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Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation.

ABSTRACT: The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction methods employed, only one neurosphere-mediated method yielded electrophysiological properties of functional neurons. Under this method, cells expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed ?III-tubulin, CNPase, GFAP, MAP-2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95, and synapsin after the second phase of differentiation to maturity. Electrophysiological experiments revealed that 8.3% of DPSC-derived neuronal cells and 21.2% of GMSC-derived neuronal cells displayed action potential, although no spontaneous excitatory/inhibitory postsynaptic action potential was observed. We conclude that DPSCs and GMSCs have the potential to become neuronal cells in vitro, therefore, these cells may be used as a source for neural regeneration.

SUBMITTER: Li D

PROVIDER: S-EPMC6358481 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation.

Li Dong D Zou Xiao-Ying XY El-Ayachi Ikbale I Romero Luis O LO Yu Zongdong Z Iglesias-Linares Alejandro A Cordero-Morales Julio F JF Huang George T-J GT

Stem cell reviews and reports 20190201 1

The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts ...[more]

PMID: 30324358

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Project description:IntroductionOdontogenic differentiation of human dental pulp stem cells (hDPSCs) is a key step of pulp regeneration. Recent studies showed that circular RNAs (circRNAs) have many biological functions and that competing endogenous RNA (ceRNA) is their most common mechanism of action. However, the role of circRNAs in hDPSCs during odontogenesis is still unclear.MethodsIsolated hDPSCs were cultured in essential and odontogenic medium. Total RNA was extracted after 14 days of culture, and then, microarray analysis was performed to measure the differential expressions of circRNAs. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was then performed to validate the microarray results. Based on microarray data from this study and available in the database, a ceRNA network was constructed to investigate the potential function of circRNAs during odontogenesis. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the potential correlation between signaling pathways and circRNAs. In addition, qRT-PCR and Western blot analysis were used to explore the function of hsa_circRNA_104101.ResultsWe found 43 upregulated circRNAs and 144 downregulated circRNAs during the odontogenic differentiation process (fold change > 1.5 and <-1.5, respectively; P < 0.05). qRT-PCR results were in agreement with the microarray results. Bioinformatic analysis revealed that the Wnt signaling pathway and the TGF-β signaling pathway, as well as the other pathways associated with odontogenic differentiation, were correlated to the differentially expressed circRNAs. hsa_circRNA_104101 was proved to promote the odontogenic differentiation of hDPSCs.ConclusionThis study reported 187 circRNAs that were differentially expressed in hDPSCs during odontogenic differentiation. Bioinformatic analysis of the expression data suggested that circRNA-miRNA-mRNA networks might act as a crucial mechanism for hDPSC odontogenic differentiation, providing a theoretical foundation for the study of pulp regeneration regulation by circRNAs.

Project description:Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Dataset Information

Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation.

Publications

Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation.

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