The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro.

DPSC-derived spheroids can mimic the developmental process of cell survival, cavitation and spontaneous multi-differentiation on matrigel under certain conditions. This work allows for functional studies to investigate organ regeneration with human DPSCs in vitro.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 µg/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

Oxidative stress causes severe tissue injury of the central nervous system in ischemic brain damage (IBD), traumatic brain injury (TBI) and neurodegenerative disorders. In this study, we used hydrogen peroxide (H2O2) to induce oxidative stress in organotypic brain slice cultures (OBSCs), and investigated the protective effects of oxidative stress-tolerant (OST) stem cells harvested from human exfoliated deciduous teeth (SHED) which were co-cultivated with OBSCs. Using presto blue assay and immunostaining, we demonstrated that both normal SHED and OST-SHED could prevent H2O2-induced cell death, and increase the numbers of mature neuron and neuronal progenitors in the hippocampus of OBSCs. During co-cultivation, OST-SHED, but not normal SHED, exhibited neuronal cell morphology and expressed neuronal markers. Results from ELISA showed that both normal SHED and OST-SHED significantly decreased oxidative DNA damage in H2O2-treated OBSCs. SHED could also produce neurotrophic factor BDNF (brain derived neurotrophic factor) and promoted the production of IL-6 in OBSCs. Although OST-SHED had lower cell viability, the neuronal protection of OST-SHED was significantly superior to that of normal SHED. Our findings suggest that SHED, especially OST-SHED, could prevent oxidative stress induced brain damage. OST-SHED can be explored as a new therapeutic tool for IBD, TBI and neurodegenerative disorders.

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