Footprint-free induced pluripotent stem cells can be successfully differentiated into mesenchymal stromal cells in the feline model

Abstract

Abstract Background Induced pluripotent stem cells (iPSCs) can propagate indefinitely and give rise to every other cell type, rendering them invaluable for disease modelling, drug development research, and usage in regenerative medicine. While feline iPSCs have been described, there are currently no reports on generating genome integration (footprint)-free iPSCs from domestic cats. Therefore, the objective of this study was to generate feline iPSCs from fetal fibroblasts using non-integrative Sendai virus (SeV) vectors carrying human transcription factors. Moreover, these iPSCs were differentiated into mesenchymal stromal cells (MSCs), which can be used as an alternative to tissue-derived MSCs. Methods Feline fetal fibroblasts were transduced with CytoTune-iPS 2.0 Sendai Reprogramming vectors at recommended multiplicity of infections (MOI) and cultured for about 6 days. At 7 days post transduction cells were dissociated, replated on inactivated feeder cells and maintained in iPSC medium for 28 days with daily medium change. Emerging iPSC colonies were mechanically passaged and transferred to fresh feeder cells and further passaged every 6–8 days. Four feline iPSC lines were generated, with two selected for further in-depth characterization. Feline iPSCs were then differentiated into MSCs using a serial plating strategy and an inhibitor of the transforming growth factor-β (TGF-β) type I receptor. Results Feline iPSCs exhibited characteristic colony morphology, high nuclear-to-cytoplasmic ratio, positive alkaline phosphatase activity, and expressed feline OCT4, SOX2, and Nanog homeobox (NANOG) stem cell markers. Expression of SeV-derived transgenes decreased during passaging to be eventually lost from the host cells and feline iPSCs could be stably maintained for over 35 passages. Feline iPSCs differentiated into embryoid bodies in vitro and did not form fully differentiated teratomas; instead, they generated in vivo masses containing mesodermal tissue derivatives when injected into immunodeficient mice. Feline iPSC-derived MSCs were plastic adherent, displayed MSC-like morphology, expressed MSC-specific surface markers, and differentiated into cells from the mesodermal lineage in vitro. RNA deep sequencing identified 1,189 differentially expressed genes in feline iPSC-derived MSCs compared to feline iPSCs. Conclusion We demonstrated the generation of footprint-free iPSCs from domestic cats and their directed differentiation potential towards MSCs. These SeV-derived feline iPSCs and iPSC-derived MSCs will provide valuable models to study feline diseases and explore novel therapeutic strategies and can serve as translational models for human health, leading to increased knowledge on disease pathogenesis and improved therapeutic interventions.