285 GENERATION OF CANINE INDUCED PLURIPOTENT STEM CELLS FROM CANINE FETAL FIBROBLAST AND ADULT FIBROBLAST OF CLONED DOG

Abstract

Induced pluripotent stem cells (iPSC) derived from a patient’s fibroblasts have been used as fine resources for studying disease mechanisms and therapeutic strategies. The dog is considered invaluable in human disease research because its genetic diseases are strikingly similar to those of human. Therefore, we generated cloned dogs and transgenic cloned dogs via somatic cell nuclear transfer. In this study, we tried to derive canine iPSCs from canine fibroblasts to establish a way to make iPSC from skin fibroblasts of transgenic cloned dogs. We isolated canine fetal fibroblast (FF) from normal beagles and adult skin fibroblast (ASF) from cloned beagles. Both ASF and FF were infected with all-in-one retroviral vector that delivers human reprogramming factors (Oct4, Sox2, Klf4, c-Myc). Ten to twenty-one days after infection, the colony-shaped structure was picked and plated on a mouse embryonic fibroblast (MEF) feeder layer, pretreated with mitomycin C. Then, all cells were cultured with DMEM/F12 supplemented with 20% fetal bovine serum, 5 ng mL^–1 basic fibroblast growth factor (bFGF), 5 ng mL^–1 LIF, 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin. Alkaline phosphatase (AP) activity and expression of Oct4, Sox2, SSEA1, and SSEA4, were observed in the cells to characterise the iPS cell colonies. In vitro differentiation of 10th-passage canine iPSC was performed through embryonic body formation. About 50 canine iPS-like colonies were formed on a 100-mm dish. As a result, the canine iPSC from FF (iPSC-FF) and canine iPSC from ASF (iPSC-ASF) showed typical colony morphology, and both stained positively for AP. The expression of pluripotency-associated transcription factors Oct4 and Sox2 was positively displayed in iPSC-FF colonies. The stem cell markers SSEA1 and SSEA4 were negative in canine iPSC-FF. The canine iPS-FF spontaneously differentiated into all 3 germ layers in vitro, showing positive expressions of βIII-tubulin (ectoderm), α-SMA (mesoderm), and GATA6 (endoderm). As for iPS-ASF, characterisation and in vitro differentiation experiment are in progress. These results show that canine iPS-FF are similar to embryonic stem cells in terms of morphology and the ability to differentiate into 3 germ layers. Although we did not demonstrate complete verification of canine iPS-ASF of the cloned dog, their morphology, AP expression, and iPS-FF generation should indicate the possibility of iPSC production in the cloned dog. In conclusion, retroviral transduction of 4 human reprogramming factors can reprogram canine fetal fibroblasts into canine iPSC. The technique of producing canine iPSC will stimulate the utilisation of transgenic cloned dogs and expand the range of human diseases or therapeutic application.

This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311011-05-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.