40 PIG CLONING AND GENE EXPRESSION PATTERNS IN MINIPIG ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS

Abstract

In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are very interesting; they are easy to harvest and can expand to generate millions of cells from a small quantity of fat. The ASC are known as useful materials for clinical applications in human cell therapy and as a donor cell in somatic cell nuclear transfer (SCNT). In this study, we investigated the expression patterns of several genes (Oct-4, Nanog, Sox2, Dnmt1, and Dnmt3b) in minipig ASC, and whether ASC can be a suitable donor cell type for producing cloned pigs. For the study, we respectively isolated ASC, adult skin fibroblast (ASF) and fetal fibroblast (FF) from a 6-year-old female minipig. The ASC were attached to a plastic dish with a fibroblast-like morphology, expressed cell-surface marker characteristics of stem cells, and underwent osteogenic, adipogenic, and neurogenic differentiation when exposed to specific differentiation-inducing conditions. To observe gene expression, total RNA was extracted from ASC, FF, and ASF, respectively, and was used for reverse transcription (RT). After RT, real-time PCR was performed to investigate the expression of Oct-4, Sox2, Nanog, Dnmt1, and Dnmt3b. The expression of β-actin was measured and used as an endogenous control. In the following experiment, we carried out SCNT using ASC, ASF, and FF. The ratio of blastocysts to 2-cell embryos and total cell number of blastocysts were monitored as experimental parameters. Statistical analysis was performed using one-way ANOVA (GraphPad Prism version 5). As a result, the relative abundance of DNMT1 in ASC (1.9 ± 0.9) was significantly higher than that in FF and ASF (0.1 ± 0.2 and 1.0 ± 0.5, respectively; P < 0.05), but no significant difference in expression of the DNMT3b gene was observed. Interestingly, the quantity of Oct-4 was significantly higher in FF and ASC than in ASF (2.8 ± 0.4 and 2.9 ± 0.5 v. 1.0 ± 0.1, respectively; P < 0.05), and Sox2 showed significantly higher expression in ASC (3.7 ± 0.5) than in ASF and FF (1.0 ± 0.1 and 1.4 ± 0.6, respectively; P < 0.05). Nanog expression was similar in ASF, FF, and ASC. After SCNT, the developmental competence to blastocysts did not differ among the 3 groups (ASF: 7.0 ± 0.2%, FF: 16.15 ± 6.1%, and ASC: 11.1 ± 0.7%). However, total cell numbers of blastocysts derived from ASC and FF were significantly higher in ASF (89.0 ± 7.9 and 105.0 ± 5.5 v. 57.5 ± 5.2, respectively). In conclusion, the present study revealed that minipig ASC and minipig FF possess slightly different gene expression patterns and ASC have potential in terms of in vitro development and blastocyst formation ability similar to ASF and FF. This study was supported by IPET (no. 311011-05-1-SB010), RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), Institute for Veterinary Science, and the BK21 program.