Abstract: Human iPSC-derived cells have emerged as useful sources for cell therapy. After transplanting cells into hosts, observing the distribution and fate of the transplanted cells is an essential step for safety evaluation. It is even more difficult and complicated to track the human iPSC-derived cells in the context of animal models with human cancer due to the genetic homology of human cells. To assess the biodistribution of the human iPSC-derived cells, highly sensitive and specific analytical methods, which can distinguish target cells from animal cells and human cancer cells, must be developed. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method that could efficiently track human iPSC-derived cells with high sensitivity, specificity and accuracy for pre-clinical research.
By targeting human MHC DNA sequences, primer pairs and a probe were designed and synthesized. A TaqMan probed based quantitative real-time PCR approach was developed after methodology validation using a touchdown PCR protocol. After the animals bearing human cancers received human iPSC-derived cells, major organ tissue and blood samples were collected, and genomic DNA was isolated. The amount of the human genomic DNA was quantified by a Qubit fluorometer.
This assay showed high sensitivity, evidenced by its ability to detect 1 human iPSC derived cell in 1.0 x 10^5 animal cells. There was no cross-reaction with genomic DNA obtained from animal models with human cancers. Using this assay, the biodistribution of intravenous injected human iPSC-derived NK (iNK) cells was investigated in NCG mice bearing human liver cancer cells. The results showed iNK cells accumulated in the lungs first and then reentered into circulation and distributed into other organs tissues after injection. The fate of injected iNK cells was evaluated by comparing the amount of iNK DNA detected in tissues collected at different time points.
Our primer-probe set and touchdown qPCR protocol are applicable for the quantitative detection and tracking of human iPSC-derived cells transplanted into animals models of human cancers in preclinical study.
