Neurophysiology and electrophysiology of human and murine dental pulp stem cells.

Abstract

The cortical brain-machine interface has the potential to improve the quality of life for millions of patients with sensory or motor loss, however a range of limitations currently exist that restrict their long term clinical application. Primary amongst these is the low biocompatibility between electrodes and brain tissue. Injury to the central nervous system (CNS) causes a recruitment of inflammatory factors that lead to the long term upregulation of the perineuronal net (PNN) and the development of a glial scar that restrict recovery by forming an inhibitory peri-injury region. We propose that a biological layer of dental pulp stem cells (DPSC) will render the interface more compatible with cortical tissue to allow more efficient signal transduction and promote long-term success. I have approached this interface challenge in vitro to determine how DPSC may actively improve the local environment to achieve long-term biocompatibility. Microelectrode arrays (MEAs) approximate the brain-machine interface in vitro. In Chapter 3 I designed and fabricated a novel MEA with design features specific to our research goals. Initial characterisation of these MEAs in comparison with commercial MEAs demonstrated high biocompatibility with cortical cultures, however electrodes had high impedance leading to a low signal-to-noise ratio that ultimately rendered the MEAs unable to detect extracellular electrical activity from the cultured cortical neurons. Future modifications including the addition of electrode polymers on these MEAs will render them more appropriate for in vitro use. This directed us to utilise commercial MEAs for subsequent use within the studies of this thesis. In Chapter 4, human-derived DPSC (hDPSC) were seeded onto commercial MEAs to determine their long-term biocompatibility throughout neuronal differentiation and to assess the development of electrical activity within the developing cultures. DPSC had intrinsically low biocompatibility with MEAs, however long term culture was achieved. Stimulation-induced events were detected in long-term cultures yet no spontaneous activity was measured. A novel source of DPSC derived from murine incisors (mDPSC) were also characterised for their neuronal potential in vitro in Chapter 5. mDPSC developed a neuronal morphology and high expression of neuronal and glial markers identified through immunohistochemical analysis. Differentiated mDPSC networks supported electrophysiology reminiscent of early embryonic development with high expression of L-type voltage-gated Ca²⁺ channels, gap junction proteins and gamma frequency oscillatory activity following neural induction. The ability of mDPSC to differentiate into neural-like cells supports their future use in a murine model of autologous cell transplantation. The impact of DPSC on the endogenous inhibition of the brain was also investigated in Chapter 6. It was hypothesised that co-culturing DPSC with dissociated cortical neurons would downregulate the expression of the restrictive PNN around neurons. It was demonstrated that hDPSC co-culture reduced the proportion of neurons that expressed PNN in a time and dose-dependent manner. Moreover, hDPSC conditioned medium also decreased the proportion of PNN-expressing neurons, suggesting that paracrine factors released by the cells may be responsible for this effect. In conclusion, the present studies have identified a novel ability for DPSC to reduce cortical PNN expression that could improve the long-term efficacy of a brain-machine interface. However, biocompatibility of DPSC with in vitro MEAs is low and requires modification to achieve a successful interaction at the interface. Moreover, the neuronal potential of DPSC isolated from murine incisors has been demonstrated for the first time. The multifaceted characteristics of DPSC may present a viable approach to cell-based therapeutics for a range of CNS disorders.