Ps-15: Neuronal Differentiation of GFP Expressing P19 Embryonal Carcinoma Stem Cells

Objective: P19 cells are a line of pluripotent embryonal carcinoma stem cells able to grow continuously in serum-supplemented media and can be induced to differentiate along either mesodermal or ectodermal lineage. The differentiation of these cells can be controlled by nontoxic drugs. When treated with deprenyl, P19 cultures differentiate into cell types similar to those derived from neuroectoderm. The antiparkinsonian effect of deprenyl was reported by several investigators. On the other hand, there is a growing interest in the potential use of stem cell therapy in Parkinson’s disease. One of the major advantages to using P19 cells in the study of neuronal function and development is that these cells are amenable to genetic manipulation. Foreign DNA can be introduced into these cells using a standard method involving calcium phosphate precipitation. In this investigation, deprenyl was used to induce neuronal differentiation in undifferentiated pluripotent P19 embryonal carcinoma cells. We used calcium phosphate precipitation method to transfect these cells with pML8 plasmid, a vector encoding eGFP and puromycin resistance gene under the control of the murine Pgk-1 promoter. Materials and Methods: The cells were cultured using α-MEM medium that supplemented with 15% fetal bovine serum (FBS). In the treatment group, deprenyl was used to induce embryoid body (EB) differentiation to neuronal lineage. Initially, the viability test was used to select the range of nontoxic doses before transfection. The optimal inducing dose was obtained using different concentrations of deprenyl (10-6–10-11 M). The viability of untransfected P19 cells during the experiment was determined by the trypan blue dye exclusion method. The peak response was at 10-8 M, which was used for further investigation. Morphologic and immunofluorescence techniques were used to evaluate the differentiation of the P19 cells, Cresyl violet for the morphologic study, anti-synaptophysin and anti-betatubulin III antibodies for characterizing the neuronal phenotype of the cells. Results: The results show that deprenyl can induce neuronal phenotype associated with neuronal marker expression in successfully GFP-transfected P19 carcinoma cells. It induced the differentiation of the cells into neuron-like cells in a concentration-dependent manner. Conclusion: This study suggests the potential use of combined deprenyl and stem cell therapy to improve deficits in neurodegenerative diseases.