Highly sensitive and non-disruptive detection of residual undifferentiated cells by measuring miRNAs in culture supernatant

Abstract

The clinical usage of induced pluripotent stem cell (iPSC)-derived regenerative medicine products is limited by the possibility of residual undifferentiated cells forming tumours after transplantation. Most of the existing quality control tests involve crushing of cells. As a result, the cells to be transplanted cannot be directly tested, thereby increasing the cost of transplantation. Therefore, we tested a highly sensitive and non-disruptive quality-testing method that involves measuring microRNAs (miRNAs) in culture supernatants released by cells. By measuring miR-302b in the culture supernatant, residual iPSCs were detected with higher sensitivity than by measuring LIN28 (Lin-28 Homolog A) in the cells. To use this method, we also monitored the progression of differentiation. Our novel highly sensitive and non-disruptive method for detecting residual undifferentiated cells will contribute to reducing the manufacturing cost of iPSC-derived products and improving the safety of transplantation.

Estimating residual undifferentiated cells in human chemically induced pluripotent stem cell derived islets using lncRNA as biomarkers

“…In order to improve the sensitivity, we built an assay based on ddPCR platform using TaqMan probe and found that the expression of target miRNAs in hCiPSC-islets was too high to discriminate subtle transcripts of hCiPSCs (data not shown). We also tried to isolate miRNAs from cell culture supernatant which was showed to be an efficient method for detecting miRNAs 42 , 43 , but still could not reach enough detection sensitivity. This illustrates the pivotal role in selecting a proper biomarker for detecting specific iPSC derived cell product.…”

Multisite studies for optimization of a highly efficient culture assay used for in vitro detection of residual undifferentiated human pluripotent stem cells intermingled in cell therapy products

“…However, hPSCs are intrinsically tumorigenic and form teratomas [ 1 , 2 ]; therefore, the establishment of a robust and internationally harmonized methodology to evaluate the contamination of products with residual undifferentiated hPSCs is critically beneficial, not only for product developers but also for regulatory authorities and patients [ 1 , 3 ]. Among the currently available in vitro testing methodologies [ [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] ] which are based on flow cytometry [ 4 ], enzyme-linked immunosorbent assay-like sandwich assay [ 5 ], quantitative RT-PCR [ 4 , 6 ], droplet digital PCR (ddPCR) [ [7] , [8] , [9] ], reverse transcription loop-mediated isothermal amplification [ 10 ], and highly efficient culture (HEC) system [ 11 ], the HEC assay is one of the most robust and sensitive assays that can directly detect hPSCs by identifying hPSC-derived colonies under culture conditions that favor their growth [ 11 ].…”