Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells.

ABSTRACT: Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are leading candidate cells as raw materials for cell therapy products, because of their capacity for pluripotent differentiation and unlimited self-renewal. hPSC-derived products have already entered the scope of clinical application. However, the assessment and control of their tumorigenicity remains to be a critical challenge. Sensitive detection of the pluripotent cellular impurities is necessary for the safety and quality control of the hPSC-derived products. In the present study, we established a sensitive assay for detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). The ddPCR method with a probe and primers for LIN28 significantly detected as low as 0.001% undifferentiated hiPSCs in primary cardiomyocytes, which is equivalent to the ratio of a single hiPSC to 1 × 105 cardiomyocytes. The ddPCR also showed that LIN28 expression is extremely low in human tissues including liver, heart, pancreas, kidney, spinal cord, corneal epithelium and lung. These results suggest that the ddPCR method targeting LIN28 transcripts is highly sensitive and useful for the quality assessment of various cell therapy products derived from hPSCs.

SUBMITTER: Kuroda T 

PROVIDER: S-EPMC6581767 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature