Manipulation of Sonic hedgehog signaling pathway enhance trans differentiation of human dental pulp stem cells into mature oligodendrocytes and neurons

Dental pulp stem (DPS) cells, derived from the neural crest, possess multipotent properties and can be obtained through minimally invasive procedures, making them an attractive candidate for neural regeneration strategies. The Sonic Hedgehog (SHH) signaling pathway and downstream expression of GLI protein family is a key regulator of cell fate determination. GLI expression is associated with the activation of other signaling pathways induct neuronal and oligodendrocytes differentiation from neural stem cells. Therefore, we are attempting to manipulate SHH activity in DPSCs using SHH pathway agonists such as purmorphamine and its inhibitor GANT61, with the aim of identifying the role of interventions of other signaling pathways and genes to optimize the generation of neurons and oligodendrocytes cells from DPSCs.In this study, human dental pulp stem cells were used in a two-step differentiation process and exposed to different concentrations of purmorphamine and Gant61. In the first step generate olig2 cell and then mature cell are formed. After inducing of trans differentiation on each step, the cells were fixed in 4% paraformaldehyde. They were then treated overnight at 4°C with primary antibodies against Olig2+, MBP+, beta III tubulin, and GLI. Following rinses with Triton 20 in PBS, secondary antibodies and Hoechst solution were added for further incubation. Then images were captured using a fluorescent microscope, and cell counts were performed via flow cytometry. The results indicated that Purmorphamine enhanced sonic hedgehog pathway activity, increasing GLI expression, while Gant61 inhibited it. Purmorphamine raised the number of immature oligodendrocytes (Olig2+), whereas Gant61 reduced their count. The oligodendrocytes (MBP + cells) maturation increased with high dose Purmorphamine and decreased with Gant61 treatment. Additionally, the SOX10 gene expression was influenced by both Purmorphamine and Gant61. Furthermore, neurogenesis was elevated by a low dose of Purmorphamine, while Gant61 diminished it, which was accompanied by the expression of Noch1 and Hes1 was also increased by Purmorphamine treatment in a dose-dependent manner. The study showed that manipulating the Sonic hedgehog pathway can influence the differentiation of human dental pulp stem cells into oligodendrocytes and neurons. This highlights the importance of the Sonic hedgehog pathway and the cross-talk with Notch1- Hes1 signaling pathway and sox 10 expression in optimizing the fate of human dental pulp stem cells.