Reproduction Abstracts

Introduction: The α-1,3-galactosyltransferase knockout (GalT KO) pig is used as the major model for xenotransplantation studies since the knockout of the α-1,3-galactosyl epitope makes pig organ transplantation successful in primates. The homozygous GalT KO pig is produced by a combination of nuclear transfer technique and mating with a heterozygous GalT KO pig. Since most studies using GalT KO pigs have analyzed only the immunological features of their cells, we analyzed the physical and genetic features of ear fibroblasts and bone marrow mesenchymal stem cells (BM-MSCs) derived from GalT KO pig and compared them with those of the wild pig.

Material and methods: Cells were seeded at concentrations of 1X10³ or 1X10⁴cells/well in a 96-well plate. Grow rate was analyzed by ELISA using BrdU incorporation. At ∼80% cell confluence, cell size was analyzed using an automated cell counter. For immune tolerance test, activated human peripheral blood mononucleated cells and BM-MSCs were cocultured in six transwells. The expression levels of p53, Bax, Bcl2, and TERT mRNA were analyzed by real-time PCR.

Result and discussion: GalT KO cells showed lower growth rate than wild cells, regardless of the cell concentration. The cell size of GalT KO BM-MSCs was 15.2±0.2μm (size range of other cells: 13.6±0.3–14±0.4μm). GalT KO BM-MSCs showed higher immune tolerance than wild BM-MSCs, which showed the highest telomerase activity. Thus, our study showed that GalT KO BM-MSCs showed early aging compared to wild BM-MSCs in vitro.

This work was supported by the Next-Generation Biogreen 21 Program (grant no. PJ009038022014) funded by the RDA, Republic of Korea.

Keywords: α-1, 3-galactosyltransferase knockout pig, aging, telomerase activity, immune tolerance