Differentiation of human dental pulp stem cells into functional motor neuron: In vitro and ex vivo study

• Published in: Tissue & cell 2021-10, Vol.72, p.101542-101542, Article 101542
• Main Authors: Darvishi, Marzieh, Hamidabadi, Hatef Ghasemi, Bojnordi, Maryam Nazm, saeednia, Sara, Zahiri, Maria, Niapour, Ali, Alizadeh, Rafieh
• Format: Article
• Language: English
• Subjects: Biomarkers; Brain slice preparation; Brain-derived neurotrophic factor; Calcium (intracellular); Calcium ions; CD105 antigen; CD34 antigen; CD45 antigen; CD73 antigen; Cell culture; Cell Differentiation; Cell therapy; Cells, Cultured; Clinical trials; Co-cultivation; Culture; Dental pulp; Dental Pulp - cytology; Differentiation; Evaluation; Fibroblast growth factor 2; Genes; Glial cell line-derived neurotrophic factor; Glial cells; Human dental pulp stem cells (hDPSCs); Humans; Mesenchyme; Motor Neurons - cytology; Neural stem cells; Neural Stem Cells - cytology; Neurogenesis; Neuronal-glial interactions; Organotype culture; Phenotypes; Pluripotent Stem Cells - cytology; Pluripotent Stem Cells - metabolism; Reproducibility of Results; RNA, Messenger - genetics; RNA, Messenger - metabolism; Slice culture; Spheroids, Cellular - cytology; Spinal cord; Spinal Cord - cytology; Spinal cord injuries; Stem cells; Stem Cells - cytology
• ISSN: 0040-8166; 1532-3072
• DOI: 10.1016/j.tice.2021.101542

•Neuronal maturity of hDPSCs-derived motor neuron was demonstrated in our ex vivo model.•The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12.•The organotypic slice culture can be utilized for functional studies of stem cell-based therapies. There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and ; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slices.