Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation
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 βIII-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.
KeywordsOral stem cells OSCs Adult stem cells Neural stem cells NSCs Neurogenesis Neurosphere Neurons Dental pulp stem cells Gingival mesenchymal stem cells Immunocytofluorescence qPCR Electrophysiology Patch clamp Action potential
Bovine serum albumin
Human dermal fibroblasts
Dental pulp stem cells
Human foreskin fibroblasts
Gingiva-derived mesenchymal stem cells
Human embryonic stem cell
Induced pluripotent stem cells
Mesenchymal Stromal/Stem Cells
Neural differentiation medium
Neurogenic maturation medium
Neural stem/progenitor cell
Oral stem cells
Quantitative real-time polymerase chain reaction
Stem cell of apical papilla
The authors thank Dr. Kristen O’Connell (UTHSC) for her assistance during the early developmental stage of the project; Dr. Rebeca Caires Mugarra (UTHSC) for initial assistance in the patch-clamp studies; and UTHSC Biostatistic BERD Consulting Program for statistical support.
DL designed and performed the experimental work, acquired, assembled, analyzed the data, and drafted/revised the manuscript. XYZ initiated the project and along with IEA, LOR, ZY, and AIL performed some of the experimental work, assembled, analyzed the data and drafted/revised part of the manuscript. JFCM supervised some of the experimental work, assembled, analyzed the data and drafted/revised part of the manuscript. GTJH conceived, designed, performed experimental works and supervised the overall project, analyzed and interpreted the data and finalized the manuscript. All authors have read and approved the manuscript for publication.
This work was supported in part by grants from the National Institutes of Health R01 DE019156 (G.T.-J.H.), AHA 15SDG25700146 (J.F.C.), National Institutes of Health R01 GM125629–01 (J.F.C-M) and a Research Fund from the University of Tennessee Health Science Center (G.T.-J.H.).
Compliance with Ethical Standards
Ethics Approval and Consent to Participate
To be discarded extracted teeth were collected from Clinics at Boston University (BU) and University of Tennessee Health Science Center (UTHSC) based on exempt protocols approved by the respective Medical Institutional Review Board (BU: #H-28882; and UTHSC:#12–01937-XM).
Consent for Publication
This manuscript has been approved by all authors and is solely the work of the authors named.
The authors declare that they have no competing interests.
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