Defining a small molecules differentiation cocktail for generation of dopaminergic neurons from Wharton’s jelly mesenchymal stem cells

Abstract

The incidence of neurodegenerative diseases is on the rise, and yet patients remain without a cure. A suitable disease model could be applied for drug development, disease progression modelling and regenerative therapy. The study’s primary aim was to define a fully-characterised dopaminergic neuronal transdifferentiation protocol for research and therapeutic purposes, with the principal objective being the treatment of Mesenchymal Stem Cells (MSCs) with a 3-stage small molecule strategy. Using RT-qPCR, RNA transcript levels for neural markers were assessed in differentiating cells. Changes in protein lysine and arginine methylation and dopamine-release in differentiating cells were explored using Western blotting, and ELISA respectively. Changes in cell structure towards a neuronal morphology and neurite outgrowth increased considerably with each stage of differentiation. RNA transcript levels suggested that Stage 2 cells had differentiated into immature neuronal cells. Substantial changes in protein methylation levels of cells at different treatment stages were found, and dopamine release increased in Stages 2- and 3-treated cells. ELISA results suggest that the protocol was successful at specifying a dopaminergic fate, though the cells might require further maturation in culture. Mass Spectrometry results revealed hundreds of unique proteins between MSCs and Stage 3 induced neurons, with expression of many neuron-associated proteins in the final neuronal cells. More experiments are required to conclude on the cellular differentiation extent achieved, cell fate, and functionality, and to fully optimise the protocol for dopaminergic differentiation of MSCs. The results produced in this study reveal the potential of this research and warrant the study’s further investigation.