NT derived neuronal cells in vitro

NT2 derived neuronal sodium channel in vitro express a plethora of neuron-specific markers at both transcript and protein levels (Pleasure et al., 1992; Cheung et al., 1999; Megiorni et al., 2005; Couillard-Despres et al., 2008; Haile et al., 2014). Our results are roughly in line with previous studies showing increased expression of NF200 (Lee and Andrews, 1986; Pleasure et al., 1992), β-III tubulin (Megiorni et al., 2005; Couillard-Despres et al., 2008; Popovic et al., 2014), and DCX during RA-induced differentiation (Couillard-Despres et al., 2008). However, differences in culture conditions and the lack of data of the time course expression (or election of unequal time points for analysis) in these studies make it difficult a reliable comparison between their and our results. An inherent limitation for interpretation of results arises from the heterogeneous composition of cultures during RA-induced differentiation. Indeed, the neuronal phenotype represents only a small fraction over the entire cell population before mechanical isolation of RA/NT2N cells. Of the three markers used, NF200 was clearly identified as a highly neuron-specific marker in terminal cultures, in view of the high enrichment in this protein in terminally differentiated RA/NT2N cells compared to cell samples harvested before mechanical dislodging of neuronal cells. By contrast, expression levels of β-III tubulin and DCX were similar in samples from cultures after 28days of treatment and in those from terminally differentiated RA/NT2N cells, indicating that these proteins were expressed in both RA/NT2N neurons and in the highly adherent non-neuronal NT2A cells. Remarkably, β-III tubulin was already detectable in NT2 cells, reached a maximum immunoreactivity by day 7 of RA treatment, and returned to basal levels at the end of the process. This is in agreement with a similar time course analysis performed by Megiorni et al. (2005), even thought they used a cell aggregation method that shortens the time of the RA-induced differentiation to 3–4weeks. Collectively, these data support and extend previous findings of the expression of neuronal markers in RA/NT2N neurons and emphasizes the caution needed in drawing conclusions about the use of β-III tubulin for an unequivocal identification of neuronal phenotypes. In line with this conclusion, β-III tubulin is expressed in human fetal astrocytes (Dráberová et al., 2008) as well as in undifferentiated mesenchymal stem cells (Foudah et al., 2014). Our findings show that NT2N cells derived from AraC treatment not only show neuronal morphology, but also express neuronal markers, albeit with remarkable differences in the expression profiles of β-III tubulin and DCX during differentiation. At difference with that observed during RA-induced differentiation, in AraC-treated cultures, immunoreactivity for β-III tubulin increased sharply at mid-treatment and reached a maximum at day 6, whereas DCX showed an up-down regulation pattern that peaked up at 48h post-treatment. The progressive increase in expression of NeuN/Fox-3 observed during AraC-induced differentiation of NT2 progenitors, as analyzed by western blot and immunofluorescence microscopy, further confirmed the neuronal identity of AraC/NT2N neurons. The sequential and non-matching upregulation of the early (Brown et al., 2003) and late (Mullen et al., 1992) neuronal markers DCX and NeuN/Fox-3 suggests that, beside differentiation, AraC/NT2N cells have begun a maturation process after 6days of treatment. However, this suggestion should be considered with caution, as NeuN/Fox-3 expression has been found in non-neuronal cells, such as cultured rodent and human astrocytes and even 3T3 fibroblast cells (Darlington et al., 2008). Indeed, we also observed that part of the population of non-neuronal flat polygonal cells in our AraC-treated terminal cultures displayed weak to moderate nuclear staining for NeuN/Fox-3, leading us to consider morphological features to classify cells into non-neuronal or AraC/NT2N neuronal. Less than 10% of the cells in culture survived the uninterrupted 6-day period of AraC treatment, and only about 50% of them fulfilled the double criterion to be considered as neuronal (i.e., NeuN/Fox-3 positive nucleus and presence of neurite extensions) leading to a differentiation efficiency similar to that found after RA-differentiation. Our modified procedure, consisting of increasing cell density at mid-treatment, doubled overall cell survival and increased the percentage of neuronal cells with respect the total population at the end of AraC treatment to 71%, respectively. Consequently, differentiation efficiency was number augmented by almost 3-fold.