Midbrain cues dictate differentiation of human dental pulp stem cells towards functional dopaminergic neurons

Dental pulp originating from the neural crest is considered a better source of postnatal stem cells for cell-based therapies in neurodegenerative diseases. Dental Pulp Stem Cells (DPSCs) have been shown to differentiate into cell-types of cranial neural crest ontology; however, their ability to differentiate to functional neurons of the central nervous system remains to be studied. We hypothesized that midbrain cues might commit DPSCs to differentiate to functional dopaminergic cell-type. As expected, DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III, and Map2ab. On exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor), DPSCs showed upregulation of dopaminergic neuron-specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired-like homeodomain transcription factor 3 (Pitx3) as revealed by real-time RT-PCR. Immunofluorescence and flow cytometry analysis showed enhanced expression of mature neuronal marker Map2ab and dopaminergic-neuronal markers [tyrosine hydroxylase (TH), En1, Nurr1, and Pitx3], with nearly 77% of the induced DPSCs positive for TH. Functional studies indicated that the induced DPSCs could secrete dopamine constitutively and upon stimulation with potassium chloride (KCl) and adenosine triphosphate (ATP), as measured by dopamine ELISA. Additionally, the induced DPSCs showed intracellular Ca(2+) influx in the presence of KCl, unlike control DPSCs. ATP-stimulated Ca(2+) influx was observed in control and induced DPSCs, but only the induced cells secreted dopamine. Our data clearly demonstrate for the first time that DPSCs in the presence of embryonic midbrain cues show efficient propensity towards functional dopaminergic cell-type.