Effects and mechanisms of mUCMSCs on ovarian structure and function in naturally ageing C57 mice.

BACKGROUND: The ovaries are the core reproductive organs in women and are critical for maintaining normal reproductive function and endocrine system stability. An ageing C57 mouse model was used to evaluate the efficacy and mechanism of mouse umbilical cord mesenchymal stem cells (mUCMSCs) and to explore the mechanism by which mUCMSCs promote the antioxidant repair of mouse granulosa cells (mGCs). RESULTS: Eighteen-month-old C57 mice were randomly divided into a model group and a treatment group. At the same time, 2-month-old C57 mice were established as a young group (15 mice per group). The mice in the treatment group were injected via the tail vein with GFP-labelled mUCMSCs. The ovarian volume in ageing C57 mice was decreased, and there were no follicles at any stage. After mUCMSC transplantation, the mouse ovaries increased in size, follicles at various stages were observed in the cortex, and the antral follicle counts increased. The serum E2, AMH, and INH-B levels of mice in the treatment group were significantly higher than those of mice in the model control group (P < 0.05). mUCMSCs downregulated the expression of the autophagy-related gene LC3b and the apoptosis-related genes Bax and Caspase-3, upregulated the expression of SOD2 and the peroxidase gene PRDX IV, and reduced apoptosis rates and reactive oxygen species (ROS) levels in granulosa cells. CONCLUSIONS: mUCMSCs play roles in promoting the repair of ageing ovaries by regulating immunity, anti-inflammatory responses and the PI3K-Akt signalling pathway.

Establishment of a Systemic Inflammatory Response Syndrome Model and Evaluation of the Efficacy of Umbilical Cord Mesenchymal Stem Cell Transplantation.

Based on the characteristics of modern weapon injury, a repetitive model of traumatic systemic inflammatory response syndrome (SIRS) and an evaluation system were established. The models were treated with GFP-labeled tree shrew umbilical cord mesenchymal stem cells (UCMSCs). Forty out of 50 tree shrews were used to make a unilateral femoral comminuted fracture. Lipopolysaccharide was injected intravenously to create a traumatic SIRS model. The other 10 shrews were used as normal controls. After the model was established for 10 days, 20 tree shrews were injected intravenously with GFP-labeled UCMSCs, and 18 tree shrews were not injected as the model control group. The distribution of GFP-labeled cells in vivo was measured at 2 and 10 days after injection. Twenty days after treatment, the model group, the normal control group, and the treatment group were taken to observe the pathological changes in each tissue, and blood samples were taken for the changes in liver, renal, and heart function. Distribution of GFP-positive cells was observed in all tissues at 2 and 10 days after injection. After treatment, the HE staining results of the treatment group were close to those of the normal group, and the model group had a certain degree of lesions. The results of liver, renal, and heart function tests in the treatment group were returned to normal, and the results in the model group were abnormally increased. UCMSCs have a certain effect on the treatment of traumatic SIRS and provide a new technical solution for modern weapon trauma treatment.

LHPP-Mediated Histidine Dephosphorylation Suppresses the Self-Renewal of Mouse Embryonic Stem Cells.

Self-renewal of embryonic stem cells (ESCs) is orchestrated by a vast number of genes at the transcriptional and translational levels. However, the molecular mechanisms of post-translational regulatory factors in ESC self-renewal remain unclear. Histidine phosphorylation, also known as hidden phosphorylation, cannot be detected by conventional experimental methods. A recent study defined phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) as a histidine phosphatase, which regulates various biological behaviors in cells via histidine dephosphorylation. In this study, the doxycycline (DOX)-induced hLHPP-overexpressing mouse ESCs and mouse LHPP silenced mESCs were constructed. Quantitative polymerase chain reaction (qPCR), western blotting analysis, immunofluorescence, Flow cytometry, colony formation assays, alkaline phosphatase (AP) and bromodeoxyuridine (Brdu) staining were performed. We found that the histidine phosphorylation level was strikingly reduced following LHPP overexpression. Besides, the expression of Oct4 and Lefty1, indispensable genes in the process of ESCs self-renewal, was significantly down-regulated, while markers related to the differentiation were markedly elevated. Moreover, LHPP-mediated histidine dephosphorylation induced G0/G1 phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of Lhpp promoted the self-renewal of mESCs and reversed the RA induced increased expression of genes associated with differentiation. Mechanistically, our findings suggested that the enzymatic active site of LHPP was the cysteine residue at position 226, not 53. LHPP-mediated histidine dephosphorylation lowered the expression levels of ß-catenin and the cell cycle-related genes CDK4 and CyclinD1, while it up-regulated the cell cycle suppressor genes P21 and P27. Taken together, our findings reveal that LHPP-mediated histidine dephosphorylation plays a role in the self-renewal of ESCs. LHPP-mediated histidine dephosphorylation inhibited the self-renewal of ESCs by negatively regulating the Wnt/ß-catenin pathway and downstream cell cycle-related genes, providing a new perspective and regulatory target for ESCs self-renewal.

Transplantation of chicken egg white extract-induced rabbit PBMCs as a treatment for renal ischemia-reperfusion injury in rabbits.

Ischemia-reperfusion injury is an important contributor to acute kidney injury and a major factor affecting early functional recovery after kidney transplantation. We conducted this experiment to investigate the protective effect of induced multipotent stem cell transplantation on renal ischemia-reperfusion injury. Forty rabbits were divided into four groups of 10 rabbits each. Thirty rabbits were used to establish the renal ischemia-reperfusion injury model, and ten rabbits served as the model group and were not treated. Among the 30 rabbits with renal ischemia-reperfusion injury, 10 rabbits were treated with induced peripheral blood mononuclear cells (PBMCs), and 10 other rabbits were treated with noninduced PBMCs. After three weekly treatments, the serum creatinine levels, urea nitrogen levels and urine protein concentrations were quantified. The kidneys were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS) and Masson's trichrome and then sent for commercial metabolomic testing. The kidneys of the rabbits in the model group showed different degrees of pathological changes, and the recovery of renal function was observed in the group treated with induced cells. The results indicate that PBMCs differentiate into multipotent stem cells after induction and exert a therapeutic effect on renal ischemia-reperfusion injury.

Effect and mechanism of human umbilical cord mesenchymal stem cells in treating allergic rhinitis in mice.

A model of allergic rhinitis (AR) in BALB/c mice was established and evaluated to provide experimental subjects for further research. Preparation of human umbilical cord mesenchymal stem cells (hUCMSCs), including isolation, expansion culture, passaging, cryopreservation, and preparation of cell suspensions, provided materials for experimental research and clinical treatment. The mouse AR model was established by ovalbumin (OVA) intraperitoneal injection and the nasal stimulation induction method, and the model had a good effect and high repeatability. GFP-labeled hUCMSCs had good effects and were stable cells that could be used for tracking in animals. Transplantation of hUCMSCs by intraperitoneal and tail vein injections had a specific effect on the AR model of mice, and tail vein injection had a better effect. Tracking of hUCMSCs in vivo showed that the three groups of mice had the greatest number of hUCMSCs in the nose at week 2. The mouse AR model was used to evaluate the efficacy of hUCMSC transplantation via multiple methods for AR. The distribution of hUCMSCs in vivo was tracked by detecting green fluorescent protein (GFP), and the treatment mechanism of hUCMSCs was elucidated. This study provides technical methods and a theoretical basis for the clinical application of hUCMSCs.

Umbilical cord mesenchymal stem cells protect thymus structure and function in aged C57 mice by downregulating aging-related genes and upregulating autophagy- and anti-oxidative stress-related genes.

BACKGROUND: To study the effect of allogeneic umbilical cord mesenchymal stem cell transplantation on the structure and function of the thymus in aged C57 mice and provide a new method for the treatment of senile thymic atrophy. RESULTS: The changes in the thymus cortex and medulla volume and the lymphocyte ratio were analyzed by immunofluorescence. For thymus tissue sections, immunohistochemical staining was performed to detect p16, p53, SOD, becline1, LC3b, p62, sirt1, and sirt3. Changes in CK5, CK8, CD4 and CD8 expression were observed. Treatment with mUCMSCs could promote hair regeneration in aging mice and regenerate the thymus structure. CONCLUSIONS: mUCMSCs inhibited senescence of the thymus and promoted structural and functional thymus regeneration by downregulating the senescence genes p53 and p16 and upregulating the SOD, Sirt1 and Sirt3 genes, but the mechanism requires further research. METHODS: C57 mice were obtained and met the requirements of thymic aging. mUCMSCs were infused via the tail vein at a dose of 1×107 cells/kg twice per week for 3 weeks. Six weeks after the last transplantation, the thymus was weighed, and the thymus-to-body weight ratio was calculated. The thymus tissue was stained with HE.

Mechanism and therapeutic effect of umbilical cord mesenchymal stem cells in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a persistent and chronic disease that is characterized by destructive gastrointestinal (GI) inflammation. Researchers are trying to identify and develop new and more effective treatments with no side effects. Acute and chronic mouse models of IBD were established using dextran sulfate sodium (DSS) solution. To evaluate the efficacy and mechanism, umbilical cord mesenchymal stem cells (UCMSCs) were obtained from Kunming (KM) mice and humans. In the chronic IBD study, the survival rates of the normal control, model, mouse UCMSC (mUCMSC) and human UCMSC (hUCMSC) groups were 100%, 40%, 86.7%, and 100%, respectively. The histopathological scores of the normal control, intraperitoneal injection, intravenous treatment, and model groups were 0.5 ± 0.30, 5.9 ± 1.10, 8.7 ± 1.39, and 8.8 ± 1.33 (p = 0.021). UCMSCs promoted the expression of the intestinal tight junction protein occludin, downregulated the protein expression of the autophagy marker LC3A/B in colon tissue, and upregulated the expression of VEGF-A and VEGFR-1 at the injured site. This study provides an experimental model for elucidating the therapeutic effects of UCMSCs in IBD. We provide a theoretical basis and method for the clinical treatment of IBD using UCMSCs.

Relationship between senescence in macaques and bone marrow mesenchymal stem cells and the molecular mechanism.

The relationship between bone marrow mesenchymal stem cells (BMSCs) and aging, as well as the antiaging effects of BMSCs, was observed. An aging macaque BMSC model was established. We isolated BMSCs from young and aged macaques and used RT-PCR and Western blot to confirm the aging-related mRNAs and their expression, revealing that TERT, SIRT1 and SIRT6 expression was decreased in the aged BMSCs. The morphology, immunophenotype, differentiation potential, proliferation potential, and antiaging effects of aged and young BMSCs on 293T cells were compared. The expression of aging-related genes and the difference between the secreted cytokines in natural aging and induced aging BMSCs were observed. The transcriptome of peripheral blood mononuclear cells from macaques was analyzed by high-throughput sequencing. Finally, the transcriptional characteristics and regulatory mechanisms of gene transcription in aging macaques were investigated.

Umbilical cord mesenchymal stem cells are able to undergo differentiation into functional islet-like cells in type 2 diabetic tree shrews.

Islet transplantation is arguably one of the most promising strategies to treat patients suffering with diabetes mellitus. However, a combination of a lack of donors and chronic immune rejection limit clinical applications. Here, we evaluated the efficacy of cell therapy using islet-like cells differentiated from umbilical cord mesenchymal stem cells (UC-MSCs) of tree shrews for the treatment of type 2 diabetes. Enhanced green fluorescent protein (eGFP) labeled UC-MSCs were directly injected into type 2 diabetic tree shrews, where UC-MSC differentiated into functional islet-like cells and alleviated disease severity, as evidenced by improved biochemical features and reduced concentrations of inflammatory cytokines. We also demonstrated that in vitro culture of UC-MSCs for six days in a high-glucose environment (40 mmol/L or 60 mmol/L glucose) resulted in significant gene methylation. The potency of UC-MSCs differentiated into insulin-secreting cells was attributed to the activation of Notch signal pathways. This study provides evidence that cell therapy of islet-like cells differentiated from UC-MSCs is a feasible, simple and inexpensive approach in the treatment of type 2 diabetes.

Systemic delivery of human bone marrow embryonic-like stem cells improves motor function of severely affected dystrophin/utrophin-deficient mice.

BACKGROUND AIMS: Embryonic-like stem cells (ELSCs) express embryonic stem cell-specific marker genes, such as SSEA-4, Oct-4 and Nanog, and can be induced to differentiate into cells of all 3 germ layers. Our preliminary data showed that ELSCs isolated from human bone marrow express multipotent antigen markers and differentiate into multinucleated myotube-like cells more efficiently than do mesenchymal stromal cells (MSCs) isolated from the same source. We investigated the therapeutic effect of ELSCs in dystrophin/utrophin double knock-out (dko) mice, one of the Duchenne muscular dystrophy animal models, by systemically transplanting them through tail-vein injection. METHODS: ELSCs and MSCs were both isolated from human bone marrow. Two months after equal amounts of ELSCs or MSCs were injected through tail-vein injection, we evaluated skeletal muscle motor function and serum creatine kinase activity and measured dystrophin expression by means of immunostaining, Western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. RESULTS: ELSCs positive for Oct-4 and Nanog-3 expressed higher levels of SSEA-4, FZD-9 and CD105 and were induced to differentiate into myotube-like cells more efficiently than did MSCs in vitro. Transplantation of ELSCs through the tail vein improved motor function and decreased serum creatine kinase activity at 2 months after cell transplantation. In addition, dystrophin protein and messenger RNA were upregulated and the skeletal muscle histology was improved in these dko mice transplanted with ELSCs. CONCLUSIONS: ELSCs could be more efficiently induced to differentiate into myotubes than were MSCs in vitro, and systematically transplanting ELSCs improved muscle motor function and muscle histology in dko mice.

Bone-marrow mesenchymal stem cell transplantation to treat diabetic nephropathy in tree shrews.

Diabetic nephropathy (DN) is a common microvascular complication of diabetes. We used a new DN model in tree shrews to validate the use of bone-marrow mesenchymal stem cell (BM-MSC) transplantation to treat DN. The DN tree shrew model was established by a high-sugar and high-fat diet and four injections of streptozotocin. 4',6-Diamidino-2-phenylindole labelled BM-MSCs were injected into tree shrews. The DN tree shrew model was successfully established. Blood glucose was significantly increased ( p < 0.01) during the entire experiment. DN tree shrews showed dyslipidemia, insulin resistance and increased 24-h proteinuria. At 21 days after BM-MSC transplantation, glucose and levels of triglycerides, total cholesterol and 24-h urine volume were lower than in tree shrews with DN alone ( p < 0.01) but were still higher than control values ( p < 0.01). Levels of creatinine and urea nitrogen as well as 24-h proteinuria were lower for DN tree shrews with BM-MSCs transplantation than DN alone ( p < 0.05). High-sugar and high-fat diet combined with STZ injection can induce a tree shrew model of DN. BM-MSCs injection can home to damaged kidneys and pancreas, for reduced 24-h proteinuria and improved insulin resistance.

Induced autologous stem cell transplantation for treatment of rabbit renal interstitial fibrosis.

INTRODUCTION: Renal interstitial fibrosis (RIF) is a significant cause of end-stage renal failure. The goal of this study was to characterize the distribution of transplanted induced autologous stem cells in a rabbit model of renal interstitial fibrosis and evaluate its therapeutic efficacy for treatment of renal interstitial fibrosis. METHODS: A rabbit model of renal interstitial fibrosis was established. Autologous fibroblasts were cultured, induced and labeled with green fluorescent protein (GFP). These labeled stem cells were transplanted into the renal artery of model animals at 8 weeks. RESULTS: Eight weeks following transplantation of induced autologous stem cells, significant reductions (P < 0.05) were observed in serum creatinine (SCr) (14.8 ± 1.9 mmol/L to 10.1 ± 2.1 mmol/L) and blood urea nitrogen (BUN) (119 ± 22 µmol/L to 97 ± 13 µmol/L), indicating improvement in renal function. CONCLUSIONS: We successfully established a rabbit model of renal interstitial fibrosis and demonstrated that transplantation of induced autologous stem cells can repair kidney damage within 8 weeks. The repair occurred by both inhibition of further development of renal interstitial fibrosis and partial reversal of pre-existing renal interstitial fibrosis. These beneficial effects lead to the development of normal tissue structure and improved renal function.

Comparative study among three different methods of bone marrow mesenchymal stem cell transplantation following cerebral infarction in rats.

The objective of this study was to investigate the effects of transplanted bone marrow mesenchymal stem cells (BMSCs) administered via internal jugular vein injection, carotid artery injection, or intraventricular transplantation for the treatment of cerebral infarction, which was modeled in rats. The neurological scores of the treated rats and the distribution of the transplanted cells (GFP-labeled) in the infarction area were evaluated. The cerebral infarction model was produced by inserting a modified Zea-longa suture, which generated middle cerebral artery occlusion (MCAO). The GFP-labeled BMSCs were transplanted through the jugular vein or the carotid artery or by stereotactic intraventricular delivery to the infarction models 1 week after the cerebral infarction was established. The 'Nerve Function Score' of the model rats was recorded before and after BMSC transplantation. Brain tissue sections were examined under a fluorescence microscope. We determined that the transplanted BMSCs rescued brain function, which was indicated by a decrease in the neurological scores (P<0·05) following BMSC transplantation. The effect of BMSC transplantation was reflected in decreases in the neurological score in the intraventricular transplantation group, the carotid artery transplantation group, and the jugular vein graft group*. The transplanted BMSCs were able to migrate to the brain injury area and the cortex and survived the infarction; thus, BMSCs may promote the recovery of nerve function.

Induced autologous stem cell transplantation for treatment of rabbit type 1 diabetes.

We have examined the effects of induced autologous stem cells on blood sugar levels in a rabbit model of type 1 diabetes. Rabbit skin fibroblasts were induced to dedifferentiate into multipotent stem cells, and were transplanted into the treatment group via the pancreatic artery. After the fibroblasts had been induced for 72 h, some of them became multipotent stem cells. Four weeks after cell transplantation, blood glucose levels of the induced stem cell treatment group were significantly lower. The plasma insulin and plasma C-peptide levels of the treated group were significantly increased (P < 0.05). The shape and number of islets was different. In the control group, induced cell treatment group and non-induced cell treatment group. In the control group, islet ß-cell nucleoli were obvious, and cell volumes were larger with more abundant cytoplasm. The rough endoplasmic reticulum was well-developed and a large number of secretory granules could be seen within the cytoplasm. In the induced cell treatment group, islet ß cells were scattered, and their nuclei were oval and slightly irregular in shape. The cytoplasm of these cells contained a nearly normal number of secretory granules. In the non-induced cell treatment group, islet ß-cells were atrophied and cell volumes were reduced. Cytoplasmic endocrine granules were significantly reduced or absent. In conclusion, treatment with induced multipotent stem cells can reduce blood sugar levels, improve islet cell function, and repair damaged pancreas in a rabbit model of type 1 diabetes.

Treatment with chicken-egg-white or whole-egg extracts maintains and enhances the survival and differentiation of spleen cells.

The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30 min, cultured for 12 days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120 days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank's Buffered Salt Solution (HBSS) as a control. At 120 days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells.

Transient in vitro epigenetic reprogramming of skin fibroblasts into multipotent cells.

Multipotent stem cells have the potential to establish a new field of promising regenerative medicine to treat tissue damage, genetic disorders, and degenerative diseases. However, limited resource of stem cells has turned to be an evitable obstacle in clinical applications. We utilized a simple in vitro epigenetic reprogramming approach to convert skin fibroblasts into multipotent cells. After transient reprogramming, stem cell markers, including Oct4 and Nanog, became activated in the treated cells. The reprogrammed cells were multipotent as demonstrated by their ability to differentiate into a variety of cells and to form teratomas. Genomic imprinting of insulin-like growth factor II (Igf2) and H19 was not affected by this short period of cell reprogramming. This study may provide an alternative strategy to efficiently generate patient-specific stem cells for basic and clinical research, solving major hurdles of virally-induced pluripotent stem (iPS) cells that entail the potential risks of mutation, gene instability, and malignancy.

[Prokaryotic expression of N terminal fragment of mannan-binding lectin associated serine protease-1].

AIM: To express the N-terminal fragment of human mannan-binding lectin (MBL) associated serine proteases-1 (MASP1-N) in E.coli. METHODS: The target sequence in pGEM-MASP1 plasmid that contains human MBL-MASP1 cDNA was amplified by PCR, inserted into prokaryotic expression vector pGEX4T-1 and identified by restriction mapping and sequencing. The recombinant expression vector was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by GSTrap Immobilized Metal Affinity Chromatography(IMAC) and identified by SDS-PAGE and Western blot assay, its binding-activity with the collagen-like region of human MBL(MBL-CLR) and with recombinant human MBL was analyzed by an indirect enzyme-linked immunosorbent assayèELISAé. RESULTS: The DNA fragment of 860 bp, which encode the N-terminal region of human MASP1, was amplified from pGEM-MASP1 plasmid and the recombinant expression vector, pGEX4T-MASP1-N, was constructed, whose restriction maps and sequence were consistent with those expected. The component of M(r) 60 000 in the purified recombinant product was found by SDS-PAGE and could be recognized by anti-GST antibody in Western blot assay. The purified recombinant product could react with human MBL-CLR and human MBL in the indirect ELISA. CONCLUSION: The prokaryotic cell strain that expresses efficiently recombinant human MASP1-N(rhMASP1-N) protein and the purified rhMASP1-N protein were successfully obtained, which provides the basis for further research of MASP1 molecule.

[Construction of the expression vector pET32a/His MBL-CLR and its expression in E.coli].

AIM: To construct pET32a/His MBL-CLR recombinant prokaryotic expression plasmid and to express mannan-binding lectin-CLR (MBL-CLR) protein in E.coli METHODS: The human MBL-CLR gene was amplified by PCR from pGEM-MBL plasmid, and was inserted into prokaryotic expression vector pET32a. After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBL-CLR was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDS-PAGE, Western blot and indirect enzyme-linked immunosorbent assay (ELISA) using the antibody from BALB/c mice immunized with the recombinant human MBL protein. RESULTS: The cDNA fragment of 180 bp was amplified from pGEM-MBL plasmid and the recombinant expression vector pET32/His MBL-CLR was constructed. The recombinant plasmid was consistent with those expected by restriction maps and sequence. Three components of relative molecular mass 30,000, 60,000 and 120,000 in the purified recombinant product were detected by SDS-PAGE and all the components could be recognized by anti-6His antibody in Western blot assay. The three components were correspondingly with the band of the monomer and oligomer of the fusion protein. The purified recombinant product could react with the antibody against the recombinant human MBL protein in the indirect ELISA. CONCLUSION: The prokaryotic expression strains that efficiently express recombinant human MBL-CLR and the recombinant human MBL-CLR-Trx fusion protein were obtained successfully, which will help the further structure-function research of MBL molecule.