GENERATION AND CHARACTERISATION OF INDUCED PLURIPOTENT STEM CELLS (iPSCs) FROM ADULT CANINE FIBROBLASTS

Abstract

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Klf4, Sox2 and c-Myc (OKSM). In his presentation we will describe the derivation of iPS cells from adult canine fibroblast by retroviral OSKM transduction. The isolated canine iPS cells were expanded in three different iPS culture media (FGF2, LIF and FGF2 plus LIF) and only the cells cultured in FGF2 plus LIF showed strong AP activity expressed pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28 as well as ES cells-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1). In vitro differentiation by formation of embryoid bodies (EBs) and directed differentiation showed cell derivatives of all three germ layers as confirmed by expression for AFP, CXCR4 and SOX17 (endoderm), desmin (DES), vimentin (VIM), MSX1 and BMP2 (mesoderm) and glial fibrillary acidic protein (GFAP), TUJ1, NCAM and bIII-tubulin (TUBB, ectoderm). In vivo, the putative canine iPS cells formed simple teratomas that expressed markers for all three germ layers. In summary, we were able to derive induced pluripotent cells from adult somatic cells by using four transcription factors. The isolated canine iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF and LIF is still unknown. This work was funded by a grant from the America Kennel Club to JAP.