Periodontitis is a common oral disease caused by bacteria coupled with an excessive host immune response. Stem cell therapy can be a promising treatment strategy for periodontitis, but the relevant mechanism is complicated. This study aimed to explore the therapeutic potential of mitochondria from human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) for the treatment of periodontitis. The gingival tissues of periodontitis patients are characterized by abnormal mitochondrial structure. Human gingival fibroblasts (HGFs) were exposed to 5 μg/mL lipopolysaccharide (LPS) for 24 h to establish a cell injury model. When treated with hESC-MSCs or mitochondria derived from hESC-MSCs, HGFs showed reduced expression of inflammatory genes, increased adenosine triphosphate (ATP) level, decreased reactive oxygen species (ROS) production, and enhanced mitochondrial function compared to the control. The average efficiency of isolated mitochondrial transfer by hESC-MSCs was determined to be 8.93%. Besides, a therapy of local mitochondrial injection in mice with LPS-induced periodontitis showed a reduction in inflammatory gene expression, as well as an increase in both the mitochondrial number and the aspect ratio in gingival tissues. In conclusion, our results indicate that mitochondria derived from hESC-MSCs can reduce the inflammatory response and improve mitochondrial function in HGFs, suggesting that the transfer of mitochondria between hESC-MSCs and HGFs serves as a potential mechanism underlying the therapeutic effect of stem cells.
Humans ; Gingiva/cytology* ; Fibroblasts/metabolism* ; Mitochondria/physiology* ; Mesenchymal Stem Cells/cytology* ; Animals ; Periodontitis/therapy* ; Mice ; Reactive Oxygen Species/metabolism* ; Inflammation ; Lipopolysaccharides ; Human Embryonic Stem Cells/cytology* ; Cells, Cultured ; Adenosine Triphosphate/metabolism* ; Male

OBJECTIVE: To investigate the mechanism of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) in alleviating brain injury after resuscitation in swine with cardiac arrest (CA). METHODS: Twenty-nine healthy male large white swine were randomly divided into Sham group (n = 9), cardiopulmonary resuscitation (CPR) group (n = 10) and hESC-MSC group (n = 10). The Sham group only completed animal preparation. In CPR group and hESC-MSC group, the swine model of CA-CPR was established by inducing ventricular fibrillation for 10 minutes with electrical stimulation and CPR for 6 minutes. At 5 minutes after successful resuscitation, hESC-MSC 2.5×10⁶/kg was injected via intravenous micropump within 1 hour in hESC-MSC group. Venous blood samples were collected before resuscitation and at 4, 8, 24, 48 and 72 hours of resuscitation. The levels of neuron specific enolase (NSE) and S100B protein (S100B) were detected by enzyme linked immunosorbent assay (ELISA). At 24, 48 and 72 hours of resuscitation, neurological deficit score (NDS) and cerebral performance category (CPC) were used to evaluate the neurological function of the animals. Three animals from each group were randomly selected and euthanized at 24, 48, and 72 hours of resuscitation, and the hippocampus tissues were quickly obtained. Immunofluorescence staining was used to detect the distribution of hESC-MSC in hippocampus. Immunohistochemical staining was used to detect the activation of astrocytes and microglia and the survival of neurons in the hippocampus. The degree of apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: The serum NSE and S100B levels of brain injury markers in CPR group and hESC-MSC group were significantly higher than those in Sham group at 24 hours of resuscitation, and then gradually increased. The levels of NSE and S100B in serum at each time of resuscitation in hESC-MSC group were significantly lower than those in CPR group [NSE (μg/L): 20.69±3.62 vs. 28.95±3.48 at 4 hours, 27.04±5.56 vs. 48.59±9.22 at 72 hours; S100B (μg/L): 2.29±0.39 vs. 3.60±0.73 at 4 hours, 2.38±0.15 vs. 3.92±0.50 at 72 hours, all P < 0.05]. In terms of neurological function, compared with the Sham group, the NDS score and CPC score in the CPR group and hESC-MSC group increased significantly at 24 hours of resuscitation, and then gradually decreased. The NDS and CPC scores of hESC-MSC group were significantly lower than those of CPR group at 24 hours of resuscitation (NDS: 111.67±20.21 vs. 170.00±21.79, CPC: 2.33±0.29 vs. 3.00±0.00, both P < 0.05). The expression of hESC-MSC positive markers CD73, CD90 and CD105 in the hippocampus of hESC-MSC group at 24, 48 and 72 hours of resuscitation was observed under fluorescence microscope, indicating that hESC-MSC could homing to the damaged hippocampus. In addition, compared with Sham group, the proportion of astrocytes, microglia and apoptotic index in hippocampus of CPR group were significantly increased, and the proportion of neurons was significantly decreased at 24, 48 and 72 hours of resuscitation. Compared with CPR group, the proportion of astrocytes, microglia and apoptotic index in hippocampus of hESC-MSC group decreased and the proportion of neurons increased significantly at 24 hours of resuscitation [proportion of astrocytes: (14.33±1.00)% vs. (30.78±2.69)%, proportion of microglia: (12.00±0.88)% vs. (27.89±5.68)%, apoptotic index: (12.89±3.86)% vs. (52.33±7.77)%, proportion of neurons: (39.44±3.72)% vs. (28.33±1.53)%, all P < 0.05]. CONCLUSIONS Application of hESC-MSC at the early stage of resuscitation can reduce the brain injury and neurological dysfunction after resuscitation in swine with CA. The mechanism may be related to the inhibition of immune cell activation, reduction of cell apoptosis and promotion of neuronal survival.
Animals ; Heart Arrest/therapy* ; Cardiopulmonary Resuscitation ; Swine ; Humans ; Male ; Human Embryonic Stem Cells/cytology* ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology* ; Phosphopyruvate Hydratase/blood* ; Brain Injuries/therapy* ; S100 Calcium Binding Protein beta Subunit ; Apoptosis ; Disease Models, Animal

Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.
Cell Differentiation ; Consensus ; Human Embryonic Stem Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Tendons ; cytology ; Tissue Engineering

The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.
Antigens, Differentiation ; biosynthesis ; Electrophysiological Phenomena ; physiology ; Gene Expression Regulation ; physiology ; Genome-Wide Association Study ; Human Embryonic Stem Cells ; cytology ; metabolism ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Multigene Family ; physiology ; Neurons ; cytology ; metabolism ; Transcriptome ; physiology