Neurogenic induction of human dental pulp derived stem cells by hanging drop technique, basic fibroblast growth factor, and SHH factors

Background: The progressive destruction of nerve cells in nervous system will induceneurodegenerative diseases. Recently, cell‑based therapies have attracted the attention of researchersin the treatment of these abnormal conditions. Thus, the aim of this study was to provide a simpleand efficient way to differentiate human dental pulp stem cells into neural cell‑like to achieve ahomogeneous population of these cells for transplantation in neurodegenerative diseases.Materials and Methods: In this basic research, human dental pulp stem cells were isolatedand characterized by immunocytochemistry and flow cytometry techniques. In the following, thecells were cultured using hanging drop as three‑dimensional (3D) and tissue culture plate as 2Dtechniques. Subsequently, cultured cells were differentiated into neuron cell‑like in the presenceof FGF and Sonic hedgehog (SHH) factors. Finally, the percentage of cells expressing Neu N andβ tubulin III markers was determined using immunocytochemistry technique. Finally, all data wereanalyzed using the SPSS software.Results: Flow cytometry and immunocytochemistry results indicated that human dental pulp‑derivedstem cells were CD90, CD106‑positive, but were negative for CD34, CD45 markers (P ≤ 0.001).In addition, the mean percentage of β tubulin positive cells in different groups did not differsignificantly from each other (P ≥ 0.05). Nevertheless, the mean percentage of Neu N‑positivecells was significantly higher in differentiated cells with embryoid bodies’ source, especially in thepresence of SHH than other groups (P ≤ 0.05).Conclusion: It is concluded that due to the wide range of SHH functions and the facilitation ofintercellular connections in the hanging droop method, it is recommended that the use of hangingdrop method and SHH factor can be effective in increasing the efficiency of cell differentiation.Key Words: Basic fibroblast growth factor, mesenchymal stem cells, neurogenesis, SHH