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Method for the Functional identification of SVZ-derived oligodendrocytes based on selective resuponse to thrombin and utilization thereof

Publication date: 05-04-2012 10:58

Terapia e Diagnostico

Breve descrição

Neural stem cells from the subventricular zone (SVZ) give rise to progenitors endowed with the capacity to differentiate in the major cell types of the CNS, including the myelin-forming oligodendrocytes. In the present work, we examined whether living SVZ-derived oligodendrocytes could be functionally discriminated on the basis of intracellular free calcium level ([Ca2+]i) variations following 50 mM KC1, 100 [mu] M histamine or 0,1 U/ml thrombin stimulations. For this purpose, SVZ cell cultures, derived from neonatal P 1-3 C57B1/6 donor mice, were treated with the triiodo-L-thyronine (T3) hormone in order to increase the differentiation into oligodendrocytes. Our group has previously shown that SVZ-derived neurons and immature cells can be discriminated by their selective ([Ca2+]i) increase triggered by 50 mM KC1 and 100 [mu] M histamine stimulations respectively. In the present study, we demonstrate that O4-positive oligodendrocytes, as observed previously in astrocytes, do not respond to any of these stimuli. However, O4-positive oligodendrocytes display a specific increase in ([Ca2+]i) following stimulation with thrombin. ([Ca2+]i) levels are not altered by thrombin perfusion in nestin-immature cells, astrocytes and neurons. Hence, oligodendrocytes can be distinguished from other SVZ-derived cell types based on the selective ratio of response following stimulation with thrombin and histamine. Thrombin-induced calcium increase in SVZ-derived oligodendrocytes is mediated by the protease-activated receptor 1 (PAR-1) and involves the downstream activation of phospholipase C (PLC) with subsequent recruitment of calcium from the internal stores. Oligodendroglial differentiation in SVZ cell cultures is mainly analyzed by immunocyto- chemistry as there is no reliable tool allowing the evaluation of their functional differentiation. Evaluation of functional SVZ-derived oligodendrocytes in living cultures is of major interest as it allows the subsequent use of these cells for grafting strategies in demyelinated diseased brains. The method described in the present work may open new perspective for the identification of new pro-oligodendrogenic factors.

Propriedade intelectual

Patente nº WO2010046876
Data de prioridade 22-10-2008
Estado legal Pendente
Documento de patente