Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells.Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR.Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs.Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.

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The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Nestin, a currently used marker of neural stem cells, is transiently coexpressed with glial fibrillary acidic protein (GFAP) during development and is induced in reactive astrocytes following brain injury. Nestin expression has also been found in cultures of astroglial cells, but little is known about the fate and the mitotic activity of nestin-expressing cells in this in vitro model. The present study reveals a long-lasting expression of nestin in primary cultures of astroglial cells derived from the rat brain. Over 70% of the cells were nestin(+) at 12 weeks, with a large majority coexpressing the GFAP astrocytic marker. Time-course analyses supported a transition from a nestin(+)/GFAP(-) to a nestin(+)/GFAP(+) phenotype over time, which was further increased by cell cycle arrest. Interestingly, double staining with Ki67 revealed that over 90% of cycling cells were nestin(+) whereas only 28% were GFAP(+) in a population consisting of almost equivalent numbers of nestin(+) and GFAP(+) cells. These observations indicated that nestin(+)/GFAP(-) cells are actively engaged in mitotic activity, even after 2 weeks in vitro. Part of these cells might have retained properties of neural stem cells, insofar as 10% of cells in a primary culture of glial cells were able to generate neurospheres that gave rise to both neurons and astrocytes. Further studies will be necessary to characterize fully the proliferating cells in primary cultures of glial cells, but our present results reveal a major contribution of the nestin(+)/GFAP(-) cells to the increase in the number of astrocytes, even though nestin(+)/GFAP(+) cells proliferate also.