Comparative analysis of three different protocols for cholinergic neuron differentiation in vitro using mesenchymal stem cells from human dental pulp

Abstract

A 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.

Human Dental Pulp-Derived Mesenchymal Stem Cell Potential to Differentiate into Smooth Muscle-Like Cells In Vitro

“…Evaluation of these minimal features has been the basis for selecting cells for in vitro and in vivo experimental purposes. DPSCs displayed high stemness features in accordance with the previously published reports [ 6 , 16 ].…”

“…After getting donor's consent and approval from the committee for clinical research at GNUCH (GNUCH-2018-11-002), samples were aseptically transported to the laboratory within 3-4 hours. Dental pulp-derived cells were isolated according to the previously published protocol [ 16 ]. Briefly, dental pulp tissues ( n = 5) were carefully separated from the extracted wisdom tooth using a sterile scalpel, gently washed several times with 1% penicillin-streptomycin (10,000 IU and 10,000 μ g/ml, respectively; Pen-Step, Gibco Life Technologies) containing sterile Dulbecco's phosphate-buffered saline (DPBS), and then minced into small pieces using fine surgical blades.…”

Latent Tri-lineage Potential of Human Menstrual Blood–Derived Mesenchymal Stromal Cells Revealed by Specific In Vitro Culture Conditions

“…Effectively, MenSCs-derived ChLNs signi cantly expresses cholinergic markers ChAT -the enzyme that catalyzes the biosynthesis of ACh [29], and VAChT -the ACh neurotransmitter transporter [16], two unique markers for cholinergic neurons according to WB, IMF, and FC analysis. In agreement with others, who have demonstrated that MSCs from dental pulp [30], adipose tissue [31], and UC-WJ [12] can transdifferentiate into ChNs/ ChLNs, we show that MenSCs readily transdifferentiated into functional ChLNs. Similar to UC-WJ-MSCs-derived ChLNs [12], the MenSCs-derived ChLNs are responsive to ACh stimuli on days 4 and 7.…”

Latent tri-lineage potential of human menstrual blood-derived mesenchymal stromal cells revealed by specific in vitro culture conditions

“…Effectively, MenSCs-derived ChLNs signi cantly expresses cholinergic markers ChAT -the enzyme that catalyzes the biosynthesis of ACh [29], and VAChT -the ACh neurotransmitter transporter [16], two unique markers for cholinergic neurons according to WB, IMF, and FC analysis. In agreement with others, who have demonstrated that MSCs from dental pulp [30], adipose tissue [31], and UC-WJ [12] can transdifferentiate into ChNs/ ChLNs, we show that MenSCs readily transdifferentiated into functional ChLNs. Similar to UC-WJ-MSCs-derived ChLNs [12], the MenSCs-derived ChLNs are responsive to ACh stimuli on days 4 and 7.…”