Transcriptional Profiling of Xenogeneic Transplants: Examining Human Pluripotent Stem Cell-Derived Grafts in the Rodent Brain

Human pluripotent stem cells are a valuable resource for transplantation, yet our ability to profile xenografts is largely limited to low-throughput immunohistochemical analysis by difficulties in readily isolating grafts for transcriptomic and/or proteomic profiling. Here, we present a simple methodology utilizing differences in the RNA sequence between species to discriminate xenograft from host gene expression (using qPCR or RNA sequencing [RNA-seq]). To demonstrate the approach, we assessed grafts of undifferentiated human stem cells and neural progenitors in the rodent brain. Xenograft-specific qPCR provided sensitive detection of proliferative cells, and identified germ layer markers and appropriate neural maturation genes across the graft types. Xenograft-specific RNA-seq enabled profiling of the complete transcriptome and an unbiased characterization of graft composition. Such xenograft-specific profiling will be crucial for pre-clinical characterization of grafts and batch-testing of therapeutic cell preparations to ensure safety and functional predictability prior to translation. •Interspecies sequence variation allows separation of xenograft and host transcripts•Species-specific primers enable profiling of targeted xenograft genes with qPCR•Xenograft-specific RNA-seq enables genome-wide transcriptional profiling of grafts•Xenogeneic-specific profiling provides unbiased characterization of graft composition In this article, Bye and colleagues establish a simple and robust method to transcriptionally profile xenogeneic transplants. Exploiting species differences in the RNA sequence between human stem cell grafts and the rodent host, xenograft-specific qPCR or xenograft-specific RNA-seq provided a sensitive approach for characterization and exploration of transplanted cell populations that will be critical for ensuring safety and functionality.