224 DERIVATION AND IMMUNOLOGICAL CHARACTERISTICS OF PORCINE EMBRYONIC STEM-LIKE CELLS

Abstract

Pluripotent cells from porcine blastocysts can be used to serve as an in vitro model as well as an unlimited cell source for therapeutic xenotransplantation. In xenotransplantation, foreign organs and tissues are rejected because of the immune responses in the recipient. This rejection is determined by the naturally occurring cell surface antigen, major histocompatibility antigen (MHC), knowledge of which is essential to advance our understanding of the processes implicated in immune responses. In mouse and human embryonic stem (ES) cells, MHC expression patterns are changed upon their differentiation in vitro. Therefore, immunological characteristics of undifferentiated and/or differentiated pluripotent ES-like cells derived from porcine blastocysts could provide valuable information to xenotransplantation research. For such purposes, swine leukocyte antigen (SLA) isotype and its expression pattern were determined in porcine ES-like cells. Porcine inner cell masses from Day 7 in vivo-produced blastocysts were isolated by immunosurgery and seeded on a feeder layer. Isolated cells were cultured in a mixture of DMEM : HF-10 (1 : 1) with 15% FBS, 1.7 mM l-glutamine, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1000 U mL^-1 leukemia inhibitory factor, 40 ng mL^-1 stem cell factor, and 20 ng mL^-1 basic fibroblast growth factor in humidified environment of 5% CO₂ in air at 38°C. Porcine ES-like cells showing typical morphology were subpassaged by physical separation. Embryoid body (EB) formation was induced by culturing small clumps of cells by the hanging drop method. Total RNA was extracted from porcine ES-like cell and EBs. PCR amplification was performed by using locus-specific primer pairs for swine leukocyte antigen (SLA) 1 and 2, followed by sequencing of amplified DNA fragments. These sequences were compared with known SLA sequences, and their SLA allele types were determined. Porcine ES-like cells showed the typical morphology of ES cells, closer to that of human than mouse cells. They showed typical expression of pluripotent stem cell markers, determined by immunochemistry and PCR analysis. When porcine ES-like cells formed EBs, differentiation specific markers of all 3 germ layers, ±%-fetoprotein (endoderm marker), neurofilament (ectoderm marker), and cardiac troponin1 (mesoderm), were detected in the EBs. In porcine ES-like cells, two new alleles (SLA-1*06ck01 and *06ck02, and SLA-2*06ck01 and *06ck02) were identified. Therefore, our porcine ES-like cell line showed heterozygous allele isotypes in SLA-1 and SLA-2. EBs derived from porcine ES-like cells showed the same sequencing results, indicating no alteration of SLA-1 and SLA-2 isotypes in porcine ES-like cells and their differentiated derivatives. Further experiments for determining SLA-3 allele isotype in porcine ES-like cells and EBs and cell surface expression of the SLA antigens in these cells will be performed. In conclusion, porcine ES-like cells were established from in vivo-derived blastocysts and their SLA isotypes were consistent regardless of their differentiation.