Human Periapical-cysts Mesenchymal Stem Cells Differentiate into Dopaminergic Neuromelanin-Producing Neurons

Objectives: Parkinson's Disease (PD) is a neurodegenerative disease characterized by the degeneration of Dopaminergic neuromelanin-producing Neurons (DN) at midbrain; DN replacement could represent a promising approach for treating PD. The objective of this investigation was to assess the ability of the newly discovered Human-Periapical-Cysts Mesenchymal Stem Cells (hPCy-MSCs) to differentiate into functional DN. Methods: HPCy-MSCs and Dental Pulp Stem Cells (DPSCs) were isolated, as previously described, and plated with different culture media (CM). Batches of both MSCs lineages were exposed to specific neurogenic conditions (NC), on different surfaces: onto poly-L-ornithine/laminin-coated Tissue Culture Plates (TCPs) (coated group) and uncoated TCPs (uncoated group). Both groups were also grown in basal culturing conditions (BC), as control. Molecular-biology assays were performed. Both groups, under NC and BC, were then exposed to Levo-DOPA to ensure that Neuromelanin (NM) production occurred only under specific conditions. HPCy-MSCs, under NC and BC, were subjected to electrophysiological characterization of the membrane potential (Vm) with intracellular microelectrodes, to confirm whether such cells were functional DN. Lactate levels were also tested in both groups, under NC and BC. Results: Flow-cytometry and immunofluorescence assays revealed that hPCy-MSCs and DPSCs naively express neuron-specific β-III tubulin and glial-fibrillary-acidic-protein (GFAP), already under BC. Under NC, hPCy-MSCs showed an enhanced gene-expression of the dopaminergic-neuronal markers nuclear-Receptor-Related-protein-1 (Nurr1), paired-like-homeodomain-transcription-factor-3 (Pitx3), tyrosine-hydroxylase (TH) and Engrailed-1 (En1). We found NM levels significantly higher in the medium of cells exposed to NC, despite both conditions were exposed to Levo-DOPA. Interestingly, hPCy-MSCs under NC showed spontaneous oscillatory fluctuations in Vm, that we never observed in undifferentiated cells: the oscillations were regular and ranged 12,4±0,7mV (n=8). Decreased Lactate levels in medium of cells under NC confirmed neural-differentiation. Conclusions: HPCy-MSCs differentiate into functional DN, when exposed to NC. Our research identifies hPCy-MSCs as innovative source of DN for cell-based therapies to treat PD.