ANALISI DEL DIFFERENZIAMENTO NEURONALEINDOTTO DALL’ACIDO RETINOICO IN CELLULE DITERATOCARCINOMA EMBRIONALE MURINO.
2009
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.
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