ANALISI DEL DIFFERENZIAMENTO NEURONALEINDOTTO DALL’ACIDO RETINOICO IN CELLULE DITERATOCARCINOMA EMBRIONALE MURINO.

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. A first aim of the present study was to optimize culture conditions to promote the RA-induced neural differentiation of P19 cells. To this purpose, cell viability, morphology and functional activity after RA treatment have been evaluated. The results indicate that the formation of embryoid bodies from P19 cells is enhanced in presence of neuronal basal medium (NBM). Following a 48h-treatment with RA (10-6M), a massive neuronal differentiation was induced and, three-five days after the treatment, the P19 cells showed a clear expression of the neurofilament proteins NF-68 and NF-160, two markers of terminal neuronal differentiation, detected by immunofluorescence. Furthermore, 10 days after the treatment with RA, the functional activity of differentiated cells has been demonstrated by their ability to uptake [3H]GABA. Once experimental conditions favoring the RA-induced neuronal differentiation have been established, the expression of some proteins, such as protein kinase C 6 (PKCz), protein kinase C a (PKCa), extracellular signalregulated kinases 1/2 (ERK1/2) and caspase-3, at different times after RAtreatment, has been investigated. Western blot and immunocytochemical studies indicate that RA-treatment induced direct or indirect changes of the levels of the proteins under investigation, thus suggesting a possible involvement of these proteins in the signal pathways associated with RA-induced differentiation in P19 cells. Taken together, the present results suggest that the reported culture conditions favor a rapid differentiation of neural stem cells from P19 cells, and contribute to the understanding of the molecular mechanisms possibly involved in RA-induced neuronal differentiation. Thus, this approach could represent a reliable basis for studies of “regenerative therapy” of sensorineural hearing loss.