Basic Fibroblast Growth Factor Induces Cholinergic Differentiation of Tonsil-Derived Mesenchymal Stem Cells

Abstract

Background: Mesenchymal stem cells (MSCs) are considered a potential tool for regenerating damaged tissues due to their great multipotency into various cell types. Here, we attempted to find the appropriate conditions for neuronal differentiation of tonsil-derived MSCs (TMSCs) and expand the potential application of TMSCs for treating neurological diseases.

Methods: The TMSCs were differentiated in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) supplemented with various neurotrophic factors for 7-28 days to determine the optimal neuronal differentiation condition for the TMSCs. The morphologies as well as the levels of the neural markers and neurotransmitters were assessed to determine neuronal differentiation potentials and the neuronal lineages of the differentiated TMSCs.

Results: Our initial study demonstrated that DMEM/F12 supplemented with 50 ng/mL basic fibroblast growth factor with 10 μM forskolin was the optimal condition for neuronal differentiation for the TMSCs. TMSCs had higher protein expression of neuronal markers, including neuron-specific enolase (NSE), GAP43, postsynaptic density protein 95 (PSD95), and synaptosomal-associated protein of 25 kDa (SNAP25) compared to the undifferentiated TMSCs. Immunofluorescence staining also validated the increased mature neuron markers, NeuN and synaptophysin, in the differentiated TMSCs. The expression of glial fibrillar acidic protein and ionized calcium-binding adaptor molecule 1 the markers of astrocytes and microglia, were also slightly increased. Additionally, the differentiated TMSCs released a significantly higher level of acetylcholine, the cholinergic neurotransmitter, as analyzed by the liquid chromatography-tandem mass spectrometry and showed an enhanced choline acetyltransferase immunoreactivity compared to the undifferentiated cells.

Conclusion: Our study suggests that the optimized condition favors the TMSCs to differentiate into cholinergic neuron-like phenotype, which could be used as a possible therapeutic tool in treating certain neurological disorders such as Alzheimer's disease.

Keywords: Basic fibroblast growth factor; Neuronal differentiation; Tonsil-derived mesenchymal stem cells.

Fig. 1

Optimization of the neuronal inductive conditions for the TMSCs. A The TMSCs (5.0 × 10⁵) were initially seeded in 100 mm² Petri dishes, and preconditioned in either DMEM/F12 supplemented with 1% FBS or NBM supplemented with 2% B27 and 2 mM L-glutamine for 7 days. The morphology of the TMSCs was observed under the light microscope (40X) (scale bar = 200 μm). B The TMSCs in the DMEM/F12 media were treated with various neurotrophic factors including BDNF, CNTF, FGF4 and bFGF with or without 10 μM forskolin for 14 days. The protein expression of neural marker NSE was determined using western blotting analysis for 14 days. C, D The TMSCs were treated with 10–50 ng/mL of bFGF with or without 10 μM of forskolin. The neuronal morphology was observed under a light microscope (40X; C), and the protein expression of NSE was determined using western blotting analysis (D). E The protein expression of NSE in the TMSC treated with 20 or 50 ng/mL of bFGF in the presence of 10 μM forskolin for 14, 21, and 28 days. β-actin was used as the loading control for all western blotting analyses. The data in bar graphs represent mean ± standard deviation (SD) from two independent experiments. Student’s t-test was used to test the statistical significance, and denoted as *p < 0.05 or **p < 0.01

Fig. 2

Neuronal characterization of the TMSCs cultured in the differentiation media. The TMSCs were cultured in the DMEM/F12 supplemented with (D) or without (U) 50 ng/mL of bFGF and 10 μM forskolin for 7, 14, and 21 days. A The protein expression of NSE, β3-tubulin, PSD95 and SNAP25 was analyzed using western blotting analysis. α-tubulin was used as the loading control. B The immunofluorescence staining of β3-tubulin (green), NeuN (red) and synaptophysin (red). The cell nuclei were counterstained with DAPI (blue). The fluorescent images of the stained proteins were visualized using a confocal microscope at 40X (scale bar = 50 μm). The data in bar graphs represent the mean ± standard deviation (SD) from three to four independent experiments. Student’s t-test was used to test the statistical significance, and denoted as **p < 0.01 or ***p < 0.001

Fig. 3

Glial characterization of the TMSCs cultured in the differentiation media. The TMSCs were cultured in the DMEM/F12 supplemented with (D) or without (U) 50 ng/mL of bFGF and 10 μM forskolin for 7, 14 or 21 days. A The protein expression of astrocyte GFAP and microglia IBA-1 was analyzed using western blotting analysis. α-Tubulin was used as the loading control. B The immunofluorescence staining of GFAP (green), microglial marker CD11b (green), NeuN (red) and β3-tubulin (red). The cell nuclei were counterstained with DAPI (blue). The fluorescent images of the stained cells were visualized using a confocal microscope at 40X (scale bar = 50 μm). The data in bar graphs represent the mean ± standard deviation (SD) from two independent experimental trials. Student’s t-test was used to test the statistical significance

Fig. 4

Analyzing neuronal phenotypes of the differentiated TMSCs. The TMSCs were cultured in the DMEM/F12 supplemented with (D) or without (U) 50 ng/mL of bFGF and 10 μM forskolin for 21 days. A The concentration of neurotransmitters, GABA, Glu, DA, Ach and 5-HT (serotonin) in the culture media of differentiated TMSCs were analyzed using LC–MS–MS. The relative neurotransmitter concentration in the media was calculated by dividing the media concentrations of the undifferentiated TMSCs (U21) into the media concentrations of the differentiated TMSCs (D21). The data in bar graphs represent the mean ± standard deviation (SD) from two independent experiments. Student’s t-test was used to test the statistical significance, and denoted as *p < 0.05 or **p < 0.01. B The immunofluorescence staining of ChAT (green) and β3-tubulin (red). The cell nuclei were counterstained with DAPI (blue). The fluorescent images of the stained proteins were visualized using a confocal microscope at 40X (scale bar = 50 μm)