Human Progenitor Cell Quantification After Xenotransplantation in Rat and Mouse Models by a Sensitive qPCR Assay.
Xenotransplantation of human cells in animal models is an essential tool for evaluation of safety and efficacy of cell-based products for therapeutic use. Sensitive and reproducible methods are needed to detect and quantify human cells engrafted into the host tissue either in the targeted organ or in undesired locations. We developed a robust quantitative polymerase chain reaction (qPCR) assay based on amplification of human AluYb8 repeats, to assess the number of human cells present in rat or mouse tissues after transplantation. Standard curves of mixed human/rodent DNA and mixed human/rodent cells have been performed to determine the limit of detection and linear range of the assay. Standard curves from DNA mixing differed significantly from standard curves from cell mixing. We show here that the AluYb8 qPCR assay is highly reproducible and is able to quantify human cells in a rodent cell matrix over a large linear range that extends from 50% to 0.01% human cells. Short-term in vivo studies showed that human cells could be quantified in mouse liver up to 7 days after intrasplenic transplantation and in rat liver 4 h after intrahepatic transplantation.
Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique, Paediatric Hepatology and Cell Therapy, Brussels, Belgium.
Julie
J
Prigent
Astrid
A
Herrero
Jérôme
J
Ambroise
Françoise
F
Smets
Gisèle A
GA
Deblandre
Etienne M
EM
Sokal
D020087:Alu Elements / Q000235:genetics*; D000818:Animals; D019070:Cell Lineage; D004247:DNA / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000172:deficiency / Q000235:genetics; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D023421:Models, Animal; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014183:Transplantation, Heterologous
D004268:DNA-Binding Proteins; C507942:Il2rg protein, mouse; D053631:Interleukin Receptor Common gamma Subunit; C499308:Rag2 protein, mouse; D004247:DNA