179 ISOLATION AND CULTURE OF EMBRYONIC GERM-LIKE CELLS FROM PORCINE MESONEPHROS

Abstract

Many attempts to establish embryonic stem (ES) cells from pre-implantation stage embryos in pigs have failed. An alternate source of pluripotent stem cells is embryonic germ (EG) cells derived from primordial germ cells (PGCs) of the genital ridge, which is developed from the mesonephros. Mesonephros is a vestigial, transient renal organ that functions only during embryonic development. It is believed to be a source of multiple stem cells including somatic cells in the gonad, vascular endothelial cells, and hematopoietic stem cells. Therefore, we tried to obtain putative stem cells from cells isolated from porcine mesonephros under culture conditions used to establish EG cells. Porcine fetuses from crossbred gilts were collected by hysterectomy between Days 25 and 30 of pregnancy (estrus = Day 0). Mesonephros and genital ridges were separated from each other, and cells from the mesonephros and PGCs from genital ridges were isolated by a physical method. Isolated cells were cultured in PES medium [50:50 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F10 medium supplemented with 15% fetal bovine serum (FBS), l-glutamine (1.7 mM), β-mercaptoethanol (0.1 mM), 1% MEM non-essential amino acids and 1% antibiotic-antimycotic] containing the cytokines, soluble recombinant human basic fibroblast growth factor (bFGF; 20 ng/mL), and human leukemia inhibitory factor (hLIF; 10 ng/mL). Isolated cells were cultured on fresh primary murine embryonic fibroblast feeder cells (PMEF) in a humidified environment of 5% CO₂ in air at 38°C. The colonies with EG-like morphology were dissociated with 0.25% trypsin/1 mM EDTA for 10 min, and passed to fresh feeders. After 5–8 days, colonies started to grow with typical EG-like morphology. More colonies were obtained from the culture of cells from mesonephros than from culture of PGCs. Porcine EG-like cells from mesonephros (pMN-EG-like cells) were passed to fresh feeder every 6–8 days and have been cultured up to 9 passages while maintaining typical EG-like morphology. pMN-EG-like cells were stained for alkaline phosphatase throughout the culture. Furthermore, these cells reacted with antibodies against Oct-4 and SSEA-1 by immunocytochemistry, indicating that these cells have characteristics of pluripotential stem cells. In order to characterize the pMN-EG-like cells with respect to their potential for differentiation, embryoid body (EB) formation was induced. EBs started to form in 4 days and cystic structures in 2 weeks. EBs were then attached to the dish and cultured without cytokines. Spontaneously, EBs from pMN-EG-like cells could give rise to differentiated cell types such as neuronal-like, epithelial-like, and fibroblast-like cells. Further studies to characterize differentiated cells from pMN-EG-like cells by immunocytochemistry and for teratoma formation by injection into SCID mice will be performed. In conclusion, EG-like cells could be obtained from culture of mesonephric cells from porcine fetus and further characterization of these cells is required.