201 CHARACTERIZATION OF CANINE EMBRYONIC GERM CELLS: AN EXPERIMENTAL MODEL FOR CELL THERAPY

Abstract

Dogs represent excellent models to test different approaches before use for human therapy. Studies with animal models have suggested that the transplant of stem cells may have success in the treatment of degenerative diseases such as Parkinson's disease, diabetes, Duchenne muscular dystrophy (DMD), and acquired lesions. Embryonic stem cells are pluripotent and therefore have the potential to form all tissues. Our research aims to contribute to the treatment of DMD through the isolation and identification of embryonic germ cells and the development of the methodology of cellular differentiation for future transplantation into dystrophic dogs. Mongrel female dogs were ovariehysterectomized between 25 and 30 days of pregnancy. For recovery of embryos, the excised uterine horns were flushed with heparinized PBS. Samples collected from somites near the mesonephros area of four embryos recovered at 22 to 24 days of pregnancy and designated as A1 through A4 were dissociated and placed in culture. Isolated embryonic cells were allowed to plate onto monolayers of canine fibroblast cells. Flow cytometry was used to identify CD34+ markers. Isolated compact colonies of embryonic germ cells were seen growing around tissue fragments at 7 days of culture and remained in the undifferentiated stage until approximately 21 days in culture. At 14 days of explant, cell colonies were analyzed by flow cytometry. Cells from the A2 embryos contained the highest number of CD34+ cells, whereas no cells from A4 embryos showed specificity for the marker. A small proportion (2.12%) of cells from embryos A1 and A3 showed specificity for the CD34 marker. A quantity of A2 embryo cells that had maintained stem cell characteristics were frozen for future studies. Our results suggest that although spontaneous differentiation occurred, a small population of cells maintain the characteristics of stem cells. We are currently trying to improve the methodology to maintain cells undifferentiated for longer periods and to better control the specific differentiation process.