Abstract
The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.
Highlights
Cholinergic neurons have pivotal roles in cognition, locomotion, and behavioral response, which predominantly use the neurotransmitter, acetylcholine (Ach), for their message delivery [1,2]
Fibroblast-like spindle colonies were observed within the first week of culture, which became homogeneous at passage 3 upon sub-culturing (Figure 1A)
Mesenchymal stem cells (MSCs) from these cryopreserved dental follicles were subsequently shown to have immunomodulatory properties that further enhanced osteogenesis under in vivo conditions [23]. In light of these observations, the present study demonstrated that long-term preserved dental pulp tissues stored under the newly developed tissue cryopreservation protocol could safely conserve multipotent stem cells, as shown by the same MSC characteristics as those derived from fresh dental pulp tissues
Summary
Cholinergic neurons have pivotal roles in cognition, locomotion, and behavioral response, which predominantly use the neurotransmitter, acetylcholine (Ach), for their message delivery [1,2]. The loss of cholinergic neurons results in the reduction of choline acetyltransferase (ChAT) activity, leading to the absence of Ach, which results in motor nerve degeneration, as well as irreversible cognitive decline, as observed in Alzheimer’s disease [3]. Various therapeutic applications such as cell-based therapies and nerve grafts are being practiced for the treatment of a wide variety of neurological disorders, a novel treatment method for the loss of cholinergic neurons is yet to be developed [4,5]. Various stem-cell-based therapies were proposed as an alternative treatment modality to relieve the pathophysiology of cholinergic nerve disorders, including Alzheimer’s disease and motor nerve disorders [3,5,6]. Embryonic stem cells and induced pluripotent stem cells were studied for their higher in vitro and in vivo neuronal differentiation potential; these have many drawbacks for clinical application in humans, including ethical concerns, difficulty in isolating and cultivating autologous cells, and economical demerits [8,9]
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