Fibroblast Growth Factor-2 alone as an efficient inducer for differentiation of human bone marrow mesenchymal stem cells into dopaminergic neurons

Background: The reported efficiency of differentiation of human bone marrow derived Mesenchymal Stem Cells (hBM MSC) into dopaminergic neurons with different inducers is found to vary. Thus, in the current study we have investigated the response of hBM MSC to some of the neuronal inducers and their combinations. Neuronal differentiation inducing agents Fibroblastic Growth Factor 2 (FGF2), Sonic Hedge Hog (Shh), Fibroblastic Growth Factor 8 (FGF8) & All Trans Retinoic Acid (ATRA) were used either singly or in varied combinations. Results: The differentiated and undifferentiated hBM MSC were characterized in terms of morphology, expression of cell markers at transcriptional and translational levels, amount of dopamine secreted by the cells in the media and changes in cell membrane potential by calcium ions imaging. Induced hBM MSC revealed neuron like morphology and expressed cellular markers suggesting neuronal differentiation with all the inducing agents. However, upon quantitative analysis through qPCR, cells induced with FGF2 were found to show maximum expression of tyrosine hydroxylase (TH) by 47.5 folds. Immunofluorescence analysis of differentiated and undifferentiated cells also revealed expression of nestin, neurofilament, microtubule associated protein-2, beta tubulin III and TH in differentiated cells, at translational level. This data was supported by immunoblotting analysis. Further, ELISA study also supported the release of dopamine by cultures induced with FGF2. When the cells were depolarised with KCl solution, those induced with Shh & FGF8 showed maximum calcium ion trafficking, followed by the cells induced with FGF2 only. Conclusions: We conclude that hBM MSC can be coaxed to differentiate efficiently into dopaminergic neurons in the presence of a very simple media cocktail containing only one main inducer like FGF2 and thus contribute towards cellular therapy in Parkinson's and other related disorders. These dopaminergic neurons are also functionally active, as shown by calcium ion trafficking.