ISOLATION AND CULTURE OF MESONEPHRIC STEM-LIKE CELLS FROM PORCINE FETUS

Abstract

Many attempts to establish embryonic stem (ES) cells from preimplantation stage embryos in pigs have come to a failure. An alternate source of pluripotent stem cells are embryonic germ (EG) cells derived from primordial germ cells (PGCs) of the genital ridge, which is developed from mesonephros. Mesonephros is a vestige, 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 with the culture condition to establish porcine 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 each other and cells from mesonephros and PGCs from genital ridges were isolated by a physical method. Isolated cells were cultured in PES medium [50:50 mixture of Dulbecco modified Eagle medium (DMEM) and Ham F10 medium supplemented with 15% fetal bovine serum (FBS), L-glutamine (1.7mM), beta-mercaptoethanol (0.1mM), 1% MEM nonessential amino acids and 1% antibiotic-antimycotic] containing the cytokines, soluble recombinant human basic fibroblast growth factor (bFGF;20ng/ml), and human leukemia inhibitory factor (hLIF;10ng/ml). Isolated cells were cultured on fresh primary murine embryonic fibroblast feeder cells (MEF) 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 PGCs. Porcine mesonephric stem-like cells (PMSCs) from fetal mesonephros were passed to fresh feeder every 6-8 days and have been cultured up to 20th passages maintaining typical EG-like morphology. PMSCs were stained for alkaline phosphatase throughout the culture. Futhermore, these cells were reacted with antibodies against Oct-4 and SSEA-1 by immunocytochemstry, indicating that these cells have characteristics of pluripotential stem cells. In order to characterize the PMSCs in respect to their potential for differentiation, embryoid body (EB) formation was induced. EBs were started to form in 4 days and cystic structures in 2 weeks. Dissociated cells from EBs were then attached to the dish and cultured without cytokines. Spontaneously, cells from EBs could give rise to differentiate into various cell types from all three germ layers. PMSCs cultured with the medium containing bFGF grew in compact colonial groups with the ‘nests’ of cells that often have a convex, 3D shape. The PMSCs maintained in the medium containing bFGF highly expressed SSEA-1, OCT-4 and alkaline phosphatase. In addition, PMSCs with bFGF maintained the expression of oct4 and nanog by real time RTPCR at high levels compared to those without bFGF. These results show that the bFGF effectively sustained proliferation and pluripotency of undifferentiated PMSCs. (platform)