSCAN10</i>, and <i>LIN28A</i> expression in iPSCs was generally uniform. The LOD for <i>ZSCAN10</i> and <i>LIN28A</i> in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of ΔΔC_t found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC/10,000 RPE cells in both iPSC lines. <b><i>Conclusions:</i></b> qPCR for <i>ZSCAN10</i> and <i>LIN28A</i> detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.