Abstract:Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is a transcriptional co-activator involved in mitochondrial biogenesis, respiratory capacity, and oxidative phosphorylation (OXPHOS). PGC-1α plays an important role in cellular metabolism and is associated with tumorigenesis, suggesting an involvement in cell cycle progression. However, the underlying mechanisms mediating its involvement in these processes remain unclear. To elucidate the signaling pathways involved in PGC-1α function, we established a cell line, CH1 PGC-1α, which stably overexpresses PGC-1α. Using this cell line, we found that over-expression of PGC-1α stimulated extra adenosine triphosphate (ATP) and reduced reactive oxygen species (ROS) production. These effects were accompanied by up-regulation of the cell cycle checkpoint regulators CyclinD1 and CyclinB1. We hypothesized that ATP and ROS function as cellular signals to regulate cyclins and control cell cycle progression. Indeed, we found that reduction of ATP levels down-regulated CyclinD1 but not CyclinB1, whereas elevation of ROS levels down-regulated CyclinB1 but not CyclinD1. Furthermore, both low ATP levels and elevated ROS levels inhibited cell growth, but PGC-1α was maintained at a constant level. Together, these results demonstrate that PGC-1α regulates cell cycle progression through modulation of CyclinD1 and CyclinB1 by ATP and ROS. These findings suggest that PGC-1α potentially coordinates energy metabolism together with the cell cycle.

It has been demonstrated that mesenchymal stem cells (MSCs) differentiated from human embryonic stem cells (hESCs), name EMSCs, can treat a variety of autoimmune and inflammatory diseases, with similar efficacies to those achieved with MSCs derived from somatic tissues such as bone marrow (BMSCs). The chance increases even higher for EMSCs, than somatic tissue derived MSCs​, to become a cell drug as the former can be produced in large scale from an unlimited hESC line with easier quality control and less biosafety concern. We have further demonstrated that both human ESCs and EMSCs, after aggregation to form spheroids, can tolerate hypoxic and ambient conditions (AC) for over 4 and 10 days, respectively, without loss of their viability and alteration of their functions. Based on these advantages, we decided to test whether EMSC spheroids, made in large quantity and delivered through a long-term distance at AC, can treat osteoarthritis spontaneously developed in rhesus macaques (M. mulatta) monkeys as well as the allogenic MSCs.Methods: Xenogeneic AC-transported EMSC spheroids or allogenic BMSCs were injected into the articular cavity of both knees of the monkeys at 3 animals per group. Another two macaques were injected the same way with saline as controls.Results: Both EMSCs and BMSCs groups showed significant amelioration indicated by the reduction of swelling joint size and amplification of keen flare angle post-treatment, compared to the control group. Examinations via X-ray and MRI also indicated the decrease of inflammation and osteophyma, and recovery of the synovium and cartilage in both treated groups. No sign of allergy or graft versus host disease was observed in the animals.Conclusion: Our results demonstrate that human EMSC spheroids can prevent the osteoarthtitis progression and ameliorate osteoarthritis in the rhesus macaques as well as allogenic BMSCs, and this study shall help advance the clinical application of EMSCs.

Bcl-2 inhibits cell proliferation by delaying G/G to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G/G phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G/G arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1-3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.

Liver fibrosis is a disease that causes high morbidity and has become a major health problem. Liver fibrosis can lead to the end stage of liver diseases (livercirrhosisand hepatocellularcarcinoma). Currently, liver transplantation is the only effective treatment for end-stage liver disease. However, the shortage of organ donors, high cost of medical surgery, immunological rejection and transplantation complications severely hamper liver transplantation therapy. Mesenchymal stem cells (MSCs) have been regarded as promising cells for clinical applications in stem cell therapy in the treatment of liver diseases due to their unique multipotent differentiation capacity, immunoregulation and paracrine effects. Although liver fibrosis improvements by MSC transplantation in preclinical experiments as well as clinical trials have been reported, the in vivo fate of MSCs after transportation and their therapeutic mechanisms remain unclear. In this present study, we isolated MSCs from the bone marrow of rhesus macaques. The cells exhibited typical MSC markers and could differentiate into chondrocytes, osteocytes, and adipocytes, which were not affected by labeling with enhanced green fluorescent protein (EGFP). The harvested MSCs respond to interferon-γ stimulation and have the ability to inhibit lymphocyte proliferation in vitro. EGFP-labeled MSCs (1 × 106 cells) were transplanted into mice with carbon tetrachloride-induced liver fibrosis via tail vein injection. The ability of the heterogenic MSC infusion to ameliorate liver fibrosis in mice was evaluated by a blood plasma chemistry index, pathological examination and liver fibrosis-associated gene expression. Additionally, a small number of MSCs that homed and engrafted in the mouse liver tissues were evaluated by immunofluorescence analysis. Our results showed that the transplantation of heterogenic MSCs derived from monkey bone marrow can be used to treat liver fibrosis in the mouse model and that the paracrine effects of MSCs may play an important role in the improvement of liver fibrosis.

Background: Human umbilical cord-derived MSCs (hUC-MSCs) have been identified as promising seeding cells in tissue engineering and clinical applications of regenerative medicine due to their advantages of simple acquisition procedure and the capability to come from a young tissue donor over the other MSCs sources. In clinical applications, large scale production is required and optimal cryopreservation and culture conditions are essential to autologous and allogeneic transplantation in the future. However, the influence of cryopreserved post-thaw and long-term culture on hUC-MSCs remains unknown, especially in terms of specific protein expression. Therefore, biological characteristics and proteomic profiles of hUC-MSCs after cryopreserving and long-term culturing were investigated. Methods: Firstly, hUC-MSCs were isolated from human umbilical cord tissues and identified through morphology, surface markers and tri-lineage differentiation potential at passage 3, and then the biological characteristics and proteomic profiles were detected and compared after cryopreserving and long-term culturing at passage 4 and continuously cultured to passage 10 with detection occurring here as well. The proteomic profiles were tested by using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique and differential protein were confirmed by mass spectrometry. Results: The results showed no significant differences in phenotypes including morphology, surface marker and tri-lineage differentiation potential but have obvious changes in translation level, which is involved in metabolism, cell cycle and other pathways. Conclusion: This suggests that protein expression may be used as an indicator of hUC-MSCs security testing before applying in clinical settings, and it is also expected to provide the foundation or standardization guide of hUC-MSCs applications in regenerative medicine.

Di-(2-ethylhexyl) phthalate (DEHP) is one of the most commonly used plasticizers, and it is widely applied in various plastic products. DEHP is an endocrine-disrupting chemical (EDC) that has been shown to disrupt the function of reproductive system in females. Although many studies have shown that DEHP potentially causes female reproductive toxicity, including depletion of the primordial follicle and decreased sex hormone production, the specific mechanisms by which DEHP affects female reproduction remain unknown. In recent years, research focused on the intestinal flora has provided an idea to eliminate our confusion, and gut bacterial dysbiosis may contribute to female reproductive toxicity. In the present study, the feces of DEHP-exposed mice were collected and analyzed using 16S rRNA amplicon sequencing and untargeted global metabolite profiling of metabolomics. DEHP obviously causes reproductive toxicity, including the ovarian organ coefficient, estradiol level, histological features of the ovary and estrus. Furthermore, DEHP exposure alters the structure of the intestinal microbiota community and fecal metabolite profile in mice, suggesting that the reproductive toxicity may be caused by gut bacterial dysbiosis and altered metabolites, such as changes in the levels of short-chain fatty acid (SCFA). Additionally, it is well known that changes in gut microbiota and fecal metabolites cause inflammation and tissue oxidative stress, expectedly, we found oxidative stress in the ovary and systemic inflammation in DEHP exposed mice. Thus, based on our findings, DEHP exposure may cause gut bacterial dysbiosis and altered metabolite profiles, particularly SCFA profiles, leading to oxidative stress in the ovary and systemic inflammation to ultimately induce female reproductive toxicity.

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Mesenchymal stem cells (MSCs) are one of the most promising adult stem cells for clinical application in a cell therapy. The development of large-scale cryopreservation techniques, such as vitrification, for MSCs is a prerequisite for clinical therapies. Dimethyl sulfoxide (DMSO) and ethylene glycol (EG) are two types of cryoprotectants widely used for cell vitrification. However, the effects of DMSO and EG on the biological characteristics and transcriptome profiles of MSCs after cryopreservation remain unknown. In the present study, the viability, immunophenotype of cell surface markers, proliferation, differentiation potency, and global gene expression of rhesus macaque bone marrow-derived MSCs vitrified using DMSO and EG were studied. The results showed that vitrification did not affect the morphology, surface markers, and differentiation of the MSCs, and compared to DMSO, EG better protected cell viability and proliferation. Most importantly, vitrification resulted in changes in a large number of transcripts of MSCs either preserved using DMSO or EG. This report is the first to examine the effects of DMSO and EG on global gene expression in stem cells. These results will be beneficial to understanding the biological process involved in MSC vitrification and will contribute to improving cryopreservation protocols that maintain transcriptomic identity with high cryosurvival for preclinical research and clinical long-term storage.