Abstract
Background: Human mesenchymal stem cells (hMSC) can be derived from various tissue sources and differentiated into dopaminergic (DAergic) neurons using various types of inducers. There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible. However, this area is still under extensive research. In this study, we aim to compare hMSCs derived from bone marrow (BM), adipose tissue (AD) and dental pulp (DP) to generate functional dopaminergic neurons, using FGF2 and forskolin. Post-differentiation, multiple factors were used to characterize the cells at morphological, morphometric, ultra-structural, mRNA and protein levels for various markers (Nestin, NF, MAP2, Tuj1, TH, DAT, PitX3, Ngn2, Kv4.2, SCN5A). Cells’ functionality was studied by calcium ion imaging, along with the amount of dopamine secreted by the cells in the culture medium. Results: Data analysis revealed that forskolin has comparable effect on BM- and AD-derived MSC (28.43% and 29.46% DAergic neurons, respectively), whereas DP-MSC (42.78 ± 1.248% DAergic neurons) show better outcome in terms of efficient generation of DAergic neuronal cells, expression of neuronal associated markers, dopamine release and calcium ion efflux. Ultra-structural studies by SEM and TEM also revealed a substantial change in both cellular morphology and composition of cellular organelles. It was observed that AD-MSCs showed the best neuronal features, at morphological, gene, and protein levels upon induction with the above-mentioned induction cocktail. Conclusion: It may be concluded that a combination of FGF2 and forskolin yields functionally active dopaminergic neuronal cells in vitro, with highest percentage of the same from AD-MSCs, as compared to that in BM-MSCs and DP-MSCs. The outcomes and comparative evaluation provide a substantial platform for further studies on molecular pathways involved in the process of DAergic neurogenesis in individual cases.
Highlights
Human mesenchymal stem cells can be derived from various tissue sources and differentiated into dopaminergic (DAergic) neurons using various types of inducers.There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible
In the current study, we report the in vitro differentiation of human MSCs derived from bone marrow, adipose tissue and dental pulp by using FSK along with FGF2 in minimal concentration to yield dopaminergic neuronal cells
Bone Marrow Mesenchymal Stem Cells (BM-MSC) were obtained by direct plating of bone marrow on to the culture dish and adipose tissue (AD)-MSC and dental pulp (DP)-MSCs were obtained by explant culture
Summary
There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible. We aim to compare hMSCs derived from bone marrow (BM), adipose tissue (AD) and dental pulp (DP) to generate functional dopaminergic neurons, using FGF2 and forskolin. Its formation is promoted by activation of adenylyl cyclate by FSK, which further occurs after the ligation of G-protein coupled receptors by ligands like prostaglandins, hormones, and pharmacologic agents [3]. This cAMP signaling pathway plays vital role in several cellular mechanisms like cell differentiation, metabolism, and apoptosis. Neurological disorders are toilsome in management and treatment, as damaged neurons have constrained regenerative potential and the adult nervous tissue exhibit very poor neuro-regeneration
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