The neural-crest is a vertebrate-specific transient embryonic tissue that gives rise to several cell types . It has been clearly established that early neural-crest cells are multipotent at the cellular level , and perhaps in reflection of the innate multipotency, neural-crest derived cells display long lasting plasticity during embryonic development . This plasticity is also reflected in the phenomenon of transdifferentiation, whereby cells change from one unique phenotype into another without going through a developmentally less mature stage . Neural-crest derived tumors like NBT as well as NBT cell lines show many of the cell phenotypes characteristic of the developing neural crest cells: cellular heterogeneity, plasticity and transdifferentiation capacity .
Normal embryogenesis of the PNS involves a bilineal stage of cell differentiation whereby early glial and neuronal markers can be found coexpressed . In neuroblastoma, I-type cells have been described to exhibit an intermediate morphological and in vitro behavior, sharing characteristics of both N- and S-type cells, and express neuronal as well as glial lineage markers, and to reproduce a distinct and bipotential (neuronal and glial) stage of differentiation [11, 24].
Instead of neurofilaments and calcyclin, we found cytoplasm membrane-GD2 and nuclear-calcyclin as reliable NBT neuroblastic and glial lineage markers, respectively. By immunofluorescence, N- and I-type showed similar pattern of expression with high GD2 and undetectable calcyclin. On the other hand, S-type exhibited a high nuclear-membrane calcylin staining and no ganglioside GD2 expression. I-type cells represent thus, a distinct NB cell subpopulation, an intermediate state between the neuronal and glial lineages, however closer to neuroblastic lineage.
In this study, we aimed to characterize each NBT cell line subtype according to differential lineage markers from the embryonic development of PNS. Initially, lineage markers such as vimentin and GAP43 (glial and neuronal, respectively) were studied. In our hands, these proteins did not display differential expression between the different cell subtypes. An undifferentiated state of the cell lines was studied by using neural crest cells markers such as c-kit and Phox2b. These proteins displayed positive immunostaining in all the cell lines subtypes, suggesting a derivative from an immature state, comparable to neural crest cells.
NB cell lines can be induced to differentiate along many neural crest cell lineages like chromaffin cells, Schwann cells, melanocytes and neurons [33, 34]. Differentiation inducers towards neuronal lineage like ATRA or glial lineage like BrdU have been thoroughly described in NBT [22, 11, 35]. We showed that, upon ATRA treatment, both N- and I-type cell lines can be induced to differentiate towards a neuronal-like lineage, while, no neuronal-like phenotype was observed in the S-type cell line, despite the gene expression changes detected.
Furthermore, with BrdU treatment, our results for N- and I-type cell lines showed a similar shift towards a glial-like phenotype (S-type shape, reduced GD2 and increase of glial-lineage markers) for both cell subtypes. Recently, Narath et al. (2007)  described cellular senescence in MYCN amplified F-type NBT cells after 6 weeks of hydroxyurea treatment. F-type cells share some morphological features with the S-type cells. In our experiments, the abnormal aspect of BrdU treated cells and proliferation arrest after few weeks of treatment suggested that long exposition to BrdU might induce senescence. Our results, however, demonstrate that BrdU induced bona fide glial differentiation for the first two weeks of treatment. After longer exposure (3 weeks) to BrdU, senescence was induced.
In order to further correlate the embryonic development of the PNS and the in vitro model, we studied Sox10 induction. Sox10, which is expressed in NCSC and a subset of neural-crest derived lineages, plays a key role in maintaining pluripotency and inhibiting premature differentiation at the stem cell stage. In vivo, glial-lineage differentiation is a default program during the NCSC differentiation pathway towards a neuronal fate . Downregulation of SOX10 is a prerequisite for neuronal lineage differentiation and its expression is maintained in the terminally differentiated glial cell [36, 10]. Our results show that SOX10 is undetectable in all cell lines and ATRA treatment does not activate SOX10 expression. However, SOX10 expression is induced when glial differentiation is promoted by BrdU treatment with a fast increase of SOX10 expression in the S-type cell line and slower but significant increase in the N- and I-type cell lines.
Our results suggest that N- and I-type NB cells represent an immature bilineage stage, able to progress towards neuronal as well as glial fates upon induction of differentiation.
In addition to neurite elongation, neuron specific protein and gene expression induction, when N- and I-type cell lines were induced to differentiate with ATRA, two phenomena appeared concomitantly: clumps floating spheres similar to the previously described neurospheres  and newly formed S-type cells. Neurospheres formation has been previously reported to occur spontaneously in N-type cell lines . The spontaneous or induced formation of neurospheres or big floating cell clusters has been described related to neural stemness . Ongoing experiments in our laboratory are investigating whether the differentiating neurospheres contain multipotent stem-like cells.
Concurrently to neuroblastic cells clustering, S-like cells appeared in the periphery of these cell clusters. Motohashi et al., (2007)  have recently reported the presence of glial cells in ATRA treated NCSC cultures. Neuron-glia interactions control several processes in brain development, such as neurogenesis, myelination, synapse formations and neuronal migration, proliferation and differentiation [39, 40]. During PNS development neuronal and glial differentiation are interdependent and the fates of these two types of cells are inextricably entangled. Moreover, in neuroblastoma, it has been hypothesized that Schwann cells release factors that could induce neuronal differentiation, providing better prognosis . Our results, first the appearance of S-like cells during induction of differentiation with ATRA in N- and I-type cells, and second the incapacity of these cells to survive devoid of N-/I-type cells, suggest a necessary cross-talk between immature neuron-glia in neuroblastoma tumor cells upon induced differentiation.
Overall, our findings highlight how N- and I-type cells possess a similar differentiation potential, and represent an uncommitted stage between neuronal and glial fates, whereas S-type cells seem to be committed towards a glial lineage fate. These comparable characteristics display, however, a degree of variability within and amongst the N- and I-cell line subtypes suggesting a continuum process in the differentiation program reproduced by the cell lines.