Self-replicating RNA for Cellular Reprogramming in Domestic Cat

Abstract

Cat Induced Pluripotent Stem Cells have been previously established using viral vectors integrating into the host genome to deliver reprogramming factors. To aid wild felid conservation using stem cell technology, in this study we utilized non-integrative Venezuelan Equine Encephalitis-based self-replicating RNA (srRNA) to carry the reprogramming factors. This approach induced cat somatic cells towards pluripotency without genomic modification, supporting the future use of srRNA in wild felid cells. The srRNA expressing human OCT4, SOX2, KLF4, CMYC, and GLIS1 (OSKMG) could induce the formation of cat iPSC colonies after 4 rounds of transfections. Different species-specific Leukemia Inhibitory Factor (LIF) could capture the induced cells maintaining pluripotency. A combination of feline and human LIFs, could maintain cat iPSC in a pluripotent state, that was different from those seen in human or mouse embryonic stem cells (ESCs). The cat iPSCs also exhibited spontaneous differentiation towards ectodermal, mesodermal, and endodermal lineages upon the removal of LIF from the culture medium. This work demonstrates the utility of srRNA containing human reprogramming factors to induce reprogramming in somatic cat cells in the presence of serum and LIF. Further investigations into the maintenance of cat iPSC will aid in inducing wild felid stem cells for long-term conservation.