Abstract
IntroductionInterest in the use of dental pulp stem cells (DPSC) to enhance neurological recovery following stroke and traumatic injury is increasing following successful pre-clinical studies. A murine model of autologous neural stem cell transplantation would be useful for further pre-clinical investigation of the underlying mechanisms. However, while human-derived DPSC have been well characterised, the neurogenic potential of murine DPSC (mDPSC) has been largely neglected. In this study we demonstrate neuronal differentiation of DPSC from murine incisors in vitro.MethodsmDPSC were cultured under neuroinductive conditions and assessed for neuronal and glial markers and electrophysiological functional maturation.ResultsmDPSC developed a neuronal morphology and high expression of neural markers nestin, ßIII-tubulin and GFAP. Neurofilament M and S100 were found in lower abundance. Differentiated cells also expressed protein markers for cholinergic, GABAergic and glutaminergic neurons, indicating a mixture of central and peripheral nervous system cell types. Intracellular electrophysiological analysis revealed the presence of voltage-gated L-type Ca2+ channels in a majority of cells with neuronal morphology. No voltage-gated Na+ or K+ currents were found and the cultures did not support spontaneous action potentials. Neuronal-like networks expressed the gap junction protein, connexin 43 but this was not associated with dye coupling between adjacent cells after injection of the low-molecular weight tracers Lucifer yellow or Neurobiotin. This indicated that the connexin proteins were not forming traditional gap junction channels.ConclusionsThe data presented support the differentiation of mDPSC into immature neuronal-like networks.
Highlights
Interest in the use of dental pulp stem cells (DPSC) to enhance neurological recovery following stroke and traumatic injury is increasing following successful pre-clinical studies
Transmission electron microscopy demonstrated an elaborate ultrastructure of murine DPSC (mDPSC) with large irregularly shaped nuclei (Figure 1E), extensive rough endoplasmic reticula (Figure 1F, arrows) and an intricate outer membrane of microvilli-like projections
We found that during the plating and epigenetic reprogramming (ER) stages there was a significant increase in cell number from an average at plating of 20,000 cells to 43,000 by the third day of the neuronal induction protocol, likely due to continued cell proliferation (Figure 1J)
Summary
Interest in the use of dental pulp stem cells (DPSC) to enhance neurological recovery following stroke and traumatic injury is increasing following successful pre-clinical studies. A murine model of autologous neural stem cell transplantation would be useful for further pre-clinical investigation of the underlying mechanisms. Pre-differentiation of human DPSC promoted greater cell survival and neural differentiation following rat cortical lesion [7], which could be reflected therapeutically with greater functional recovery. Given their potential for autologous transplantation and therapeutic applications in dental engineering and neurological disease treatment, the focus to date has been on applications for human-derived DPSC. A murine model of autologous DPSC transplantation would, be of great utility
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