Clonal cell cultures from adult spinal cord of the amphibian urodelePleurodeles waltl to study the identity and potentialities of cells during tail regeneration

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Abstract
The urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail. The most striking feature of this model lies in the ability of the spinal cord (SC) to differentiate, within the regenerating tail, a new ependymal tube from which the SC and the peripheral nervous system originate. A fundamental question is whether, in response to tail excision, the ependymoglia of the old SC stump behaves as an embryonic neuroepithelium. To evaluate this possibility, cell lines from primary cell cultures of adult SC were established for the first time in newts, and two cell clones, immunochemically characterized as ependymoglial cell populations, could be obtained. To analyze the potentialities of these clonal cells, after transplantation in tail regenerates, cell-marking experiments, using either in vitro transfection with lacZ gene or the lineage tracer lysinated rhodamine dextran (LRD), were performed. One to 2 weeks postimplantation, most of labeled derivatives were identified as melanocytes. Interestingly, labeled cells were also seen integrated in the ependymoglia of the regenerating SC. Two to 6 weeks after implantation in young regenerates, we also observed LRD-labeled elongated cells close to nerves or myofibers which were unambiguously identified as Schwann cells by galactocerebroside staining. Taken together, these findings showed that clonal cells derived from adult newt SC cultures could largely find, in regenerate mesenchyme, suitable environmental conditions to differentiate into melanocytes or Schwann cells. Because these two cell types arise from neural crest cells during embryo-genesis, this supports the interesting view that multipotent cells are still present in the SC of adult urodeles.