Abstract
The development of dopaminergic (DA) neurons is a very complex process, and a combination of extrinsic and intrinsic factors involves their differentiation. Transcription factor, Nurr1 plays an essential role in the differentiation and maintenance of midbrain DA neurons. Nurr1-based therapies may restore DA function in Parkinson's disease (PD) by replacing damaged cells with differentiated cells derived from stem cells. Providing tissue-specific microenvironments such as brain extract can effectively induce dopaminergic gene expression in stem cells. The present study aimed to investigate the combined effects of Nurr1 gene overexpression and a neonatal rat brain extract (NRBE) induction on dopaminergic differentiation of P19 stem cells. In order to neural differentiation induction, stably Nurr1-transfected cells were treated with 100 μg/ml of NRBE. The differentiation potential of the cells was then evaluated during a period of 1–3 weeks via various methods. The initial evaluation of the cells by direct observation under a light microscope and cresyl violet specific staining, confirmed neuron-like morphology in the differentiated cells. In addition, different molecular and cellular techniques, including real-time PCR, immunofluorescence, and flow cytometry, demonstrated that the treated cells expressed pan-neuronal and dopaminergic markers. In all experimental groups, neuronal phenotype with dopaminergic neuron-like cells characteristics mainly appeared in the second week of the differentiation protocol. Overall, the results of the present study revealed for the first time the synergistic effects of Nurr1 gene overexpression and possible soluble factors that existed in NRBE on the differentiation of P19 stem cells into dopaminergic neuron-like cells.
Highlights
In vitro production of midbrain dopamine neurons from stem cells is a promising approach for managing and treating Parkinson’s disease (PD)
To induce neural differentiation of P19 cells, they were stably transfected with a vector containing Nurr1 gene and GFP reporter gene under the control of cytomegalovirus (CMV)
We have shown that combined molecular cloning and tissue-specific induction strategies could efficiently enhance the neural differentiation of stem cells with dopaminergic neuron-like phenotypes
Summary
In vitro production of midbrain dopamine neurons from stem cells is a promising approach for managing and treating Parkinson’s disease (PD). Previous studies have demonstrated that orphan nuclear receptor Nurr (nuclear receptor-related 1 protein, known as NR4A2; nuclear receptor 4A2), as a transcriptional factor, plays an essential role in the differentiation and maintenance of midbrain DA neurons. Exogenous Nurr inserted in neural stem cells (NSCs) induced TH expression in a concentration-dependent manner (Kim et al, 2007). It has been previously reported that injection of Nurr1-overexpressing cells in combination with glial-derived neurotrophic factor (GDNF) or neurturin into the midbrain might protect DA neurons against toxicity of α-synuclein (Decressac et al, 2012; Bartus et al, 2013)
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