Single-nucleus Atlas of Sevoflurane-induced Hippocampal Cell Type- and Sex-specific Effects during Development in Mice.

BACKGROUND: Multiple neonatal exposures to sevoflurane induce neurocognitive dysfunctions in rodents. The lack of cell type-specific information after sevoflurane exposure limits the mechanistic understanding of these effects. In this study, the authors tested the hypothesis that sevoflurane exposures alter the atlas of hippocampal cell clusters and have neuronal and nonneuronal cell type-specific effects in mice of both sexes. METHODS: Neonatal mice were exposed to 3% sevoflurane for 2 h at postnatal days 6, 8, and 10 and analyzed for the exposure effects at postnatal day 37. Single-nucleus RNA sequencing was performed in the hippocampus followed by in situ hybridization to validate the results of RNA sequencing. The Morris Water Maze test was performed to test neurocognitive function. RESULTS: The authors found sex-specific distribution of hippocampal cell types in control mice alongside cell type- and sex-specific effects of sevoflurane exposure on distinct hippocampal cell populations. There were important changes in male but not in female mice after sevoflurane exposure regarding the proportions of cornu ammonis 1 neurons (control vs. sevoflurane, males: 79.9% vs. 32.3%; females: 27.3% vs. 24.3%), dentate gyrus (males: 4.2% vs. 23.4%; females: 36.2% vs. 35.8%), and oligodendrocytes (males: 0.6% vs. 6.9%; females: 5.9% vs. 7.8%). In male but not in female mice, sevoflurane altered the number of significantly enriched ligand-receptor pairs in the cornu ammonis 1, cornu ammonis 3, and dente gyrus trisynaptic circuit (control vs. sevoflurane, cornu ammonis 1-cornu ammonis 3: 18 vs. 42 in males and 15 vs. 21 in females; cornu ammonis 1-dentate gyrus: 21 vs. 35 in males and 12 vs. 20 in females; cornu ammonis 3-dentate gyrus: 25 vs. 45 in males and 17 vs. 20 in females), interfered with dentate gyrus granule cell neurogenesis, hampered microglia differentiation, and decreased cornu ammonis 1 pyramidal cell diversity. Oligodendrocyte differentiation was specifically altered in females with increased expressions of Mbp and Mag. In situ hybridization validated the increased expression of common differentially expressed genes. CONCLUSIONS: This single-nucleus RNA sequencing study reveals the hippocampal atlas of mice, providing a comprehensive resource for the neuronal and nonneuronal cell type- and sex-specific effects of sevoflurane during development.

Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs).

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC) characterized by multi-lineage differentiation making it an attractive choice for tissue regeneration. However, before DPSCs can be used for cell-based therapy, we have to understand their biological properties in response to intrinsic and extrinsic stimuli such as lipopolysaccharide (LPS). DPSCs were therefore stimulated with LPS and senescence was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining, with cell number and cell-cycle arrest being examined by BrdU assay and flow cytometry, respectively. The morphology of DPSCs was characterized by their flat shape, increased size and increased SA-ß-gal activity after repeated stimulation (3 or 6 times) with LPS. Reactive oxygen species (ROS) staining showed that the number of ROS-stained cells and the DCFH fluorescent level were higher in the LPS-treated DPSCs compared with those in the untreated DPSCs. Protein and mRNA expression levels of γ-H2A.X and p16(INK4A) were also increased in DPSCs with repeated LPS stimulation. We found that the LPS bound with Toll-like receptor 4 (TLR4) and that TLR4 signaling accounted for p16(INK4A) expression. Further results indicated that the senescence of DPSCs stimulated repeatedly with LPS was reversed by p16(INK4A) short interfering RNA. The DNA damage response and p16(INK4A) pathways might be the main mediators of DPSC senescence induced by repeated LPS stimulation. Thus, DPSCs tend to undergo senescence after repeated activation, implying that DPSC senescence starts after many inflammatory challenges. Ultimately, these findings should lead to a better understanding of DPSC-based clinical therapy.

Age-dependent impaired neurogenic differentiation capacity of dental stem cell is associated with Wnt/ß-catenin signaling.

Two kinds of dental stem cells (DSCs), dental pulp stem cells (DPSCs) and stem cells from human-exfoliated deciduous teeth (SHED), have been identified as novel populations of mesenchymal stem cells that can be induced to differentiate into osteoblasts, chondrocytes, adipocytes, and neuron-like cells in vitro. As we know, both of them originate from the neural crest, but have distinct characteristics and functions in vitro and in vivo. The regeneration potential of DSCs declines with advanced age; however, the mechanism of the impaired potential in DSCs has not been fully explored. In this study, we investigated whether declined neurogenic differentiation capacity is associated with an altered expression of Wnt signaling-related proteins in vitro. We compared stem cells isolated from human dental pulp in two age groups: the exfoliated deciduous teeth (5-12 years), and the third permanent teeth (45-50 years). We found that the expression levels of neuron markers, such as ßIII-tubulin, microtubule-associated protein 2(MAP2), tyrosine hydroxylase (TH), and Nestin were lower in the DPSCs group compared with that in the SHED group; however, in supplementation with human recombinant Wnt1 in the medium, the DPSCs were prone to neural differentiation and expressed higher levels of neurogenic markers. In summary, our study demonstrated that Wnt/ß-catenin signaling may play a vital role in the age-dependent neural differentiation of DSCs. Therefore, DSCs may provide an ideal source of stem cells that can further extend their therapeutic application in nerve injury and neurodegenerative diseases.

TNF-α triggers osteogenic differentiation of human dental pulp stem cells via the NF-κB signalling pathway.

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) characterised by self-renewal and multi-lineage differentiation, including chondrocytes, adipocytes, neural cells and osteoblasts, which make it an attractive choice for tissue engineering purposes. Tumour necrosis factor α (TNF-α) had the positive effect on the mineralisation of bone marrow MSCs and stromal cells derived from human adipose tissue. However, the effect of TNF-α on DPSCs is unclear. We found that TNF-α activated the NF-κB pathway during the osteogenic differentiation of DPSCs. TNF-α also increased mineralisation and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and collagen type I (COL I) during this process. PDTC, an NF-κB inhibitor, blocked the osteogenic differentiation induced by TNF-α. No effect of TNF-α on proliferation of DPSCs or cell cycle was detected. In summary, TNF-α promotes mineralisation and mineralisation-related gene expression through the NF-κB signalling pathway in DPSCs, which may provide a foundation for autologous transplantation of DPSCs.