RETINOIC ACID-INDUCED NEURAL DIFFERENTIATION OF MOUSE P19 EMBRYONIC CARCINOMA CELLS

In humans, the loss of sensory hair cells is an irreversible process leading to hearing loss. Regenerative medicine, that is the replacement of degenerating cells with neural stem cells, has been proposed for treatment of inner ear sensorineural damage. The pluripotent mouse P19 embryonal carcinoma (EC) cell line usually grows into an epithelial monolayer and, after aggregation and treatment with retinoic acid (RA), differentiates into neural cells, including neurons and glial cells. Thus, the mouse P19 cells have been extensively used as a model to study molecular mechanisms of neural differentiation in vitro and might represent an useful tool in regenerative medicine. The aim of the present study was to optimize culture conditions to promote the RA-induced neural differentiation of P19 cells. Cell morphology and expression of molecular markers involved in signal pathways associated with RA-induced differentiation in P19 cells were also evaluated. The results indicate for the first time that the formation of embryoid bodies from P19 cells is enhanced in presence of a specific neural serum free medium. Following a 48h-treatment with RA (10-6M), a massive neuronal differentiation was induced and, five days after the treatment, the P19 cells showed a clear expression of the neurofilament protein NF160, a marker of terminal neuronal differentiation, detected by immunofluorescence. Furthermore, an increased expression of protein kinase C ζ, involved in cell differentiation, was observed by Western blot during RA treatment. Finally, the expression in P19 cells of other biological markers involved in the apoptotic pathway associated with RA-induced differentiation was also observed. The results suggest that the reported culture conditions favour a rapid differentiation of neural stem cells from P19 cells, and could be a reliable basis for studies of therapy of sensorineural hearing loss.