StemCell Therapy

Abstract Introduction

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. 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.

mDPSC were cultured under neuroinductive conditions and assessed for neuronal and glial markers and electrophysiological functional maturation.

mDPSC developed a neuronal morphology and high expression of neural markers nestin, III-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.

The data presented support the differentiation of mDPSC into immature neuronal-like networks.

Since their discovery as a source of multipotent adult human stem cells by Gronthos et al.[1], numerous groups have confirmed the potential of dental pulp stem cells (DPSC) to differentiate into multiple neural crest-lineage cell types [2-4]. Previous studies in our laboratory and others have demonstrated the neural potential of human-derived DPSC in vitro[2,5] and in vivo[6-8]. Human DPSC were found to express neural markers following injection into the rat and embryonic chick brain [7,8] and also induced endogenous responses through paracrine effects [6,9,10]. In the chick embryo, human DPSC induced neuroplasticity of the highly structured trigeminal ganglion [6] and promoted the recruitment, proliferation and neural differentiation of endogenous precursors in the mouse brain [9]. Interestingly, 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. The cellular and molecular mechanisms underlying recovery in pre-clinical studies of varied animal models of disease are poorly understood. Xenotransplantation is often problematic (that is, human DPSC injected into rodents) due to immune rejection. The mouse is a fundamentally important animal model in relation to understanding human disease, pre-clinical testing, and transgenic potential to gain better knowledge of mechanisms of action. A murine model of autologous DPSC transplantation would, therefore, be of great utility.

Like their human counterparts, rodent DPSC show neural crest multipotentiality [11-14]. However, a distinction has emerged between DPSC from murine molar and incisor teeth. While they both possess osteo-dentin and adipocyte differentiation potential, erupted murine molars, but not incisors, have been found to have chondrocytic potential [11-13,15]. Janebodin et al. [13] have described the expression of neuronal, oligodendrocyte and glial markers after in vitro differentiation of murine molar DPSC. To the best of our knowledge neural differentiation of incisor murine DPSC (mDPSC) has not yet been attempted and could offer an easily accessible source of DPSC for pre-clinical studies. Work by two other groups suggests that rodent incisor DPSC do have neurogenic potential through the successful formation of cells with neuronal-like multipolar morphology that expressed neuronal markers in vitro[16,17] and the promotion of nerve regeneration in vivo using rat incisor DPSC [18]. Neither study reported electrophysiological properties of the rat DPSC after neuronal differentiation.

Herein, we report the in vitro neuronal development of DPSC isolated from murine incisors using a neural differentiation methodology found to generate functional neurons from human DPSC [5]. We found species-specific differences between human and mouse cells and demonstrated that mDPSC develop characteristics suggesting their differentiation into immature neural-like cells. Unique to our study is the interrogation of the neuronal characteristics of mDPSC-derived cells using electrophysiological methodologies, which is fundamental to understanding neuronal function.

Incisors from adult BalbC mice were removed and their pulp exposed to enzymatic digestion with 3 mg/mL collagenase type I and 4 mg/mL dispase in PBS for one to two hours at 37C with 5% CO2. The resulting solution was centrifuged at 200g for five minutes, the supernatant and enzymes removed and the remaining cells cultured in mesenchymal stem cell medium [19] containing alpha-modified Eagles medium (-MEM) supplemented with 10% foetal bovine serum (FBS, Invitrogen, Mulgrave, Victoria, Australia), 1x GlutaMAX (Gibco, Mulgrave, Victoria, Australia), 100 M L-ascorbate-2-phosphate (Wako, Neuss, Germany), 50 U/mL penicillin and 50 g/mL streptomycin (Invitrogen), and dental pulp stem cells were allowed to adhere to the plastic base. Floating debris could subsequently be removed.

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Neurogenic potential of dental pulp stem cells isolated ...

This entry was posted on June 15, 2015 at 8:46 am and is filed under Dental Stem Cells. You can follow any responses to this entry through the RSS 2.0 feed. Both comments and pings are currently closed. |