Alteration of the gut microbiota in rhesus monkey with spontaneous osteoarthritis.

BACKGROUND: The spontaneous osteoarthritis (OA) in rhesus macaque is similar to OA in human, which maintains an upright body posture and shows very similar biomechanical properties of bones to humans. At present, there is no good treatment for OA. This study aims to explore relationship between OA and intestinal microbiota, and provide a reference for the treatment of clinical OA. RESULTS: We collected colonic contents of the 20 rhesus macaque (6-15 years old, female) for intestinal microbiota analysis by metagenomics sequencing, of which 10 were spontaneous OA monkeys and 10 were normal monkeys. Our results showed the diversity of gut microbiota in monkeys with OA was decreased compared to the normal monkeys (p = 0.16). Mollicutes, Tenericutes, Coprobacillus and Faecalitalea may be biomarkers for the monkeys of OA. Lactobacillus found significantly increased in OA monkeys. Prevotella and Ruminococcus were higher in the normal group than OA group. Zinc/manganese transport system permease protein (p = 0.0011) and Cyclopropane-fatty-acyl-phospholipid synthase (p = 0.0012) are a microbiota metabolic pathway related to cartilage production. CONCLUSIONS: Our results indicate that the diversity and composition of intestinal microbiota in monkeys with OA are different compared to the normal monkeys. we have found microbes that may be a biomarker for the diagnosis of osteoarthritis. Functional analysis of the microbiota also predicts cartilage damage in the monkeys with osteoarthritis. Non-human primates are closely related to humans, so this study can provide a reference for the development of drugs for the treatment of OA.

Improving Sperm Cryopreservation With Type III Antifreeze Protein: Proteomic Profiling of Cynomolgus Macaque (Macaca fascicularis) Sperm.

Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p < 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo-AFP) (p < 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 µg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.

Effects of cryopreservation and long-term culture on biological characteristics and proteomic profiles of human umbilical cord-derived mesenchymal stem cells.

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.

Improvement of sperm cryo-survival of cynomolgus macaque (Macaca fascicularis) by commercial egg-yolk-free freezing medium with type III antifreeze protein.

When nonhuman primate sperm undergoes cryopreservation in an egg yolk medium there is an increased risk that the egg yolk might adversely affect the sperm due to containing of avian pathogens. Although commercial egg-yolk-free medium for human sperm cryopreservation has been used for macaque sperm, the cryo-survival remains less than optimal. The present study, therefore, was conducted to determine the optimal concentration of antifreeze protein (AFP) III supplemented in a commercial egg-yolk-free medium for cynomolgus macaque (Macaca fascicularis) sperm cryo-survival. The function of frozen-thawed sperm was evaluated by post-thaw sperm motility, acrosome integrity, and mitochondrial function. Results indicate that the sperm motilities were greater when 0.1, 1, and 10 µg/ml of AFP III were supplemented into the sperm freezing medium (P < 0.05). In addition, the mitochondrial membrane potential was greater in the sperm cryopreserved with the medium that was supplemented with 0.1 µg/ml of AFP III (P < 0.05). The addition of AFP III at any of the concentrations, however, did not have any cryoprotection effect on the sperm acrosome, and the greatest concentrations of AFP III at 100 and 200 µg/ml had detrimental effects on acrosomal integrity (P < 0.05). Results of the present study indicated the methods used are effective for the cryopreservation of cynomolgus monkey sperm while reducing associated health risks due to avian pathogens being present in egg yolk-based extenders.

Transplantation of human ESC-derived mesenchymal stem cell spheroids ameliorates spontaneous osteoarthritis in rhesus macaques.

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.