Generation of Putative Induced Pluripotent Stem Cells (iPS) from Adult Canine Fibroblast.

Pluripotent stem cells such as embryonic stem (ES) and iPS cells can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. Unfortunately, isolation of ES cells requires availability of embryos. Moreover, it is not possible to genetically match ES cells and patients resulting in immune rejection. iPS cells, in contrast, can be generated from somatic cells from the patients being treated, solving the issue of genetic match and availability of embryos. 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, Sox2, Klf4 and c-Myc. Here we report the derivation of putative iPS cells from adult canine fibroblast. VSV-G pseudotyped retroviruses containing transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) were transduced into skin fibroblasts isolated from a 3-year old dog. The transduced cells were cultured in DMEM containing 10% FBS for 6 days. The cells were then replated onto γ–irradiated mouse embryonic fibroblasts (MEFs) and the media was replaced with mTeSR1. A few granulated colonies appeared at day 14 and ES-like colonies were picked at day 21 post-infection. Colonies were expanded in three different ES culture media containing either FGF (10 ng/mL), LIF (103 units/mL) or both FGF and LIF, and supplemented with two chemical inhibitors (3 μM CHIR99021 and 20 μM PD98059). The isolated canine iPS cells were very similar to dog ES cells in morphology and alkaline phosphatase (AP) expression. Though the morphology and self-renewal of canine iPS cells was maintained over 12 passages in all three conditions, only the cells cultured in (FGF plus LIF) showed strong AP activity. The cells cultured in FGF/LIF were further analyzed by RT-PCR and immunocytochemistry (ICC) and found to express the pluripotency markers, Oct3/4 and Nanog, as well as human ES cell- (Podxl, FGF5 and Rex1) and mouse ES cell- (LAMP1, dppa5 and Brachyury) specific genes. However, the viral transcription factors transduced in the iPS cells were not silenced. Spontaneous differentiation by withdrawal of growth factors led to formation of embryoid bodies (EB) composed of cells representative of all three germ layers as confirmed by both immunostaining and RT-PCR. The EB cells were positive for CXCR4 and Sox17 (endoderm), desmin, vimentin and Nkx2.5 (mesoderm) and GFAP, TuJ1, NCAM and bIII-tubulin (ectoderm). Similar results were seen by directed In vitro differentiation. Subcutaneous injection of the putative canine iPS cells into SCID mice created immature teratomas which failed to reach epithelial structure formation, but expressed markers for all three germ layer as confirmed by both RT-PCR and ICC. In summary, we were able to isolate induced pluripotent cells from cultured adult somatic cells by using four transcription factors. The isolated reprogrammed cells have similar characteristics to ES cells from other species, but the exact cellular mechanisms behind its unique co-dependency on both FGF and LIF is still unknown. Availability of stable canine iPS cells would have significant implications for regenerative medicine in both veterinary and human clinical applications. This work was funded by a grant to JP and NO from the Canine Health Foundation, grant #01272. (poster)