A highly efficient culture method for growth and detection of undifferentiated human pluripotent stem cells present as impurities in cell-processed therapeutic products

      Human pluripotent stem cells (hPSCs), i.e. induced pluripotent stem cells (hiPSCs) and embryonic stem cells, have properties of indefinite proliferation and pluripotency, offering the possibility of renewable sources of various types of cells for production of cell-processed therapeutic products (CTPs). For the clinical application of CTPs derived from hPSCs, quality assessment is critical to ensure their safety and efficacy. The presence of residual undifferentiated hPSCs is one of concerned quality issues associated with tumorigencity of hPSCs-derived CTPs. Therefore, simple, quantitative and highly sensitive methods are necessary to detect a small amount of undifferentiated hPSCs present as impurities in hPSC-derived CTPs. In the present study, we developed a novel method for detection of undifferentiated hiPSCs by amplification using a combination of an extracellular matrix component and a defined xeno-free media. The new culture system allowed robust proliferation of hiPSCs dissociated into single cells without apoptosis, whereas the dissociated hiPSCs easily underwent apoptosis under the conventional culture condition. We next spiked dissociated hiPSCs into primary human somatic cells and examined whether our culture system is applicable to detection of undifferentiated hPSCs in CTPs. As an example of the somatic cells, we employed human mesenchymal stem cells (hMSCs), because “off-the-shelf” hMSCs derived from hPSCs are one of promising CTPs. Our new culture system detected as low as 0.001% hiPSCs, which were spiked into hMSCs, as hiPSC colonies within a week. These results suggest that our culture system is useful for sensitive and quantitative detection of residual undifferentiated hPSCs in CTPs and contributes to the quality control of hPSC-derived products during manufacturing processes.
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