Abstract
Highly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.
Highlights
Sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells
We have developed several methods for detecting a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) in cell-processed therapeutic products (CTPs), such as flow cytometry assay for tumor rejection antigens (TRA)-1-6010, droplet digital polymerase chain reaction (PCR) assays for LIN28A11, and the highly efficient culture (HEC) assay using a cell culture system optimized for hiPSCs12
Consistent with the levels of iCasp[9] expression, when the transduced cells were treated with AP1903, a reagent that induces apoptosis via the dimerization of iCasp[9], the number of Ad vector-transduced cells decreased to less than 10% of the number of untreated cells (P < 0.005, two-way analysis of variance (ANOVA) and Student–Newman–Keuls (SNK) post-hoc test), whereas the numbers of cells transduced with the four AAV vectors were not decreased (Fig. 1B)
Summary
Sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adenoassociated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In upcoming clinical trials to treat SCI, 2.0 × 1 06 hiPSC-NPCs are expected to be transplanted into patients[13]; undifferentiated hiPSCs present in the transplanted cells could remain undetected by the aforementioned test methods To overcome their limitation in sensitivity, we constructed adenovirus serotype 5 (Ad) and adenoassociated virus serotype 1, 2, 5, and 6 (AAV1, AAV2, AAV5, and AAV6, respectively) vectors expressing a suicide gene, inducible Caspase 9 (iCasp9), under the control of the cytomegalovirus (CMV) promoter, which is dormant in iPSCs14,15. We investigated whether these vectors are applicable to the concentration and sensitive detection of hiPSCs intermingled in cultures of hiPSC-NPCs
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