Functional identification of neurons and oligodendrocytes differentiating from neural stem cells cultures

João O. Malva

Neuroprotection and Neurogenesis in Brain Repair Group, Center for Neurocience of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal

A major problem faced by research groups and companies dedicated to regenerative medicine of the brain resides on the lack of good methods to functionally identify neural stem cell differentiation and novel drug targets. We describe a unique single cell calcium imaging-based method allowing the functional discrimination of different cell types, derived from neural stem cell cultures. The unique functional profile of each cell type was correlated at single cell level with the immunodetection of phenotypic markers including nestin (immature cells), GFAP (astrocytes), doublecortin (neuronal progenitors), MAP 2 (neurons) and O4 (oligodendrocytes) in subventricular zone (SVZ) cell cultures. This platform was raised on the basis of our previous work where we described the unique functional response of neurons and immature cells to depolarizing agents and to histamine, respectively (Agasse et al, 2008 Rejuvenation Research; Agasse et al, 2008 Stem Cells; Bernardino et al, 2008 Stem Cells).

The unique development of this novel strategy to functionally identify oligodendrocyte differentiation resides on the specific stimulation of this cell type with thrombin, acting through PAR-1 receptors. Moreover, we describe that PAR-1 receptor activation triggers the oligodendroglial increase of the intracellular calcium concentration, involving PLC-induced recruitment of calcium from internal stores. As a proof of concept we treated SVZ cultures with thyroid hormone, stem cell factor or cilliary neurotrophic factor to enrich the cultures in oligodendrocytes, neurons or astrocytes, respectively. In agreement with our working hypothesis, the resulting profile of population responses revealed the efficiency of the procedure.

In conclusion, the present technological platform is extremely relevant in the context of drug discovery and development in general area of regenerative medicine of the brain.

Acknowledgments: PTDC/SAU-NEU/68465/2006, Fundação para a Ciência e a Tecnologia and FEDER.