Breast cancer is the predominant form of carcinoma among women worldwide,with 70%of advanced patients developing bone metastases,with a high mortality rate.In this sense,the bone marrow(BM)mesenchymal stem/stromal cel...Breast cancer is the predominant form of carcinoma among women worldwide,with 70%of advanced patients developing bone metastases,with a high mortality rate.In this sense,the bone marrow(BM)mesenchymal stem/stromal cells(MSCs)are critical for BM/bone homeostasis,and failures in their functionality,transform the BM into a premetastatic niche(PMN).We previously found that BM-MSCs from advanced breast cancer patients(BCPs,infiltrative ductal carcinoma,stage III-B)have an abnormal profile.This work aims to study some of the metabolic and molecular mechanisms underlying MSCs shift from a normal to an abnormal profile in this group of patients.A comparative analysis was undertaken,which included self-renewal capacity,morphology,proliferation capacity,cell cycle,reactive oxygen species(ROS)levels,and senescence-associatedβ‑galactosidase(SA‑β‑gal)staining of BMderived MSCs isolated from 14 BCPs and 9 healthy volunteers(HVs).Additionally,the expression and activity of the telomerase subunit TERT,as well as telomere length,were measured.Expression levels of pluripotency,osteogenic,and osteoclastogenic genes(OCT-4,SOX-2,M-CAM,RUNX-2,BMP-2,CCL-2,M-CSF,and IL-6)were also determined.The results showed that MSCs from BCPs had reduced,self-renewal and proliferation capacity.These cells also exhibited inhibited cell cycle progression and phenotypic changes,such as an enlarged and flattened appearance.Additionally,there was an increase in ROS and senescence levels and a decrease in the functional capacity of TERT to preserve telomere length.We also found an increase in pro-inflammatory/pro-osteoclastogenic gene expression and a decrease in pluripotency gene expression.We conclude that these changes could be responsible for the abnormal functional profile that MSCs show in this group of patients.展开更多
BACKGROUND Bone marrow mesenchymal stromal cells(BMSCs)are the commonly used seed cells in tissue engineering.Aryl hydrocarbon receptor(AhR)is a transcription factor involved in various cellular processes.However,the ...BACKGROUND Bone marrow mesenchymal stromal cells(BMSCs)are the commonly used seed cells in tissue engineering.Aryl hydrocarbon receptor(AhR)is a transcription factor involved in various cellular processes.However,the function of constitutive AhR in BMSCs remains unclear.AIM To investigate the role of AhR in the osteogenic and macrophage-modulating potential of mouse BMSCs(mBMSCs)and the underlying mechanism.METHODS Immunochemistry and immunofluorescent staining were used to observe the expression of AhR in mouse bone marrow tissue and mBMSCs.The overexpression or knockdown of AhR was achieved by lentivirus-mediated plasmid.The osteogenic potential was observed by alkaline phosphatase and alizarin red staining.The mRNA and protein levels of osteogenic markers were detected by quantitative polymerase chain reaction(qPCR)and western blot.After coculture with different mBMSCs,the cluster of differentiation(CD)86 and CD206 expressions levels in RAW 264.7 cells were analyzed by flow cytometry.To explore the underlying molecular mechanism,the interaction of AhR with signal transducer and activator of transcription 3(STAT3)was observed by co-immunoprecipitation and phosphorylation of STAT3 was detected by western blot.RESULTS AhR expressions in mouse bone marrow tissue and isolated mBMSCs were detected.AhR overexpression enhanced the osteogenic potential of mBMSCs while AhR knockdown suppressed it.The ratio of CD86+RAW 264.7 cells cocultured with AhR-overexpressed mBMSCs was reduced and that of CD206+cells was increased.AhR directly interacted with STAT3.AhR overexpression increased the phosphorylation of STAT3.After inhibition of STAT3 via stattic,the promotive effects of AhR overexpression on the osteogenic differentiation and macrophage-modulating were partially counteracted.CONCLUSION AhR plays a beneficial role in the regenerative potential of mBMSCs partially by increasing phosphorylation of STAT3.展开更多
Transplantation of bone marrow stromal cells(BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury(SCI).BMSCs did not survive long-term,disappearing fr...Transplantation of bone marrow stromal cells(BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury(SCI).BMSCs did not survive long-term,disappearing from the spinal cord within 2–3 weeks after transplantation.Astrocyte-devoid areas,in which no astrocytes or oligodendrocytes were found,formed at the epicenter of the lesion.It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas.Regenerating axons were associated with Schwann cells embedded in extracellular matrices.Transplantation of choroid plexus epithelial cells(CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI.Although CPECs disappeared from the spinal cord shortly after transplantation,an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas,as in the case of BMSC transplantation.These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord,including axonal regeneration and reduced cavity formation.This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate.The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI.It should be emphasized that the generally anticipated long-term survival,proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.展开更多
BACKGROUND:Bone marrow cells can differentiate into hepatocytes in a suitable microenvironment.This study was undertaken to investigate the effects of transplanted bone marrow stromal cells (BMSCs) on liver fibrosis i...BACKGROUND:Bone marrow cells can differentiate into hepatocytes in a suitable microenvironment.This study was undertaken to investigate the effects of transplanted bone marrow stromal cells (BMSCs) on liver fibrosis in mice. METHODS:BMSCs were harvested and cultured from male BALB/c mice, then transplanted into female syngenic BALB/c mice via the portal vein. After partial hepatectomy, diethylnitrosamine (DEN) was administered to induce liver fibrosis. Controls received BMSCs and non-supplemented drinking water, the model group received DEN with their water, and the experimental group received BMSCs and DEN. Mice were killed after 3 months, and ALT, AST, hyaluronic acid (HA), and laminin (LN) in serum and hydroxyproline (Hyp) in the liver were assessed. Alpha-smooth muscle actin (α-SMA) in the liver was assessed by immunohistochemistry. Bone marrow- derived hepatocytes were identified by fluorescent in situ hybridization (FISH) in liver sections. RESULTS:BMSCs were shown to differentiate into hepatocyte-like phenotypes after hepatocyte growth factor treatment in vitro. Serum ALT, AST, HA, and LN were markedly reduced by transplanted BMSCs. Liver Hyp content and α-SMA staining in mice receiving BMSCs were lower than in the model group, consistent with altered liver pathology. FISH analysis revealed the presence of donor- derived hepatocytes in the injured liver after cross-gender mouse BMSC transplantation. After three months, about 10% of cells in the injured liver were bone marrow-derived. CONCLUSION:BMSCs transplanted via the portal vein can convert into hepatocytes to repair liver injury induced by DEN, restore liver function, and reduce liver fibrosis.展开更多
Objective To investigate the differentiation of bone marrow stromal cells (BMSC) into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultu...Objective To investigate the differentiation of bone marrow stromal cells (BMSC) into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC (hBMSC) to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilament1 (NF1), nestin and neuron-specific enolase (NSE) in rat BMSC (rBMSC). Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NF1 and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated. rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.展开更多
Objective To investigate the effects of Panax notoginseng saponins(PNS)on hydrogen peroxide(H2O2)-induced apoptosis in cultured rabbit bone marrow stromal cells(BMSCs).Methods BMSCs from 3-month-old New Zealand rabbit...Objective To investigate the effects of Panax notoginseng saponins(PNS)on hydrogen peroxide(H2O2)-induced apoptosis in cultured rabbit bone marrow stromal cells(BMSCs).Methods BMSCs from 3-month-old New Zealand rabbits were isolated and cultured by the density gradient centrifugation combined with adherent method.The cultured BMSCs were divided into three groups:normal control,H2O2 treatment(100μmol/L),and PNS pretreatment(0.1g/L).Intracellular reactive oxygen species(ROS)levels as the index of oxidative stress were measured by using 2'7'-dichlorodihydrofluorescein diacetate.Flow cytometry was used to observe the apoptosis of BMSCs by staining with annexinV-FITC/PI.The protein expression of Bax in BMSCs was analyzed by Western blotting.Activity of caspase-3 enzyme was measured by spectrofluorometry.Results Pretreatment with PNS significantly decreased intracellular ROS level induced by H2O2(P<0.01).PNS markedly attenuated H2O2-induced apoptosis rate from 38.68% to 19.24%(P<0.01).PNS reversed H2O2-induced augmentation of Bax expression.Furthermore,PNS markedly reduced the altered in activity of caspase-3 enzyme induced by H2O2(P<0.01).Conclusion PNS has a protective effect on hydrogen peroxide-induced apoptosis in cultured rabbit BMSCs by scavenging ROS and decreasing Bax expression and caspase-3 activity.展开更多
Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analys...Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison, the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.展开更多
We previously demonstrated that overexpression of tropomyosin receptor kinase A(TrkA)promotes the survival and Schwann celllike differentiation of bone marrow stromal stem cells in nerve grafts,thereby enhancing the r...We previously demonstrated that overexpression of tropomyosin receptor kinase A(TrkA)promotes the survival and Schwann celllike differentiation of bone marrow stromal stem cells in nerve grafts,thereby enhancing the regeneration and functional recovery of the peripheral nerve.In the present study,we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts.Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA,TrkA-shRNA or the respective control.The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect.Then,8 weeks after surgery,hematoxylin and eosin staining showed that compared with the control groups,the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged,whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group.Western blot assay showed that compared with the control groups,the TrkA overexpressing group had higher expression of the myelin marker,myelin basic protein and the axonal marker neurofilament 200.The TrkA overexpressing group also had higher levels of various signaling molecules,including TrkA,pTrkA(Tyr490),extracellular signal-regulated kinases 1/2(Erkl/2),pErk1/2(Thr202/Tyr204),and the anti-apoptotic proteins Bcl-2 and Bcl-xL.In contrast,these proteins were downregulated,while the pro-apoptotic factors Bax and Bad were upregulated,in the TrkA-shRNA group.The levels of the TrkA effectors Akt and pAkt(Ser473)were not different among the groups.These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway.All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University,China in December 2014(approval No.AEWC-2014-001219).展开更多
AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were ...AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were isolated and cultured. Their morphological and phenotypic characteristics, as well as their multiple differentiation capacity were compared. A new culture system was established to induce ADSCs and BMSCs into functional hepatocytes. Reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to identify the induced hepatocytelike cells. CM-Dil-labeled ADSCs and BMSCs were then transplanted into a mouse model of CCl4-induced acute liver failure. fluorescence microscopy was used to track the transplanted MSCs. Liver function was tested by an automatic biochemistry analyzer, and liver tissue histology was observed by hematoxylin and eosin(HE) staining.RESULTS ADSCs and BMSCs shared a similar morphology and multiple differentiation capacity, as well as a similar phenotype(with expression of CD29 and CD90 and no expression of CD11 b or CD45). Morphologically, ADSCs and BMSCs became round and epithelioid following hepatic induction. These two cell types differentiated into hepatocyte-like cells with similar expression of albumin, cytokeratin 18, cytokeratin 19, alpha fetoprotein, and cytochrome P450. fluorescence microscopy revealed that both ADSCs and BMSCs were observed in the mouse liver at different time points. Compared to the control group, both the function of the injured livers and HE staining showed significant improvement in the ADSC-and BMSC-transplanted mice. There was no significant difference between the two MSC groups.CONCLUSION ADSCs share a similar hepatic differentiation capacity and therapeutic effect with BMSCs in an acute liver failure model. ADSCs may represent an ideal seed cell type for cell transplantation or a bio-artificial liver support system.展开更多
This study investigated the ability of millimeter-wave(MMW)to promote the differen-tiation of bone marrow stromal cells(BMSCs)into cells with a neural phenotype.The BMSCs were primarily cultured.At passage 3,the cells...This study investigated the ability of millimeter-wave(MMW)to promote the differen-tiation of bone marrow stromal cells(BMSCs)into cells with a neural phenotype.The BMSCs were primarily cultured.At passage 3,the cells were induced byβ-mercaptoethanol(BME)in combination with MMW or BME alone.The expressions of nucleostemin(NS)and neuron-specific enolase(NSE) were detected by immunofluorescent staining and Western blotting respectively to identify the differentiation.The untreated BMSCs predominately expressed NS.After induced by BME and MMW,the BMSCs exhibited a dramatic decrease in NS expression and increase in NSE expression.The differentiation rate of the cells treated with BME and MMW in combination was significantly higher than that of the cells treated with BME alone(P<0.05).It was concluded that MMW exposure enhanced the inducing effect of BME on the differentiation of BMSCs into cells with a neural phenotype.展开更多
Objective To explore the possibilities of bone marrow stromal cells (MSCs) to adopt Schwann cell phenotype in vitro and in vivo in SD rats. Methods MSCs were obtained from tibia and femur bone marrow and cultured in c...Objective To explore the possibilities of bone marrow stromal cells (MSCs) to adopt Schwann cell phenotype in vitro and in vivo in SD rats. Methods MSCs were obtained from tibia and femur bone marrow and cultured in culture flasks. Beta-mercaptoethanol followed by retinoic acid, forskolin, basic-FGF, PDGF and heregulin were added to induce differentiation of MSCs’. Schwann cell markers, p75, S-100 and GFAP were used to discriminate induced properties of MSCs’ by immunofluorescent staining. PKH-67-labelled MSCs were transplanted into the mechanically injured rat sciatic nerve, and laser confocal microscopy was performed to localize the PKH67 labelled MSCs in the injured sciatic nerve two weeks after the operation. Fluorescence PKH67 attenuation rule was evaluated by flow cytometry in vitro. Results MSCs changed morphologically into cells resembling primary cultured Schwann cells after their induction in vitro. In vivo, a large number of MSCs were cumulated within the layer of epineurium around the injured nerve and expressed Schwann cell markers, p75, S-100, and GFAP. Conclusion MSCs are able to support nerve fiber regeneration and re-myelination by taking on Schwann cell function, and can be potentially used as possible substitutable cells for artificial nerve conduits to promote nerve regeneration.展开更多
Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isola...Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.展开更多
BACKGROUND:Studies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinic...BACKGROUND:Studies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinical application.The effects of vascular endothelial growth factor (VEGF) on in vitro neuronal differentiation of BMSCs remain poorly understood.OBJECTIVE:To investigate the effect of VEGF on neuronal differentiation of BMSCs in vitro,and to determine the best VEGF concentration for experimental induction.DESIGN,TIME AND SETTING:In vitro comparative study was performed at the Central Laboratory and Laboratory of Male Reproductive Medicine,Shenzhen Hospital of Peking University from October 2008 to August 2009.MATERIALS:Recombinant human VEGF165 was purchased from Peprotech Asia,Rehovot,Israel.Neuron-specific enolase (NSE) was purchased from Beijing Biosynthesis Biotechnology,China.METHODS:BMSCs were harvested from adult Sprague Dawley rats.The passaged cells were pre-induced with 10 ng/mL basic fibroblast growth factor for 24 hours,followed by differentiation induction with 0,5,10,and 20 ng/mL VEGF,respectively.MAIN OUTCOME MEASURES:Morphological changes in BMSCs prior to and following VEGF induction.Expression of NSE following induction was determined by immunocytochemistry.RESULTS:Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20 ng/mL VEGF.However,these changes were not observed in the control group.At 10 days after induction,the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group (P < 0.05).The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction (P < 0.05).Moreover,the number of NSE-positive cells was greater at 10 days compared with at 3 days after induction (P < 0.05).CONCLUSION:Of the VEGF concentrations tested,10 ng/mL induced the greatest number of neuronal-like cells in vitro from BMSCs.展开更多
BACKGROUND: Transplantation of fetal cell suspension or blocks of fetal tissue can ameliorate the nerve function after the injury or disease in the central nervous system, and it has been used to treat neurodegenerati...BACKGROUND: Transplantation of fetal cell suspension or blocks of fetal tissue can ameliorate the nerve function after the injury or disease in the central nervous system, and it has been used to treat neurodegenerative disorders induced by Parkinson disease. OBJECTIVE: To observe the effects of the transplantation of neuron-like cells derived from bone marrow stromal cells (rMSCs) into the brain in restoring the dysfunctions of muscle strength and balance as well as learning and memory in rat models of cerebral infarction. DESIGN: A randomized controlled experiment. SETTING: Department of Pathophysiology, Zhongshan Medical College of Sun Yat-sen University. MATERIALS: Twenty-four male SD rats (3-4 weeks of age, weighing 200-220 g) were used (Certification number:2001A027). METHODS: The experiments were carried out in Zhongshan Medical College of Sun Yat-sen University between December 2003 and December 2004. ① Twenty-four male SD rats randomized into three groups with 8 rats in each: experimental group, control group and sham-operated group. Rats in the experiment al group and control group were induced into models of middle cerebral artery occlusion. After in vitro cultured, purified and identified with digestion, the Fischer344 rMSCs were induced to differentiate by tanshinone ⅡA, which was locally injected into the striate cortex (18 area) of rats in the experimental group, and the rats in the control group were injected by L-DMEM basic culture media (without serum) of the same volume to the corresponding brain area. In the sham-operated group, only muscle and vessel of neck were separated. ② At 2 and 8 weeks after the transplantation, the rats were given the screen test, prehensile-traction test, balance beam test and Morris water-maze test. ③ The survival and distribution of the induced cells in corresponding brain area were observed with Nissl stained with toluidine blue and hematoxylin and eosin (HE) staining in the groups. MAIN OUTCOME MEASURES: ① Results of the behavioral tests (time of the Morris water-maze test screen test, prehensile-traction test, balance beam test); ② Survival and distribution of the induced cells. RESULTS: All the 24 rats were involved in the analysis of results. ① Two weeks after transplantation, rats with neuron-like cells grafts in the experimental group had significant improvement on their general muscle strength than those in the control group [screen test: (9.4±1.7), (4.7±1.0) s, P < 0.01]; forelimb muscle strength [prehensile-traction test: (7.6±1.4), (5.2±1.2) s, P < 0.01], ability to keep balance [balance beam test: (7.9±0.74), (6.1±0.91) s, P < 0.01] and abilities of learning and memory [latency to find the platform: (35.8±5.9), (117.5±11.6) s, P < 0.01; distance: (623.1±43.4), (1 902.3±98.6) cm, P < 0.01] as compared with those in the control group. The functional performances in the experimental group at 8 weeks were better than those at two weeks, which were still obviously different from those in the sham-operated group (P < 0.05). ② The HE and Nissl stained brain tissue section showed that there was nerve cell proliferation at the infarcted cortex in the experiment group, the density was higher than that in the control group, plenty of aggregative or scattered cells could be observed at the site where needle was inserted for transplantation, the cells migrated directively towards the area around them, the cerebral vascular walls were wrapped by plenty of cells; In the control group, most of the cortices were destroyed, karyopyknosis and necrosis of neurons were observed, normal nervous tissue structure disappeared induced by edema, only some nerve fibers and glial cells remained. CONCLUSION: The rMSCs transplantation can obviously enhance the motor function and the abilities of learning and memory in rat models of cerebral infarction.展开更多
Carboxyl terminus of Hsp70-interacting protein(CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that ...Carboxyl terminus of Hsp70-interacting protein(CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal(BMS) cells derived from Chip^(-/-) mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip^(-/-) mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.展开更多
BACKGROUND: Bone marrow stromal cells (BMSCs) or Schwann cells (SCs) transplantation alone can treat spinal cord injury. However, the transplantation either cell-type alone has disadvantages. The co-transplantation of...BACKGROUND: Bone marrow stromal cells (BMSCs) or Schwann cells (SCs) transplantation alone can treat spinal cord injury. However, the transplantation either cell-type alone has disadvantages. The co-transplantation of both cells may benefit structural reconstruction and functional recovery of spinal nerves. OBJECTIVE: To verify spinal cord repair and related mechanisms after co-transplantation of BMSCs and SCs in a rat model of hemisected spinal cord injury. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Histology and Embryology, Mudanjiang Medical College from January 2008 to May 2009. MATERIALS: Rabbit anti-S-100, glial fibrillary acidic protein, neuron specific enolase and neurofilament-200 monoclonal antibodies were purchased from Sigma, USA. METHODS: A total of 100 Wistar rats were used in a model of hemisected spinal cord injury. The rats were randomly assigned to vehicle control, SCs transplantation, BMSCs transplantation, and co-transplantation groups; 25 rats per group. At 1 week after modeling, SCs or BMSCs cultured in vitro were labeled and injected separately into the hemisected spinal segment of SCs and BMSCs transplantation groups through three injection points [5 μL (1 × 107 cells/mL)] cell suspension in each point). In addition, a 15 μL 1 × 107 cells/mL SCs suspension and a 15 μL 1 × 107 cells/mL BMSC suspension were injected into co-transplantation group by the above method. MAIN OUTCOME MEASURES: The Basso-Beattie-Bresnahan (BBB) locomotor rating scale and somatosensory evoked potential (SEP) tests were used to assess the functional recovery of rat hind limbs following operation. Structural repair of injured nerve tissue was observed by light microscopy, electron microscopy, immunohistochemistry, and magnetic resonance imaging (MRI). In vivo differentiation, survival and migration of BMSCs were evaluated by immunofluorescence. RESULTS: BBB scores were significantly greater in all three transplantation groups compared with vehicle control group 8 weeks after transplantation. In particular, the co-transplantation group displayed the highest scores among the groups (P < 0.05). Moreover, recovery of SEP latency and amplitude was observed in all the transplantation groups, particularly after 8 weeks. Again, the co-transplantation group exhibited the greatest improvement (P < 0.05). In the co-transplantation group, imaging showed a smooth surface and intact inner structure at the injury site, with no scar formation, and a large number of orderly cells at the injured site. Axonal regeneration, new myelination, and a large amount of cell division were detected in the co-transplantation group by electron microscopy. Neuron specific enolase (NSE)- and glial fibrillary acidic protein (GFAP)-positive cells were observed in the spinal cord sections 1 week following co-transplantation by immunofluorescence staining. CONCLUSION: Co-transplantation of SCs and BMSCs effectively promoted functional recovery of injured spinal cord in rats compared with SCs or BMSCs transplantation alone. This repair effect is probably achieved because of neuronal-like cells derived from BMSCs to supplement dead neurons in vivo.展开更多
Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells (BMSCs) into cardi...Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells (BMSCs) into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract (IMTE), normal myocardial tissue extract (NMTE) and cultured neonatal myocardial lysate (NML)] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction (RT-PCR) detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.展开更多
Summary: To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24...Summary: To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random. In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.展开更多
Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to ...Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.展开更多
基金Supported by the FONCYT,Argentina(PICT 2016-#1093)CONICET,Argentina(PIP2014-2016,#300)Fundación Florencio Fiorini(Subsidio 2021-2022),Argentina.
文摘Breast cancer is the predominant form of carcinoma among women worldwide,with 70%of advanced patients developing bone metastases,with a high mortality rate.In this sense,the bone marrow(BM)mesenchymal stem/stromal cells(MSCs)are critical for BM/bone homeostasis,and failures in their functionality,transform the BM into a premetastatic niche(PMN).We previously found that BM-MSCs from advanced breast cancer patients(BCPs,infiltrative ductal carcinoma,stage III-B)have an abnormal profile.This work aims to study some of the metabolic and molecular mechanisms underlying MSCs shift from a normal to an abnormal profile in this group of patients.A comparative analysis was undertaken,which included self-renewal capacity,morphology,proliferation capacity,cell cycle,reactive oxygen species(ROS)levels,and senescence-associatedβ‑galactosidase(SA‑β‑gal)staining of BMderived MSCs isolated from 14 BCPs and 9 healthy volunteers(HVs).Additionally,the expression and activity of the telomerase subunit TERT,as well as telomere length,were measured.Expression levels of pluripotency,osteogenic,and osteoclastogenic genes(OCT-4,SOX-2,M-CAM,RUNX-2,BMP-2,CCL-2,M-CSF,and IL-6)were also determined.The results showed that MSCs from BCPs had reduced,self-renewal and proliferation capacity.These cells also exhibited inhibited cell cycle progression and phenotypic changes,such as an enlarged and flattened appearance.Additionally,there was an increase in ROS and senescence levels and a decrease in the functional capacity of TERT to preserve telomere length.We also found an increase in pro-inflammatory/pro-osteoclastogenic gene expression and a decrease in pluripotency gene expression.We conclude that these changes could be responsible for the abnormal functional profile that MSCs show in this group of patients.
基金Supported by National Natural Science Foundation of China,No.82001014,No.82071090Hubei Provincial Natural Science Foundation of China,No.2022CFB115.
文摘BACKGROUND Bone marrow mesenchymal stromal cells(BMSCs)are the commonly used seed cells in tissue engineering.Aryl hydrocarbon receptor(AhR)is a transcription factor involved in various cellular processes.However,the function of constitutive AhR in BMSCs remains unclear.AIM To investigate the role of AhR in the osteogenic and macrophage-modulating potential of mouse BMSCs(mBMSCs)and the underlying mechanism.METHODS Immunochemistry and immunofluorescent staining were used to observe the expression of AhR in mouse bone marrow tissue and mBMSCs.The overexpression or knockdown of AhR was achieved by lentivirus-mediated plasmid.The osteogenic potential was observed by alkaline phosphatase and alizarin red staining.The mRNA and protein levels of osteogenic markers were detected by quantitative polymerase chain reaction(qPCR)and western blot.After coculture with different mBMSCs,the cluster of differentiation(CD)86 and CD206 expressions levels in RAW 264.7 cells were analyzed by flow cytometry.To explore the underlying molecular mechanism,the interaction of AhR with signal transducer and activator of transcription 3(STAT3)was observed by co-immunoprecipitation and phosphorylation of STAT3 was detected by western blot.RESULTS AhR expressions in mouse bone marrow tissue and isolated mBMSCs were detected.AhR overexpression enhanced the osteogenic potential of mBMSCs while AhR knockdown suppressed it.The ratio of CD86+RAW 264.7 cells cocultured with AhR-overexpressed mBMSCs was reduced and that of CD206+cells was increased.AhR directly interacted with STAT3.AhR overexpression increased the phosphorylation of STAT3.After inhibition of STAT3 via stattic,the promotive effects of AhR overexpression on the osteogenic differentiation and macrophage-modulating were partially counteracted.CONCLUSION AhR plays a beneficial role in the regenerative potential of mBMSCs partially by increasing phosphorylation of STAT3.
基金supported in part by grants from the Japanese Ministry of Education,Culture,Sports,Science,and Technology(No.2300125 to CI,No.15K10957 to NN,and No.26870744 to KK)
文摘Transplantation of bone marrow stromal cells(BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury(SCI).BMSCs did not survive long-term,disappearing from the spinal cord within 2–3 weeks after transplantation.Astrocyte-devoid areas,in which no astrocytes or oligodendrocytes were found,formed at the epicenter of the lesion.It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas.Regenerating axons were associated with Schwann cells embedded in extracellular matrices.Transplantation of choroid plexus epithelial cells(CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI.Although CPECs disappeared from the spinal cord shortly after transplantation,an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas,as in the case of BMSC transplantation.These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord,including axonal regeneration and reduced cavity formation.This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate.The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI.It should be emphasized that the generally anticipated long-term survival,proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.
文摘BACKGROUND:Bone marrow cells can differentiate into hepatocytes in a suitable microenvironment.This study was undertaken to investigate the effects of transplanted bone marrow stromal cells (BMSCs) on liver fibrosis in mice. METHODS:BMSCs were harvested and cultured from male BALB/c mice, then transplanted into female syngenic BALB/c mice via the portal vein. After partial hepatectomy, diethylnitrosamine (DEN) was administered to induce liver fibrosis. Controls received BMSCs and non-supplemented drinking water, the model group received DEN with their water, and the experimental group received BMSCs and DEN. Mice were killed after 3 months, and ALT, AST, hyaluronic acid (HA), and laminin (LN) in serum and hydroxyproline (Hyp) in the liver were assessed. Alpha-smooth muscle actin (α-SMA) in the liver was assessed by immunohistochemistry. Bone marrow- derived hepatocytes were identified by fluorescent in situ hybridization (FISH) in liver sections. RESULTS:BMSCs were shown to differentiate into hepatocyte-like phenotypes after hepatocyte growth factor treatment in vitro. Serum ALT, AST, HA, and LN were markedly reduced by transplanted BMSCs. Liver Hyp content and α-SMA staining in mice receiving BMSCs were lower than in the model group, consistent with altered liver pathology. FISH analysis revealed the presence of donor- derived hepatocytes in the injured liver after cross-gender mouse BMSC transplantation. After three months, about 10% of cells in the injured liver were bone marrow-derived. CONCLUSION:BMSCs transplanted via the portal vein can convert into hepatocytes to repair liver injury induced by DEN, restore liver function, and reduce liver fibrosis.
基金This work was supported by Natural Science Foundation of Guangdong Province (No. 012452, No. 020001).
文摘Objective To investigate the differentiation of bone marrow stromal cells (BMSC) into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC (hBMSC) to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilament1 (NF1), nestin and neuron-specific enolase (NSE) in rat BMSC (rBMSC). Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NF1 and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated. rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.
基金supported by the National Natural Science Foundation of China(No.30600624)
文摘Objective To investigate the effects of Panax notoginseng saponins(PNS)on hydrogen peroxide(H2O2)-induced apoptosis in cultured rabbit bone marrow stromal cells(BMSCs).Methods BMSCs from 3-month-old New Zealand rabbits were isolated and cultured by the density gradient centrifugation combined with adherent method.The cultured BMSCs were divided into three groups:normal control,H2O2 treatment(100μmol/L),and PNS pretreatment(0.1g/L).Intracellular reactive oxygen species(ROS)levels as the index of oxidative stress were measured by using 2'7'-dichlorodihydrofluorescein diacetate.Flow cytometry was used to observe the apoptosis of BMSCs by staining with annexinV-FITC/PI.The protein expression of Bax in BMSCs was analyzed by Western blotting.Activity of caspase-3 enzyme was measured by spectrofluorometry.Results Pretreatment with PNS significantly decreased intracellular ROS level induced by H2O2(P<0.01).PNS markedly attenuated H2O2-induced apoptosis rate from 38.68% to 19.24%(P<0.01).PNS reversed H2O2-induced augmentation of Bax expression.Furthermore,PNS markedly reduced the altered in activity of caspase-3 enzyme induced by H2O2(P<0.01).Conclusion PNS has a protective effect on hydrogen peroxide-induced apoptosis in cultured rabbit BMSCs by scavenging ROS and decreasing Bax expression and caspase-3 activity.
基金the Plan Program of Shenyang Science and Technology Bureau, No. 1091161-0-00
文摘Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison, the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.
基金supported by the National Natural Science Foundation of China,No.81372041(to DW),and No.81801220(to MGZ)
文摘We previously demonstrated that overexpression of tropomyosin receptor kinase A(TrkA)promotes the survival and Schwann celllike differentiation of bone marrow stromal stem cells in nerve grafts,thereby enhancing the regeneration and functional recovery of the peripheral nerve.In the present study,we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts.Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA,TrkA-shRNA or the respective control.The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect.Then,8 weeks after surgery,hematoxylin and eosin staining showed that compared with the control groups,the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged,whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group.Western blot assay showed that compared with the control groups,the TrkA overexpressing group had higher expression of the myelin marker,myelin basic protein and the axonal marker neurofilament 200.The TrkA overexpressing group also had higher levels of various signaling molecules,including TrkA,pTrkA(Tyr490),extracellular signal-regulated kinases 1/2(Erkl/2),pErk1/2(Thr202/Tyr204),and the anti-apoptotic proteins Bcl-2 and Bcl-xL.In contrast,these proteins were downregulated,while the pro-apoptotic factors Bax and Bad were upregulated,in the TrkA-shRNA group.The levels of the TrkA effectors Akt and pAkt(Ser473)were not different among the groups.These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway.All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University,China in December 2014(approval No.AEWC-2014-001219).
基金Supported by the National Natural Science foundation of China,No.30900669 and No.81473271Technology Nova Plan of Beijing City,No.2011117China Postdoctoral Science foundation,No.2016T90994
文摘AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were isolated and cultured. Their morphological and phenotypic characteristics, as well as their multiple differentiation capacity were compared. A new culture system was established to induce ADSCs and BMSCs into functional hepatocytes. Reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to identify the induced hepatocytelike cells. CM-Dil-labeled ADSCs and BMSCs were then transplanted into a mouse model of CCl4-induced acute liver failure. fluorescence microscopy was used to track the transplanted MSCs. Liver function was tested by an automatic biochemistry analyzer, and liver tissue histology was observed by hematoxylin and eosin(HE) staining.RESULTS ADSCs and BMSCs shared a similar morphology and multiple differentiation capacity, as well as a similar phenotype(with expression of CD29 and CD90 and no expression of CD11 b or CD45). Morphologically, ADSCs and BMSCs became round and epithelioid following hepatic induction. These two cell types differentiated into hepatocyte-like cells with similar expression of albumin, cytokeratin 18, cytokeratin 19, alpha fetoprotein, and cytochrome P450. fluorescence microscopy revealed that both ADSCs and BMSCs were observed in the mouse liver at different time points. Compared to the control group, both the function of the injured livers and HE staining showed significant improvement in the ADSC-and BMSC-transplanted mice. There was no significant difference between the two MSC groups.CONCLUSION ADSCs share a similar hepatic differentiation capacity and therapeutic effect with BMSCs in an acute liver failure model. ADSCs may represent an ideal seed cell type for cell transplantation or a bio-artificial liver support system.
文摘This study investigated the ability of millimeter-wave(MMW)to promote the differen-tiation of bone marrow stromal cells(BMSCs)into cells with a neural phenotype.The BMSCs were primarily cultured.At passage 3,the cells were induced byβ-mercaptoethanol(BME)in combination with MMW or BME alone.The expressions of nucleostemin(NS)and neuron-specific enolase(NSE) were detected by immunofluorescent staining and Western blotting respectively to identify the differentiation.The untreated BMSCs predominately expressed NS.After induced by BME and MMW,the BMSCs exhibited a dramatic decrease in NS expression and increase in NSE expression.The differentiation rate of the cells treated with BME and MMW in combination was significantly higher than that of the cells treated with BME alone(P<0.05).It was concluded that MMW exposure enhanced the inducing effect of BME on the differentiation of BMSCs into cells with a neural phenotype.
基金This research was supported by the 863 Program (2002AA205071) and National Natural Foundation of China (30271306)
文摘Objective To explore the possibilities of bone marrow stromal cells (MSCs) to adopt Schwann cell phenotype in vitro and in vivo in SD rats. Methods MSCs were obtained from tibia and femur bone marrow and cultured in culture flasks. Beta-mercaptoethanol followed by retinoic acid, forskolin, basic-FGF, PDGF and heregulin were added to induce differentiation of MSCs’. Schwann cell markers, p75, S-100 and GFAP were used to discriminate induced properties of MSCs’ by immunofluorescent staining. PKH-67-labelled MSCs were transplanted into the mechanically injured rat sciatic nerve, and laser confocal microscopy was performed to localize the PKH67 labelled MSCs in the injured sciatic nerve two weeks after the operation. Fluorescence PKH67 attenuation rule was evaluated by flow cytometry in vitro. Results MSCs changed morphologically into cells resembling primary cultured Schwann cells after their induction in vitro. In vivo, a large number of MSCs were cumulated within the layer of epineurium around the injured nerve and expressed Schwann cell markers, p75, S-100, and GFAP. Conclusion MSCs are able to support nerve fiber regeneration and re-myelination by taking on Schwann cell function, and can be potentially used as possible substitutable cells for artificial nerve conduits to promote nerve regeneration.
文摘Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.
基金the Science and Technology Research Program of Shenzhen,No. 200802005
文摘BACKGROUND:Studies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinical application.The effects of vascular endothelial growth factor (VEGF) on in vitro neuronal differentiation of BMSCs remain poorly understood.OBJECTIVE:To investigate the effect of VEGF on neuronal differentiation of BMSCs in vitro,and to determine the best VEGF concentration for experimental induction.DESIGN,TIME AND SETTING:In vitro comparative study was performed at the Central Laboratory and Laboratory of Male Reproductive Medicine,Shenzhen Hospital of Peking University from October 2008 to August 2009.MATERIALS:Recombinant human VEGF165 was purchased from Peprotech Asia,Rehovot,Israel.Neuron-specific enolase (NSE) was purchased from Beijing Biosynthesis Biotechnology,China.METHODS:BMSCs were harvested from adult Sprague Dawley rats.The passaged cells were pre-induced with 10 ng/mL basic fibroblast growth factor for 24 hours,followed by differentiation induction with 0,5,10,and 20 ng/mL VEGF,respectively.MAIN OUTCOME MEASURES:Morphological changes in BMSCs prior to and following VEGF induction.Expression of NSE following induction was determined by immunocytochemistry.RESULTS:Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20 ng/mL VEGF.However,these changes were not observed in the control group.At 10 days after induction,the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group (P < 0.05).The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction (P < 0.05).Moreover,the number of NSE-positive cells was greater at 10 days compared with at 3 days after induction (P < 0.05).CONCLUSION:Of the VEGF concentrations tested,10 ng/mL induced the greatest number of neuronal-like cells in vitro from BMSCs.
基金the National Natural Science Foundation of China, No. 03030307 the Great Special Fund of Guangdong Province, No. 2004A30201002
文摘BACKGROUND: Transplantation of fetal cell suspension or blocks of fetal tissue can ameliorate the nerve function after the injury or disease in the central nervous system, and it has been used to treat neurodegenerative disorders induced by Parkinson disease. OBJECTIVE: To observe the effects of the transplantation of neuron-like cells derived from bone marrow stromal cells (rMSCs) into the brain in restoring the dysfunctions of muscle strength and balance as well as learning and memory in rat models of cerebral infarction. DESIGN: A randomized controlled experiment. SETTING: Department of Pathophysiology, Zhongshan Medical College of Sun Yat-sen University. MATERIALS: Twenty-four male SD rats (3-4 weeks of age, weighing 200-220 g) were used (Certification number:2001A027). METHODS: The experiments were carried out in Zhongshan Medical College of Sun Yat-sen University between December 2003 and December 2004. ① Twenty-four male SD rats randomized into three groups with 8 rats in each: experimental group, control group and sham-operated group. Rats in the experiment al group and control group were induced into models of middle cerebral artery occlusion. After in vitro cultured, purified and identified with digestion, the Fischer344 rMSCs were induced to differentiate by tanshinone ⅡA, which was locally injected into the striate cortex (18 area) of rats in the experimental group, and the rats in the control group were injected by L-DMEM basic culture media (without serum) of the same volume to the corresponding brain area. In the sham-operated group, only muscle and vessel of neck were separated. ② At 2 and 8 weeks after the transplantation, the rats were given the screen test, prehensile-traction test, balance beam test and Morris water-maze test. ③ The survival and distribution of the induced cells in corresponding brain area were observed with Nissl stained with toluidine blue and hematoxylin and eosin (HE) staining in the groups. MAIN OUTCOME MEASURES: ① Results of the behavioral tests (time of the Morris water-maze test screen test, prehensile-traction test, balance beam test); ② Survival and distribution of the induced cells. RESULTS: All the 24 rats were involved in the analysis of results. ① Two weeks after transplantation, rats with neuron-like cells grafts in the experimental group had significant improvement on their general muscle strength than those in the control group [screen test: (9.4±1.7), (4.7±1.0) s, P < 0.01]; forelimb muscle strength [prehensile-traction test: (7.6±1.4), (5.2±1.2) s, P < 0.01], ability to keep balance [balance beam test: (7.9±0.74), (6.1±0.91) s, P < 0.01] and abilities of learning and memory [latency to find the platform: (35.8±5.9), (117.5±11.6) s, P < 0.01; distance: (623.1±43.4), (1 902.3±98.6) cm, P < 0.01] as compared with those in the control group. The functional performances in the experimental group at 8 weeks were better than those at two weeks, which were still obviously different from those in the sham-operated group (P < 0.05). ② The HE and Nissl stained brain tissue section showed that there was nerve cell proliferation at the infarcted cortex in the experiment group, the density was higher than that in the control group, plenty of aggregative or scattered cells could be observed at the site where needle was inserted for transplantation, the cells migrated directively towards the area around them, the cerebral vascular walls were wrapped by plenty of cells; In the control group, most of the cortices were destroyed, karyopyknosis and necrosis of neurons were observed, normal nervous tissue structure disappeared induced by edema, only some nerve fibers and glial cells remained. CONCLUSION: The rMSCs transplantation can obviously enhance the motor function and the abilities of learning and memory in rat models of cerebral infarction.
基金supported by National Institutes of Health Grants, R01 AR054465, R01 AR070222, and R01 AR070222supported by the grants of Natural Science Foundation of China (NSFC) to TW (grants No. 81301531 and 81572104)+1 种基金supported by the grant from Shenzhen Science and Technology Innovation Committee, China (grant No. JCYJ20160331114205502)the grant from Shenzhen Development and Reform Committee, China for Shenzhen Engineering Laboratory of Orthopedic Regenerative Technologies
文摘Carboxyl terminus of Hsp70-interacting protein(CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal(BMS) cells derived from Chip^(-/-) mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip^(-/-) mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.
基金the National Natural Science Foundation of China, No. C010602the Natural Science Foundation of Heilongjiang Province, No. D200559the Scientific Research Program of Educa-tion Department of Heilong-jiang Province, No. 11511428
文摘BACKGROUND: Bone marrow stromal cells (BMSCs) or Schwann cells (SCs) transplantation alone can treat spinal cord injury. However, the transplantation either cell-type alone has disadvantages. The co-transplantation of both cells may benefit structural reconstruction and functional recovery of spinal nerves. OBJECTIVE: To verify spinal cord repair and related mechanisms after co-transplantation of BMSCs and SCs in a rat model of hemisected spinal cord injury. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Histology and Embryology, Mudanjiang Medical College from January 2008 to May 2009. MATERIALS: Rabbit anti-S-100, glial fibrillary acidic protein, neuron specific enolase and neurofilament-200 monoclonal antibodies were purchased from Sigma, USA. METHODS: A total of 100 Wistar rats were used in a model of hemisected spinal cord injury. The rats were randomly assigned to vehicle control, SCs transplantation, BMSCs transplantation, and co-transplantation groups; 25 rats per group. At 1 week after modeling, SCs or BMSCs cultured in vitro were labeled and injected separately into the hemisected spinal segment of SCs and BMSCs transplantation groups through three injection points [5 μL (1 × 107 cells/mL)] cell suspension in each point). In addition, a 15 μL 1 × 107 cells/mL SCs suspension and a 15 μL 1 × 107 cells/mL BMSC suspension were injected into co-transplantation group by the above method. MAIN OUTCOME MEASURES: The Basso-Beattie-Bresnahan (BBB) locomotor rating scale and somatosensory evoked potential (SEP) tests were used to assess the functional recovery of rat hind limbs following operation. Structural repair of injured nerve tissue was observed by light microscopy, electron microscopy, immunohistochemistry, and magnetic resonance imaging (MRI). In vivo differentiation, survival and migration of BMSCs were evaluated by immunofluorescence. RESULTS: BBB scores were significantly greater in all three transplantation groups compared with vehicle control group 8 weeks after transplantation. In particular, the co-transplantation group displayed the highest scores among the groups (P < 0.05). Moreover, recovery of SEP latency and amplitude was observed in all the transplantation groups, particularly after 8 weeks. Again, the co-transplantation group exhibited the greatest improvement (P < 0.05). In the co-transplantation group, imaging showed a smooth surface and intact inner structure at the injury site, with no scar formation, and a large number of orderly cells at the injured site. Axonal regeneration, new myelination, and a large amount of cell division were detected in the co-transplantation group by electron microscopy. Neuron specific enolase (NSE)- and glial fibrillary acidic protein (GFAP)-positive cells were observed in the spinal cord sections 1 week following co-transplantation by immunofluorescence staining. CONCLUSION: Co-transplantation of SCs and BMSCs effectively promoted functional recovery of injured spinal cord in rats compared with SCs or BMSCs transplantation alone. This repair effect is probably achieved because of neuronal-like cells derived from BMSCs to supplement dead neurons in vivo.
基金This work was supported by the National Natural Science Foundation of China (No. 30570722)
文摘Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells (BMSCs) into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract (IMTE), normal myocardial tissue extract (NMTE) and cultured neonatal myocardial lysate (NML)] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction (RT-PCR) detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.
文摘Summary: To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random. In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.
文摘Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.
