Human Genome-Specific Real-Time PCR Method for Sensitive Detection and Reproducible Quantitation of Human Cells in Mice

Abstract

Xenotransplantation of human cells into immunodeficiency mice has been frequently used to study stem cells in tissue repair and regeneration and cancer cell metastasis. However, a sensitive and reproducible method to quantify cell engraftment lacks. Here, we developed a Real-Time PCR-based method which facilitated consistent detection and quantification of small amounts of human cells distributed in mouse organs after infusion. The principle of the method was to directly detect a humans-specific sequence in the human-murine genomic DNA mixture. In a mouse myocardial infarction model, the Real-Time PCR-based method consistently determined the amounts of human mesenchymal stem cells (hMSCs) engrafted into the heart and other organs 7 days after infusion of as little as 2.5 × 10(5) cells, indicating a high sensitivity, and the amounts of hMSCs detected in mice highly correlated to the numbers of hMSCs transplanted. Importantly, different from previous PCR-based methods, our method produced highly consistent and reproducible results. The reliability of the method was further proven by parallel analyses of DiI-labeled hMSCs in tissue sections and in single cell suspensions of mice. Our data show that the present human genomic DNA-specific primers-based Real-Time PCR method is sensitive and highly reproducible in determining the amount of xenotransplanted human cells in murine tissues.

Biodistribution and retention of locally administered human mesenchymal stromal cells: Quantitative polymerase chain reaction–based detection of human DNA in murine organs

“…Although many groups have described PCR-based techniques for the detection of human cells in xenotransplantation systems, each qPCR protocol differs in its degree of sensitivity. Using the primer targeting strategy described herein with SYBR Green qPCR detection, we were able to detect 1 hMSC in 100 murine cells, superior to the detection limits reported by Song et al [17] of 1 human cells in 20 murine cells, but inferior to those described by Toupet et al [18] and Prigent et al [10], detecting 1 adipose-derived hMSC in 41,000 murine cells or one hMSC in 200,000 murine cells, respectively. Although used to detect circulating human tumor cells, the SYBR Green-based protocol described by Schneider et al [19] was far superior to our initial experimentation, detecting 1 human cell in 1 x 10 6 murine cells.…”

Validation of a Quantitative Polymerase Chain Reaction Method for Human Alu Gene Detection in Microchimeric Pigs Used as Donors for Xenotransplantation

“…Earlier studies suggest that real-time PCR-based methods may facilitate high throughput detection and quantification of minimal amounts of human donor cells in mice recipient tissues [15]. However, previously recorded experiments based on this technique have shown considerable limitations, namely, high technical variations and poor reproducibility [16,17].…”

Calcium-binding protein S100A4 confers mesenchymal progenitor cell fibrogenicity in idiopathic pulmonary fibrosis

“…Of note, the engraftment of human IPF MPCs treated with S100A4 shRNA or scrambled shRNA in the lungs of the immune-deficient mice was confirmed utilizing a realtime PCR method that has been shown to be sensitive for the detection and quantitation of human cells in mice (Supplemental Figure 7; ref. 38). This indicates that -compared with IPF cells in which S100A4 levels were intact -the reduced number of human IPF cells in the mouse lungs, in which S100A4 had been knocked down‚ cannot be explained by a lack of engraftment.…”