Maduración y funcionalidad de neuronas dopaminérgicas derivadas de células troncales neurales humanas de mesencéfalo ventral. Efectos de bcl-xl

Resumen

Parkinson¿s disease is one of the major neurodegenerative disorders characterized by a progressive degeneration and loss of nigrostriatal dopaminergic neurons. Experimental therapies based on cell replacement of the lost Substatia Nigra pars compacta dopaminergic neurons using human ventral mesencephalic fetal tissue, provided the proof of principle that replacement therapies had a therapeutic effect on Parkinson¿s disease patients. However, ethical and logistic problems related to fetal tissue procurement, made necessary to found an alternative source of dopaminergic neurons which could be used routinely in cell replacement therapies. Our group generated a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) using v-myc immortalization. hVM1 cells expressed neural stem cell and radial glial markers under proliferation conditions, and after withdrawal of growth factors, differentiated into astrocytes, oligodendrocytes and neurons. The hVM1 cells yield a large number of dopaminergic neurons (12%), but lose their neurogenic ability after long time of in vitro expansion. The over-expression of Bcl-XL in hVM1 cell line solves this problem and increases the number of dopaminergic neuron generated up to 18%. In the present work, both cell lines (hVM1 and hVM1 over-expressing Bcl-XL) were characterized in detail, to understand the expression of genes and proteins involved in neurogenesis, development of neurons and specifically dopaminergic neurons, maturation and functionality of the long term differentiated cultures. Both cell lines express the proper genes related with acquisition of a dopaminergic A9 phenotype. Interestingly, Bcl-XL over-expressing cultures showed an increase in the number of neuronal progenitors and generated a higher percentage of dopaminergic neurons, which arise earlier during differentiation, and present higher levels of proteins related with maturation and functionality. Moreover, we report here that cells differentiated from hVM1 and hVM1 high Bcl-XL cultures express electrophysiological properties of functional neurons and are able to respond to neurotransmitters in vitro. Finally, we investigated if hVM1 high Bcl-XL cells could be labelled in vitro with SPIOs (superparamagnetic iron oxide nanoparticles) without changing their phenotypical properties. The hVM1 high Bcl-XL SPIO labelled cells could be easily detected by RMI after transplantation in the striatum of hemiparkinsonian rats. Now, we started the studies needed to co-localize RMI with PET images, and develop the protocols to study in parallel the anatomical and functional properties of the transplanted cells in a non-invasive manner.