Presenter Chandrindu Abeykoon
Authors Chandrindu Abeykoon, Melany Jackson, Louise Adamson, Tom Watson, Stephen Meek, Amy Findlay, Gura Bergkvist, Tom Burdon, Jeffrey J. Schoenebeck
Affiliations The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh.
Presentation Type Talk
Abstract
Induced pluripotent stem cells (iPSCs) are multifaceted tools used in regenerative medicine, drug discovery and disease modelling. However, the generation of canine iPSCs has not been efficient compared with their human counterparts. Ethical constraints in obtaining embryonic material and technical challenges in maintaining adult somatic cells in culture complicate canine iPSC generation. Furthermore, compared with well-established human iPSC systems, canine iPSC generation often shows lower reprogramming efficiency, incomplete pluripotency, continual dependence on reprogramming transgenes, and poor directed differentiation outcomes.
To enable efficient reprogramming of canine somatic cells while reducing dependence on limited primary material, we immortalised canine fibroblasts using SV40 Large T antigen (SV40Tag). Previous studies on other species show that SV40Tag enhances reprogramming efficiency by antagonising p53 and Rb pathways, suppressing stress-induced apoptosis, and extending the proliferative lifespan of somatic cells.
In our study, a doxycycline-inducible SV40Tag system enhanced the proliferative capacity of canine fibroblasts, allowing them to be maintained for extended passages in continuous culture. Two immortalised fibroblast cell lines were established, and their transfection efficiencies were evaluated using a GFP reporter plasmid, yielding an average efficiency of approximately 88%.
To assess the dependence of SV40Tag-mediated immortalisation, doxycycline was withdrawn from the culture medium, and cells were monitored over 96 hours. A significant reduction in proliferation was observed as early as 12 hours after doxycycline removal. Moreover, a decline in SV40Tag expression and a gradual increase in the p53 downstream marker MDM2 gene expression were observed. These findings confirm that SV40Tag activity can be modulated in this system.
To explore the transcriptomic landscape of reprogrammed cells and evaluate the effects of SV40Tag in propagated canine iPSCs, an immortalised cell line was reprogrammed in parallel with primary fibroblasts using a doxycycline-inducible PiggyBac system carrying eight transcription factors, each with three technical replicates. Following transfection, iPSC-like colonies emerged by Day 11. Cell pellets from untransfected (Day 0) and transfected cells (Days 2, 4, 6, 8 and 11) were collected and processed for bulk RNA sequencing, and data were analysed using the nf-core RNA-seq and differential abundance workflows.
Preliminary analyses revealed a clear and consistent pattern highlighting the strong transcriptional impact of SV40Tag-mediated immortalisation. Principal component analysis (PCA) showed that PC1 (76.4%) accounted for most variance, distinguishing SV40Tag-immortalised cells from primary fibroblasts. PC2 (8.1%) captured shifts linked to reprogramming, separating earlier from later stages in both cell lines. The heatmap of top variable genes showed two distinct clusters, with SV40Tag-immortalised samples displaying upregulation of genes involved in cell-cycle regulation and DNA replication, while primary fibroblasts exhibited higher expression of extracellular matrix associated genes.
To further investigate these differences, datasets from Day 0 and Day 2 are under analysis to investigate the molecular pathways influenced by SV40Tag during early reprogramming, focusing on p53 downstream targets, apoptotic and cell cycle regulators, and the expression of transgenes and endogenous pluripotency markers. Data from a second biological replicate are currently being generated to strengthen these analyses.