Analysis of three characterization assays reveals ddPCR of LIN28A as the most sensitive for the detection of residual pluripotent stem cells in cellular therapy products

With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. Due to limitations in both time and cost, it is impractical to test each batch of PSC-derived products for tumorigenicity using in vivo animal studies. To mitigate these limitations, in vitro analytical methods can be utilised as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared three in vitro assays: qPCR, ddPCR and RT-LAMP using spike-in samples from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68), to determine their detection limit in measuring a commonly used PSC marker, LIN28A. The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts.