Basic fibroblast growth factor-induced cholinergic neuronal differentiation of tonsil-derived mesenchymal stem cells

Abstract

Background & Aim Mesenchymal stem cells (MSCs) have been considered as a potential tool in regenerative medicine for their great multipotency characteristics into various cell types. In past years, our group have been studying tonsil-derived MSCs (TMSCs) for their application and ease of obtaining large number and low immunogencity. Recently we attempted to expand potential regenerative implications of TMSCs not only for mesoderms but also for ectoderms, specifically neurons. Methods, Results & Conclusion Our initial study demonstrated that TMSCs incubated in the presence of 25 ng/mL of basic fibroblast growth factor and 10 μM of forskolin for 22 days resulted in large number of cells containing neurites which are morphologically similar with those observed with neurons. Western blot analyses determined that the TMSCs expressed the various neuronal markers, including neuron-specific enolase, growth-associated protein-43 (GAP43), postsynaptic density protein 95, synaptosomal nerve-associated protein 25, compared with the undifferentiated TMSC group. The expression of these markers dramatically increased at day 8 of the neuronal differentiation and further increased thereafter until day 22. In contrast, glial fibrillary acidic protein, a marker of neural stem cell/astrocyte, gradually declined as the neuronal differentiation progresses. In contrast, neither of oligodendrocyte marker O4 nor microglia marker CD11b was detected. Further studies revealed that the differentiated TMSCs showed enhanced immunoreactivities of mature neuronal markers such as neuronal nuclei, neuron-specific class III β tubulin, GAP43, and synaptophysin. Additionally, the differentiated TMSCs exhibited cholinergic neuronal phenotype as evidenced with the increased expression of choline acetyltransferase, a cholinergic neuronal marker but not vesicular glutamate transporter 1, a glutamatergic neuronal marker. Taken altogether, the findings suggest that TMSCs have a potential to differentiate into cholinergic neuron-like cells under the established differentiation condition. Although further optimization to increase neuronal cell population within heterogenous TMSC pools and functional validation are required, our study provides a glimpse of evidence that TMSC-derived cholinergic neurons could be used as a possible therapeutic tool in treating certain neurodegenerative disorders such as Alzheimer's diseases. The present study was supported by a research grant of the National Research Foundation of Korea (2019R1A2C108676711).