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Objective:To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on the apoptosis of retinal ganglion cells (RGCs) in diabetic retinopathy (DR) model rats and on the regulation of p38 mitogen-activated protein kinase (p38MAPK) pathway.Methods:Forty-five SPF male 8-week old SD rats were selected.The DR rat model was established by intraperitoneal injection of streptozotocin (STZ) combined with a high-sugar and high-fat diet.The fasting blood glucose (FBG) and body weight of the rats were measured every week during the high-sugar and high-fat diet, and fundus angiography was used to observe the circulation and leakage of retinal blood vessels.Forty rats with successful modeling were randomly divided into model group and hUC-MSC injection group according to the random number table method, with 20 rats in each group.Another 20 normal rats fed routinely were served as control group, and intraperitoneally injected with the same amount of citric acid buffer.The hUC-MSC injection group was injected intravitreously with hUC-MSC, and the control group and model group were injected intravitreously with the same amount of phosphate buffer saline (PBS). Fluoro gold (FG) retrograde tracer labeling RGCs was used to observe the number of survived RGCs.Hematoxylin-eosin staining was used to observe the pathological changes of retina.TUNEL method was used to observe the apoptosis of RGCs.Western blot was used to detect B cell lymphoma /leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax), p38MAPK and phosphorylated (p-) p38MAPK protein expression in retinal tissues.The use and care of the rats complied with the ARVO statement.The study protocol was approved by an Animal Ethics Committee of Zhengzhou central Hospital Affiliated to Zhengzhou University (NO.2980316).Results:The FBG of control rats was maintained at a normal level, and the body weight gradually increased over time, and was gradually decreased as the course of disease prolonged.The retinal blood vessels ran normally without fluorescein leakage in the control group.In the modeling group, the FBG was maintained at a high level, and the body weight increased slowly and gradually decreased with the prolongation of the disease course since the second week after modeling.The distal retinal vessels were found twisted with large area of capillary fluorescein leakage in the modeling group.The density of RGCs in the control group, model group and hUC-MSC injection group were (2 136.10±215.17), (849.40±167.82), (1 549.20±183.26) cells/mm 2, with significant overall differences ( F=115.218, P<0.01). The density of RGCs in the model group and the hUC-MSC injection group were significantly lower than that of the control group, and the density of RGCs in the hUC-MSC injection group was significantly higher than that of the model group, and the differences were statistically significant (all at P<0.05). The retina in the control group was with clear structure, distinct layers, and a large number of complete RGCs.The number of RGCs in the model group was significantly reduced with nuclear pyknosis, thinned and atrophied RGC layer.The retinal structure was relatively complete, and there were more RGCs in the hUC-MSC injection group than the model group.The apoptosis rates of RGCs in the control group, model group and hUC-MSC injection group were (2.16±1.11)%, (43.47±2.26)%, (20.75±2.18)%, with significant overall difference ( F=445.159, P<0.01). The apoptosis rates of RGCs in the model group and hUC-MSC injection group were significantly higher than that of the control group, and the apoptosis rate of RGCs in the hUC-MSC injection group was lower than that of the model group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of Bax, Bcl-2 and p-p38MAPK proteins in the retina tissues among the three groups ( F=30.982, 12.526, 73.158, all at P<0.01). The relative expression of Bax and p-p38MAPK protein were significantly higher, and the relative expression of Bcl-2 protein was significantly lower in the hUC-MSC injection group and the hUC-MSC injection group than those of the control group, and the differences were statistically significant (all at P<0.05). The relative expression of Bax and p-p38MAPK protein was significantly lower, and the relative expression of Bcl-2 protein was significantly higher in the hUC-MSC injection group than those in the model group, and the differences were statistically significant (all at P<0.05). There was no significant difference in the relative expression of p38MAPK protein among the three groups ( F=1.182, P=0.322). Conclusions:Intravitreal injection of hUC-MSC can inhibit the apoptosis of RGCs in DR model rats and protect the retinal structure of rats, which may play an anti-apoptotic effect by inhibiting the p38MAPK signaling pathway.
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Objective:To investigate the long-term effect of the implantation of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) for type 1 diabetes mellitus.Methods:Fifteen patients with type 1 diabetes mellitus were treated with HUC-MSCs from September 2009 to December 2011 at Department of Endocrinology and Metabolism, the Second People′s Hospital. Patients were followed-up for 10 years and the parameters were collected including fasting blood glucose, HbA 1C, mean amplitude of glycemic excursions (MAGE), fasting C-peptide, daily insulin doses and glutamic acid decarboxylase antibody (GADA). Results:Among 15 patients, 1 patient (6.67%) was found with breast cancer. All patients with type 1 diabetes mellitus decreased daily insulin doses due to frequent hypoglycemia one week later. Six months later, 4 patients (26.67%) stopped insulin injection. While among the 4 patients, 1 patient (6.67%) had not yet used insulin until today and GADA was negative, the other 3 patients (20.00%) restarted insulin within 3-5 years after implantation with significantly less daily insulin doses [(18.00±1.00)U vs (29.00±1.73)U, P<0.01]. The remaining 11 patients (73.33%) with type 1 diabetes mellitus who did not stop insulin also had significantly lower daily insulin doses [(18.09±0.83)U vs (29.64±0.89)U, P<0.01]. The level of MAGE was signicantly decreased compared to those of pre-implantation [(6.14±0.25)mmol/L vs (9.72±0.32)mmol/L, P <0.01], while fasting C-peptide level was significantly improved[(0.91±0.03)nmol/L vs (0.11±0.01)nmol/L, P <0.01]. There were no significant differences in fasting blood glucose and HbA 1C before and after implantation. Conclusions:The implantation of HUC-MSCs for the treatment of type 1 diabetes mellitus can restore the function of islet β cells, decrease daily insulin doses and reduce blood glucose fluctuations in the long term. Although precise mechanisms are unknown, this therapy is expected to be an effective strategy for treatment of type 1 diabetes mellitus.
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BACKGROUND: Mesenchymal stem cells have been widely applied in autoimmune diseases and graft-versus-host diseases because of their immunomodulatory capabilities. However, mesenchymal stem cells have plasticity in immunomodulation, which leads to heterogeneity and instability when used in vivo. OBJECTIVE: To investigate the polarization of human umbilical cord derived mesenchymal stem cells to an immunosuppressive phenotype (MSC2) in the inflammatory microenvironment induced by interferon-γ and lipopolysaccharide. METHODS: Human umbilical cord-derived mesenchymal stem cells were isolated and cultured in vitro, and then were identified by morphological characteristics, surface markers, adipogenesis and osteoinduction activity. Human umbilical cord-derived mesenchymal stem cells were treated with interferon-γ (10 μg/L), lipopolysaccharide (100 μg/L), or their combination for 24 hours, respectively, and were then co-cultured with phytohemagglutinin pre-treated peripheral blood mononuclear cells for 5 days under direct or Transwell indirect contact. The percentages of regulatory T cells and T helper 1 cells were analyzed by flow cytometry at different times. The mRNA expression levels of Toll-like receptors 2, 3 and 4 were detected by real-time fluorescence quantitative PCR. RESULTS AND CONCLUSION: (1) Human umbilical cord derived mesenchymal stem cells exhibited spindle-shaped or fibroblast-like morphology, highly expressed CD73, CD90 and CD105, and lacked expression of CD34, CD45 and HLA-DR. (2) Under direct or indirect co-culture, human umbilical cord-derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide could promote the generation of regulatory T cells, which was superior to the interferon-γ, lipopolysaccharid, un-treated and control groups (P < 0.05). (3) The percentage of T helper 1 cells gradually decreased over time. (4) Under indirect co-culture, human umbilical cord derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide were polarized into immunosuppressive MSC2 phenotype at an earlier period and highly expressed Toll-like receptor 3 (P < 0.05). (5) In conclusion, the combination of interferon-γ (10 μg/L) and lipopolysaccharide (100 μg/L) results in the high-efficient polarization of mesenchymal stem cells toward the MSC2 phenotype under indirect co-culture, and the immunosuppressive capability of MSC2 is independent of intercellular contact, which provides clinical evidence for the MSC2-derived exosome therapy in the future.
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Objective:To compare the ability between bone marrow-derived mesenchymal stem cells (MSCs) (BM-MSCs) and adipose-derived MSCs (AD-MSCs) or umbilical cord-derived MSCs (UC-MSCs) on promotion of vessels formation and vessds stabilization relevant to the functions of EPCs.Methods:In vitro,co-culture blood vessel test was performed to compare the angiogenic ability between BM-MSCs,AD-MSCs or UC-MSCs.In vivo,angiogenic assay dependent on basement membrane matrix Matrigel and immunohistochemistry were performed to compare the ability of vessels formation functions between BM-MSCs and AD-MSCs or UC-MSCs.Results:The lengths and dots of vascular structures formed by EPCs on AD-MSCs layer are greater than those by EPCs on BM-MSCs layer and UC-MSCs layer in angiogenic assay in vitro.The stability of the capillary-like structures formed by EPCs with AD-MSCs on Matrigel was more stable than that by the BM-MSCs,UC-MSCs or EPCs.AD-MSCs and EPCs could form abundant functional vessels with blood perfusion in Matrigelin vivo;UC-MSCs and EPCs could form a few functional vessels with blood perfusion in Matrigelin vivo;BM-MSCs and EPCs could form broken vessels with hemocytes leakage in Matrigel in vivo.Conclusion:AD-MSCs have the stronger ability to promote the angiogenesis and stabilize the vessels compared with BM-MSCs or UC-MSCs ex vivo and in vivo.
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AIM: To investigate the effects of human umbilical cord-derived mesenchymal stem cells ( hUC-MSCs) on the proliferation and migration of osteosarcoma cells ( Saos-2 ) and the underlying molecular mechanism. METHODS:hUC-MSCs were isolated and cultured by tissue explants adherent method.The cell surface markers on hUC-MSCs were identified by flow cytometry.The effects of conditioned medium ( CM) from hUC-MSCs ( hUC-MSCs-CM) , re-combinant human interleukin-6 (rhIL-6) and IL-6 neutralizing antibody on the proliferation of Saos-2 cells were detected by CCK-8 assay and cell counting.IL-6 secretion of hUC-MSCs was assayed by ELISA.RT-PCR was used to assess the tran-scription level of proliferation-related genes proliferating cell nuclear antigen ( PCNA) , cyclin D1 and survivin.The migra-tion potential of hUC-MSCs and Saos-2 cells was measured by Transwell assay.RESULTS:hUC-MSCs migrated to Saos-2 cells.hUC-MSCs-CM contained a high concentration of IL-6, up to (1 835.5 ±134.1) ng/L.hUC-MSCs-CM and rhIL-6 promoted the proliferation and migration of Saos-2 cells.Addition of neutralizing antibody against IL-6 in the hUC-MSCs-CM impaired this proliferation and migration of Saos-2 cells.The mRNA expression of PCNA, cyclin D1 and survivin was up-regulated by hUC-MSCs-CM and rhIL-6, while this effect was dramatically attenuated by treatment with IL-6 neutralizing antibody.CONCLUSION:hUC-MSCs migrate to osteosarcoma cells and promote the proliferation and migration of osteo-sarcoma cells through secreting IL-6 in vitro.
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Objective To observe the intervention effect of Ginkgo biloba extract EGb761 on tert-butyl hydroperoxide ( t-BHP)-induced injury in human umbilical cord-derived mesenchymal stem cells ( hUC-MSCs) . Methods Proliferation of hUC-MSCs after primary culture was detected by methyl thiazolyl tetrazolium (MTT) assay, and then the optimal concentrations of t-BHP and EGb761 for oxidative stress injured MSC model were screened. Cell apoptosis was assessed by flow cytometry after Annexin V-fluorescein isothiocyanatel propidium iodide ( Annexin V-FITC/PI) staining. Content of malondialdehyde ( MDA) and superoxide dismutase (SOD) activity in hUC-MSCs were evaluated, and the expression levels of p53 and p21Cip1/Waf1 were analyzed by real-time fluorescence PCR. Results Pretreatment with 10~200 mg/L of EGb761 for 3 hours reduced the sensitivity of hUC-MSCs to t-BHP ( 100 μmol/L) induced proliferation inhibition, while EGb761 over 100 mg/L had no significant effect on enhancing the protection of hUC-MSCs . EGb761 at 100 mg/L prohibited hUC-MSCs apoptosis and MDA accumulation in hUC-MSCs induced by 100μmol/L of t-BHP acting for 6 hours, maintained the enzymatic activity of SOD, and decreased the expression of p53 and p21Cip1/Waf1 in hUC-MSCs with t-BHP-induced injury. Conclusion EGb761 is capable of protecting hUC-MSCs against oxidative stress injury, and its mechanism is probably related with the modulation of p53/p21 signal pathway.
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Objective:To investigate the impact of human umbilical cord-derived mesenchymal stem cells on the activation ,the survival of human peripheral blood mononuclear cell ( hPBMC) and the proportions of each human lymphoid subgroup .Methods:PB-MC were isolated from healthy donors by density gradient centrifugation , then cultured in MSC-CM as treatment group after being acti-vated by OKT3.Each lymphoid subgroup proportion was analyzed by flow cytometry to observe the difference between treatment and control group .The effect of MSC-CM on activated PBMC for the production of IFN-γand IL-10 were tested by ELISA .The level of ap-optosis was assessed by flow cytometry with Annexin-V/PI as fluorescent marker .Results:Compared with the control group , MSC-CM down-regulated the ratio of CD4 +T cell to CD8 +T cell, and increased the proportion of CD4 +CD25 +CD127low Treg cell, thus other subgroup had no significant difference .MSC-CM inhibited the production of IFN-γby PBMC, but promoted the secretion of IL-10, and protected PBMCs from apoptosis when activated with OKT 3.Conclusion:hUC-MSC may play a role of immunosuppression by promo-ting the proliferation and activation of Treg cell .This kind of inhibitory activity is neither relied direct or indirect contact with the lym -phocytes , nor influenced by inducing immune cells apoptosis .
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The use of human umbilical cord blood-derived mesenchymal stem cells for cell transplantation therapy holds great promise for repairing spinal cord injury. Here we report the first clinical trial transplantation of human umbilical cord (hUCB)-derived mesenchymal stem cells (MSCs) into the spinal cord of a dog suspected to have fibrocartilaginous embolic myelopathy (FCEM) and that experienced a loss of deep pain sensation. Locomotor functions improved following transplantation in a dog. Based on our findings, we suggest that transplantation of hUCB-derived MSCs will have beneficial therapeutic effects on FCEM patients lacking deep pain sensation.
Subject(s)
Animals , Dogs , Female , Humans , Cartilage Diseases/etiology , Cord Blood Stem Cell Transplantation/veterinary , Dog Diseases/etiology , Embolism/etiology , Mesenchymal Stem Cells/cytology , Spinal Cord Diseases/etiology , Treatment OutcomeABSTRACT
The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cordderived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we selected four types of novel small molecule candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.
Subject(s)
Humans , Cell Count , Cell Culture Techniques , Cell Cycle , Fetal Stem Cells , Hydrazines , Mass Screening , Mesenchymal Stem Cells , Seeds , Stem Cell Research , Stem Cells , Umbilical CordABSTRACT
Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.