Abstract
ABSTRACTA decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer’s disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of β-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 µg/ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 µM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 µM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuron-like morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.
Highlights
Neurons are the basic functional units of the nervous system facilitating the transmission of electrical impulses
Under protocol III, the DPSCs were cultured in serum-free advanced Dulbecco’s modified Eagle’s medium (ADMEM) containing 10 ng/ml of basic fibroblast growth factor, 50 μM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 μM of retinoic acid (RA) for 7 days
After isolation of DPSCs from dental pulp tissues, the cells were cultured in 10% ADMEM and maintained in a humidified incubator with 5% CO2
Summary
Neurons are the basic functional units of the nervous system facilitating the transmission of electrical impulses. MSCs when treated with neuron specific inductions can result into the development of functionally distinct types of neurons such as cholinergic neurons, amino acidergic neurons (γ-aminobutyrinergic neurons), and aminergic neurons (dopaminergic, 5-hydroxytryptaminergic, and norepinephrinergic neurons) (Sun et al 2013) These different types of neurons have different capabilities to release specific neurotransmitters and play specialized roles. Strategies concerning the protection or development of these valuable cells are very much needed to overcome such problems which can be made possible by using cell based therapies In one such example of a pathological condition in a monkey model of Huntington’s disease, degeneration of amino acidergic neurons has been lowered by using encapsulated cells delivering neuroprotective effects by producing neurotrophic factor CNTF (Cilliary neurotrophic factor) (Emerich et al 1997). The loss or dysfunction of cholinergic neurons leads to the reduction of acetylcholine (Ach) resulting in the motor nerve degeneration and irreversible cognitive decline as observed in
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