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
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
Several crucial stromal cell populations regulate hematopoiesis and malignant diseases in bone marrow niches.Precise regulation of these cell types can remodel niches and develop new therapeutics.Multiple nanocarriers...Several crucial stromal cell populations regulate hematopoiesis and malignant diseases in bone marrow niches.Precise regulation of these cell types can remodel niches and develop new therapeutics.Multiple nanocarriers have been developed to transport drugs into the bone marrow selectively.However,the delivery efficiency of these nanotherapeutics into crucial niche cells is still unknown,and there is no method available for predicting delivery efficiency in these cell types.Here,we constructed a three-dimensional bone marrow niche composed of three crucial cell populations:endothelial cells(ECs),mesenchymal stromal cells(MSCs),and osteoblasts(OBs).Mimetic niches were used to detect the cellular uptake of three typical drug nanocarriers into ECs/MSCs/OBs in vitro.Less than 5%of nanocarriers were taken up by three stromal cell types,and most of themwere located in the extracellular matrix.Delivery efficiency in sinusoidal ECs,arteriole ECs,MSCs,and OBs in vivo was analyzed.The correlation analysis showed that the cellular uptake of three nanocarriers in crucial cell types in vitro is positively linear correlated with its delivery efficiency in vivo.The delivery efficiency into MSCs was remarkably higher than that into ECs and OBs,no matterwhat kind of nanocarrier.The overall efficiency into sinusoidal ECswas greatly lower than that into arteriole ECs.All nanocarriers were hard to be delivered into OBs(<1%).Our findings revealed that cell tropisms of nanocarriers with different compositions and ligand attachments in vivo could be predicted via detecting their cellular uptake in bone marrow niches in vitro.This study provided the methodology for niche-directed nanotherapeutics development.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
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: Under induction of retinoic acid (RA), bone marrow stromal cells (BMSCs) can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an applica...BACKGROUND: Under induction of retinoic acid (RA), bone marrow stromal cells (BMSCs) can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor (BDNF) can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE: To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN: Randomized controlled animal study.SETTING: Department of Neurology, Affiliated Hospital of Xuzhou Medical College. MATERIALS: The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification: SYXK (su) 2002-0038]. Materials and reagents: low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein (GFAP) antibody, SABC kit, and diaminobenzidine (DAB) color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS: Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group (primary culture), BDNF group (20 μg/L BDNF) and BDNF+RA group (20 μg/L BDNF plus 20 μg/L RA). On the 3rd and the 7th days after induction, BMSCs were stained immunocytochemically with nestin (sign of nerve stem cells), neuron-specific enolase (NSE, sign of diagnosing neurons) and GFAP (diagnosing astrocyte), and evaluated cellular property. MAIN OUTCOME MEASURES: Induction and differentiation in vitro of BMSCs in 3 groups. RESULTS: ① Induction and differentiation of BMSCs: Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. ② Results of immunocytochemical detection: Three days after induction, rate of positive cells in BDNF+RA group was higher than that in BDNF group and control group [(86.15±4.58)%, (65.43±4.23)%, (4.18±1.09)%, P < 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF+RA group than that in both groups at 3 days after induction [(31.12±3.18)%, (29.35±2.69)%, P < 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction (P < 0.01); meanwhile, the amount in BDNF+RA group was remarkably higher than that in BDNF group (P < 0.01). CONCLUSION: Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.展开更多
BACKGROUND Multipotent bone marrow stromal cells(BMSCs)are adult stem cells that form functional osteoblasts and play a critical role in bone remodeling.During aging,an increase in bone loss and reduction in structura...BACKGROUND Multipotent bone marrow stromal cells(BMSCs)are adult stem cells that form functional osteoblasts and play a critical role in bone remodeling.During aging,an increase in bone loss and reduction in structural integrity lead to osteoporosis and result in an increased risk of fracture.We examined age-dependent histological changes in murine vertebrae and uncovered that bone loss begins as early as the age of 1 mo.AIM To identify the functional alterations and transcriptomic dynamics of BMSCs during early bone loss.METHODS We collected BMSCs from mice at early to middle ages and compared their selfrenewal and differentiation potential.Subsequently,we obtained the transcriptomic profiles of BMSCs at 1 mo,3 mo,and 7 mo.RESULTS The colony-forming and osteogenic commitment capacity showed a comparable finding that decreased at the age of 1 mo.The transcriptomic analysis showed the enrichment of osteoblastic regulation genes at 1 mo and loss of osteogenic features at 3 mo.The BMSCs at 7 mo showed enrichment of adipogenic and DNA repair features.Moreover,we demonstrated that the WNT and MAPK signaling pathways were upregulated at 1 mo,followed by increased pro-inflammatory and apoptotic features.CONCLUSION Our study uncovered the cellular and molecular dynamics of bone aging in mice and demonstrated the contribution of BMSCs to the early stage of age-related bone loss.展开更多
To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β 1 and Lipof...To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β 1 and Lipofectamine Reagent in vitro . The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF β 1 gene could promote the osteogenic potential of cultured BMSCs.展开更多
Objective:In this study,bone marrow stromal cells(BMSCs)were transplanted in situ after spinal cord injury(SCI)in rats.It was proved that the implanted BMSCs could differentiate into neuron-like cells in the injured s...Objective:In this study,bone marrow stromal cells(BMSCs)were transplanted in situ after spinal cord injury(SCI)in rats.It was proved that the implanted BMSCs could differentiate into neuron-like cells in the injured spinal cord,and the long-term motor ability test was carried out to investigate the recovery of neurological dysfunction after transplantation.Methods:(1)An improved Alien’s SCI rat model(weight 10 g,height 30 mm)was made by Alien weight drop method.Methyl eosin(Haematoxyli-n/eosin,HE)was used to identify with SCI.(2)The BMSCs were identified by the method of bone marrow adherent culture in vitro.The morphology of the cells was observed by cell staining,and CD44 was detected by BMSCs.After three cultures,the cells were transfected with lentiviral vectors carrying Green Fluorescent Protein(GFP)gene.(3)Cell survival after passage and transplantation:the GFP labeled BMSCs in situ(injury zone)were implanted into the injury model.The spinal cord sections were sacrificed after 2 weeks(2w),4 weeks(4w)and 6 weeks(6w)respectively,and the cells expressing GFP were observed under immunofluorescence microscope.(4)The modified Rivlin oblique plate test and spinal motor function blood brain barrir(BBB)score method were used to compare the changes of exercise ability in the simple injury group,2w after transplantation group,4w after transplantation group and 6w after transplantation group.Results:(1)The Alien’s SCI rat model was made by using Alien’s heavy drop method,and the effect was reliable and stable.(2)The proliferation of BMSCs after 3 passages has stabilized.After culture and amplification in vitro,the cell morphology changed to be conical,protruding interwoven into a network.Therefore,it could be used as a source of cell transplantation after SCI.The morphology of the cells was observed by staining,and the positive CD44 was detected.(3)BMSCs were transfected into the SCI model by lentiviral vector carrying GFP gene,and the expression of GFP in 6w was observed.(4)The results of modified inclined plane test and Rivlin locomotor BBB score of rats in simple injury group were significantly worse than those in transplantation control group(p<.05).2w,4w and 6w after transplantation groups could improve long-term motor function.The recovery of neurological dysfunction after 6w was the best,and the difference was statistically significant(p<.05).Conclusions:BMSCs transplantation could promote the recovery of neurological dysfunction after SCI,and the mechanism may be related to the differentiation of BMSCs into neurons and glial cells,and the BMSCs induced by transplantation could interact with peripheral nerve cells and produce some cytokines.展开更多
BACKGROUND: Embryonic neural stem cells (NSCs) have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone ma...BACKGROUND: Embryonic neural stem cells (NSCs) have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells (BMSCs) have been proposed for the treatment of glioma. OBJECTIVE: To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS: The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine (BrdU) monoclonal antibody and Cy3-labeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS: A total of 95 Sprague Dawley rats were randomly assigned to three groups: NSC (n = 35), transplanted with > 6 × 106 NSCs via left medial hind limb; BMSC (n = 35), transplanted with > 1 × 106 BMSCs via left medial hind limb; model group (n = 25), injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES: Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS: The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups (P > 0.05). However, gliomal diameter was significantly decreased in the NSC group compared with the model group (P < 0.05). At 4 weeks, there was no statistical difference between the groups (P > 0.05). BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION: NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.展开更多
Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors ...Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.展开更多
The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering.Wnt3a activates the classicWnt/β-catenin signaling pathway which effects bone growth and ...The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering.Wnt3a activates the classicWnt/β-catenin signaling pathway which effects bone growth and development by the accumulation ofβ-catenin in the nucleus.In this study,we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix(Wnt3a-ST2-dCM-PCL)and ST2 decellularized matrix(ST2-dCM-PCL)by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized>90%DNA while preserved most protein.Compared to ST2-dCM-PCL,Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation,osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase(Alp),Runx2,type I collagen(Col 1)and osteocalcin(Ocn)mRNA expression.After 14 days of osteogenic induction,Wnt3a-ST2-dCM-PCL promoted ST2 mineralization.These results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials’osteoinductivity and osteoconductivity.展开更多
Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mese...Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential;however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow- and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid- and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid- and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.展开更多
Diabetic peripheral neuropathy(DPN)is a long-term complication associated with nerve dysfunction and uncontrolled hyperglycemia.In spite of new drug discoveries,development of effective therapy is much needed to cure ...Diabetic peripheral neuropathy(DPN)is a long-term complication associated with nerve dysfunction and uncontrolled hyperglycemia.In spite of new drug discoveries,development of effective therapy is much needed to cure DPN.Here,we have developed a combinatorial approach to provide biochemical and electrical cues,considered to be important for nerve regeneration.Exosomes derived from bone marrow mesenchymal stromal cells(BMSCs)were fused with polypyrrole nanoparticles(PpyNps)containing liposomes to deliver both the cues in a single delivery vehicle.We developed DPN rat model and injected intramuscularly the fused exosomal system to understand its long-term therapeutic effect.We found that the fused system along with electrical stimulation normalized the nerve conduction velocity(57.60±0.45 m/s)and compound muscle action potential(16.96±0.73 mV)similar to healthy control(58.53±1.10 m/s;18.19±1.45 mV).Gastrocnemius muscle morphology,muscle mass,and integrity were recovered after treatment.Interestingly,we also observed paracrine effect of delivered exosomes in controlling hyperglycemia and loss in body weight and also showed attenuation of damage to the tissues such as the pancreas,kidney,and liver.This work provides a promising effective treatment and also contribute cutting edge therapeutic approach for the treatment of DPN.展开更多
Background and Aims: Androgenetic alopecia (AGA) is a common form of hair loss in both men and women. Despite its high prevalence and associated patient morbidity, the approved therapeutic options are limited to finas...Background and Aims: Androgenetic alopecia (AGA) is a common form of hair loss in both men and women. Despite its high prevalence and associated patient morbidity, the approved therapeutic options are limited to finasteride and minoxidil. The present study is aimed at assessing the efficacy of hair serum formulation, Trichosera<sup>®</sup>containing Bone marrow-derived mesenchymal stromal cells conditioned media as an active ingredient, for hair fall control and hair regrowth in healthy Indian human volunteers. Methods: The product was made using a 20% concentration of 10X Conditioned Media along with excipients. The final product was tested for physicochemical parameters, biomarkers, total protein content and microbial limits as per our in-house specifications. Results: The primary irritation patch test showed that the product is non-irritant and dermatologically safe. A clinical study on 40 subjects was conducted to evaluate the effectiveness of the bioactive formulation in hair fall control and hair regrowth in healthy volunteers. Phototrichogram measurement showed hair density and hair growth rate increased significantly by 11.54% and 18.66% at week 24. Hair tensile strength also increased significantly by 41.10% at 12 weeks follow-up. Hair pull test, to see a reduction in pulled hair and comb’s test to show a decrease in hair fall significantly improved from week 4 onwards. There were no significant adverse events in response to the product application. Conclusion: It is concluded that the hair serum product is completely safe on direct application to the scalp and showed significant improvement in the hair growth rate, hair density, scalp condition and reduction in hair fall. .展开更多
Based on studies over the last several decades,the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering.However,the markers for osteogenic precursors...Based on studies over the last several decades,the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering.However,the markers for osteogenic precursors;i.e.,bone marrow-derived skeletal stem cells(SSCs),in association with other cells of the marrow stroma(bone marrow stromal cells,BMSCs)and their heterogeneous nature both in vivo and in vitro remain to be clarified.This review aims to highlight:i)the importance of distinguishing BMSCs/SSCs from other“mesenchymal stem/stromal cells”,and ii)factors that are responsible for their heterogeneity,and how these factors impact on the differentiation potential of SSCs towards bone.The prospective role of SSC enrichment,their expansion and its impact on SSC phenotype is explored.Emphasis has also been given to emerging single cell RNA sequencing approaches in scrutinizing the unique population of SSCs within the BMSC population,along with their committed progeny.Understanding the factors involved in heterogeneity may help researchers to improvise their strategies to isolate,characterize and adopt best culture practices and source identification to develop standard operating protocols for developing reproducible stem cells grafts.However,more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations.展开更多
Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial f...Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.展开更多
Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of...Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of cell surface markers and transcription factors.However,it has been challenging to define micro-heterogeneity within“stem cell”populations,each of which stands at a different level of differentiation.As stem cells become defined at a single-cell level,their differentiation path becomes clearly defined.Here,this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches,with an emphasis on practical considerations in stem cell biology.展开更多
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.
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support from the National Natural Science Foundation of China(81703713,82174095,82274364)Natural Science Foundation of Zhejiang Province grants(LZ23H290001,LZ22H290001)internal support from Zhejiang Chinese Medical University(2022GJYY011).
文摘Several crucial stromal cell populations regulate hematopoiesis and malignant diseases in bone marrow niches.Precise regulation of these cell types can remodel niches and develop new therapeutics.Multiple nanocarriers have been developed to transport drugs into the bone marrow selectively.However,the delivery efficiency of these nanotherapeutics into crucial niche cells is still unknown,and there is no method available for predicting delivery efficiency in these cell types.Here,we constructed a three-dimensional bone marrow niche composed of three crucial cell populations:endothelial cells(ECs),mesenchymal stromal cells(MSCs),and osteoblasts(OBs).Mimetic niches were used to detect the cellular uptake of three typical drug nanocarriers into ECs/MSCs/OBs in vitro.Less than 5%of nanocarriers were taken up by three stromal cell types,and most of themwere located in the extracellular matrix.Delivery efficiency in sinusoidal ECs,arteriole ECs,MSCs,and OBs in vivo was analyzed.The correlation analysis showed that the cellular uptake of three nanocarriers in crucial cell types in vitro is positively linear correlated with its delivery efficiency in vivo.The delivery efficiency into MSCs was remarkably higher than that into ECs and OBs,no matterwhat kind of nanocarrier.The overall efficiency into sinusoidal ECswas greatly lower than that into arteriole ECs.All nanocarriers were hard to be delivered into OBs(<1%).Our findings revealed that cell tropisms of nanocarriers with different compositions and ligand attachments in vivo could be predicted via detecting their cellular uptake in bone marrow niches in vitro.This study provided the methodology for niche-directed nanotherapeutics development.
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.
文摘BACKGROUND: Under induction of retinoic acid (RA), bone marrow stromal cells (BMSCs) can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor (BDNF) can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE: To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN: Randomized controlled animal study.SETTING: Department of Neurology, Affiliated Hospital of Xuzhou Medical College. MATERIALS: The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification: SYXK (su) 2002-0038]. Materials and reagents: low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein (GFAP) antibody, SABC kit, and diaminobenzidine (DAB) color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS: Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group (primary culture), BDNF group (20 μg/L BDNF) and BDNF+RA group (20 μg/L BDNF plus 20 μg/L RA). On the 3rd and the 7th days after induction, BMSCs were stained immunocytochemically with nestin (sign of nerve stem cells), neuron-specific enolase (NSE, sign of diagnosing neurons) and GFAP (diagnosing astrocyte), and evaluated cellular property. MAIN OUTCOME MEASURES: Induction and differentiation in vitro of BMSCs in 3 groups. RESULTS: ① Induction and differentiation of BMSCs: Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. ② Results of immunocytochemical detection: Three days after induction, rate of positive cells in BDNF+RA group was higher than that in BDNF group and control group [(86.15±4.58)%, (65.43±4.23)%, (4.18±1.09)%, P < 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF+RA group than that in both groups at 3 days after induction [(31.12±3.18)%, (29.35±2.69)%, P < 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction (P < 0.01); meanwhile, the amount in BDNF+RA group was remarkably higher than that in BDNF group (P < 0.01). CONCLUSION: Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.
基金Supported by the National Natural Science Foundation of China,No.81573992.
文摘BACKGROUND Multipotent bone marrow stromal cells(BMSCs)are adult stem cells that form functional osteoblasts and play a critical role in bone remodeling.During aging,an increase in bone loss and reduction in structural integrity lead to osteoporosis and result in an increased risk of fracture.We examined age-dependent histological changes in murine vertebrae and uncovered that bone loss begins as early as the age of 1 mo.AIM To identify the functional alterations and transcriptomic dynamics of BMSCs during early bone loss.METHODS We collected BMSCs from mice at early to middle ages and compared their selfrenewal and differentiation potential.Subsequently,we obtained the transcriptomic profiles of BMSCs at 1 mo,3 mo,and 7 mo.RESULTS The colony-forming and osteogenic commitment capacity showed a comparable finding that decreased at the age of 1 mo.The transcriptomic analysis showed the enrichment of osteoblastic regulation genes at 1 mo and loss of osteogenic features at 3 mo.The BMSCs at 7 mo showed enrichment of adipogenic and DNA repair features.Moreover,we demonstrated that the WNT and MAPK signaling pathways were upregulated at 1 mo,followed by increased pro-inflammatory and apoptotic features.CONCLUSION Our study uncovered the cellular and molecular dynamics of bone aging in mice and demonstrated the contribution of BMSCs to the early stage of age-related bone loss.
文摘To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β 1 and Lipofectamine Reagent in vitro . The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF β 1 gene could promote the osteogenic potential of cultured BMSCs.
文摘Objective:In this study,bone marrow stromal cells(BMSCs)were transplanted in situ after spinal cord injury(SCI)in rats.It was proved that the implanted BMSCs could differentiate into neuron-like cells in the injured spinal cord,and the long-term motor ability test was carried out to investigate the recovery of neurological dysfunction after transplantation.Methods:(1)An improved Alien’s SCI rat model(weight 10 g,height 30 mm)was made by Alien weight drop method.Methyl eosin(Haematoxyli-n/eosin,HE)was used to identify with SCI.(2)The BMSCs were identified by the method of bone marrow adherent culture in vitro.The morphology of the cells was observed by cell staining,and CD44 was detected by BMSCs.After three cultures,the cells were transfected with lentiviral vectors carrying Green Fluorescent Protein(GFP)gene.(3)Cell survival after passage and transplantation:the GFP labeled BMSCs in situ(injury zone)were implanted into the injury model.The spinal cord sections were sacrificed after 2 weeks(2w),4 weeks(4w)and 6 weeks(6w)respectively,and the cells expressing GFP were observed under immunofluorescence microscope.(4)The modified Rivlin oblique plate test and spinal motor function blood brain barrir(BBB)score method were used to compare the changes of exercise ability in the simple injury group,2w after transplantation group,4w after transplantation group and 6w after transplantation group.Results:(1)The Alien’s SCI rat model was made by using Alien’s heavy drop method,and the effect was reliable and stable.(2)The proliferation of BMSCs after 3 passages has stabilized.After culture and amplification in vitro,the cell morphology changed to be conical,protruding interwoven into a network.Therefore,it could be used as a source of cell transplantation after SCI.The morphology of the cells was observed by staining,and the positive CD44 was detected.(3)BMSCs were transfected into the SCI model by lentiviral vector carrying GFP gene,and the expression of GFP in 6w was observed.(4)The results of modified inclined plane test and Rivlin locomotor BBB score of rats in simple injury group were significantly worse than those in transplantation control group(p<.05).2w,4w and 6w after transplantation groups could improve long-term motor function.The recovery of neurological dysfunction after 6w was the best,and the difference was statistically significant(p<.05).Conclusions:BMSCs transplantation could promote the recovery of neurological dysfunction after SCI,and the mechanism may be related to the differentiation of BMSCs into neurons and glial cells,and the BMSCs induced by transplantation could interact with peripheral nerve cells and produce some cytokines.
基金Hubei Provincial Education Department Foundation, No. Q20092405Hubei Provincial Science and Technology Agency Foundation, No. 2005AA301C28Hubei Provincial Health Department Foundation, No. QJX2005-15
文摘BACKGROUND: Embryonic neural stem cells (NSCs) have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells (BMSCs) have been proposed for the treatment of glioma. OBJECTIVE: To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS: The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine (BrdU) monoclonal antibody and Cy3-labeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS: A total of 95 Sprague Dawley rats were randomly assigned to three groups: NSC (n = 35), transplanted with > 6 × 106 NSCs via left medial hind limb; BMSC (n = 35), transplanted with > 1 × 106 BMSCs via left medial hind limb; model group (n = 25), injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES: Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS: The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups (P > 0.05). However, gliomal diameter was significantly decreased in the NSC group compared with the model group (P < 0.05). At 4 weeks, there was no statistical difference between the groups (P > 0.05). BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION: NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.
文摘Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.
基金This work was supported by the National Natural Science Foundation of China(Grant No.U1601220)the National Natural Science Foundation of China(Grant No.82002310)+1 种基金the Chongqing Postgraduate Research and Innovation Project(Grant No.CYB20167)the Chongqing Postdoctoral Science Foundation(Grant No.csts2019jcyj-bsh0068).
文摘The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering.Wnt3a activates the classicWnt/β-catenin signaling pathway which effects bone growth and development by the accumulation ofβ-catenin in the nucleus.In this study,we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix(Wnt3a-ST2-dCM-PCL)and ST2 decellularized matrix(ST2-dCM-PCL)by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized>90%DNA while preserved most protein.Compared to ST2-dCM-PCL,Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation,osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase(Alp),Runx2,type I collagen(Col 1)and osteocalcin(Ocn)mRNA expression.After 14 days of osteogenic induction,Wnt3a-ST2-dCM-PCL promoted ST2 mineralization.These results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials’osteoinductivity and osteoconductivity.
文摘Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential;however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow- and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid- and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid- and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.
基金The work was funded by the Department of Biotechnology (DBT) (#BT/PR13561/MED/32/392/2016 and #DBT/IN/SWEDEN/08/AK/ 2017-1)Department of Science and Technology (DST) (#DST/NM/NT- 2018/48 and #DST/INT/SWD/P-11/2016)Ministry of Human Resource Development- IMPRINT (MHRD_6714/Healthcare), Ministry of Human Resource Development- SPARC (SPARC/2018-2019/P612/ SL), and Ministry of Human Resource Development-UAY (MHRD_IITK_006), Govt. of India. AS and PAS would like to acknowledge IIT Kanpur for fellowship for the Ph.D. program. The authors would like to acknowledge Sophisticated Analytical Instrumentation Facility (SAIF), Council for Scientific and Industrial Research- Central Drug Research Institute, Lucknow for the LC-MS/MS analysis.
文摘Diabetic peripheral neuropathy(DPN)is a long-term complication associated with nerve dysfunction and uncontrolled hyperglycemia.In spite of new drug discoveries,development of effective therapy is much needed to cure DPN.Here,we have developed a combinatorial approach to provide biochemical and electrical cues,considered to be important for nerve regeneration.Exosomes derived from bone marrow mesenchymal stromal cells(BMSCs)were fused with polypyrrole nanoparticles(PpyNps)containing liposomes to deliver both the cues in a single delivery vehicle.We developed DPN rat model and injected intramuscularly the fused exosomal system to understand its long-term therapeutic effect.We found that the fused system along with electrical stimulation normalized the nerve conduction velocity(57.60±0.45 m/s)and compound muscle action potential(16.96±0.73 mV)similar to healthy control(58.53±1.10 m/s;18.19±1.45 mV).Gastrocnemius muscle morphology,muscle mass,and integrity were recovered after treatment.Interestingly,we also observed paracrine effect of delivered exosomes in controlling hyperglycemia and loss in body weight and also showed attenuation of damage to the tissues such as the pancreas,kidney,and liver.This work provides a promising effective treatment and also contribute cutting edge therapeutic approach for the treatment of DPN.
文摘Background and Aims: Androgenetic alopecia (AGA) is a common form of hair loss in both men and women. Despite its high prevalence and associated patient morbidity, the approved therapeutic options are limited to finasteride and minoxidil. The present study is aimed at assessing the efficacy of hair serum formulation, Trichosera<sup>®</sup>containing Bone marrow-derived mesenchymal stromal cells conditioned media as an active ingredient, for hair fall control and hair regrowth in healthy Indian human volunteers. Methods: The product was made using a 20% concentration of 10X Conditioned Media along with excipients. The final product was tested for physicochemical parameters, biomarkers, total protein content and microbial limits as per our in-house specifications. Results: The primary irritation patch test showed that the product is non-irritant and dermatologically safe. A clinical study on 40 subjects was conducted to evaluate the effectiveness of the bioactive formulation in hair fall control and hair regrowth in healthy volunteers. Phototrichogram measurement showed hair density and hair growth rate increased significantly by 11.54% and 18.66% at week 24. Hair tensile strength also increased significantly by 41.10% at 12 weeks follow-up. Hair pull test, to see a reduction in pulled hair and comb’s test to show a decrease in hair fall significantly improved from week 4 onwards. There were no significant adverse events in response to the product application. Conclusion: It is concluded that the hair serum product is completely safe on direct application to the scalp and showed significant improvement in the hair growth rate, hair density, scalp condition and reduction in hair fall. .
基金This work was supported in part by the Fulbright-Nehru Postdoctoral Research Fellowship,United States-India Educational Foundation,the Biosystem and Biomaterials Division,National Institute of Standards and Technology,Department of Commerce,and the Shobhit Institute of Engineering and Technology,Meerut,India(to DA),and by the Division of Intramural Research,National Institute of Dental and Craniofacial Research,a part of the Intramural Research Program,the National Institute of Health,Department of Health and Human Services(1ZIA DE000380 to PGR).
文摘Based on studies over the last several decades,the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering.However,the markers for osteogenic precursors;i.e.,bone marrow-derived skeletal stem cells(SSCs),in association with other cells of the marrow stroma(bone marrow stromal cells,BMSCs)and their heterogeneous nature both in vivo and in vitro remain to be clarified.This review aims to highlight:i)the importance of distinguishing BMSCs/SSCs from other“mesenchymal stem/stromal cells”,and ii)factors that are responsible for their heterogeneity,and how these factors impact on the differentiation potential of SSCs towards bone.The prospective role of SSC enrichment,their expansion and its impact on SSC phenotype is explored.Emphasis has also been given to emerging single cell RNA sequencing approaches in scrutinizing the unique population of SSCs within the BMSC population,along with their committed progeny.Understanding the factors involved in heterogeneity may help researchers to improvise their strategies to isolate,characterize and adopt best culture practices and source identification to develop standard operating protocols for developing reproducible stem cells grafts.However,more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations.
基金the National Natural Science Foundation of China(Nos.U1601220,82072450,and 81672118)Chongqing Science and Technology Commission-Basic Science and Frontier Technology Key Project(No.cstc2015jcyjBX0119)Chongqing Medical University Intelligent Medicine Research Project(No.ZHYX202115).
文摘Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.
基金This research was supported by National Institute of Health Grants R01DE026666 and R01DE030630(to NO)and R01DE029181(to WO).
文摘Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of cell surface markers and transcription factors.However,it has been challenging to define micro-heterogeneity within“stem cell”populations,each of which stands at a different level of differentiation.As stem cells become defined at a single-cell level,their differentiation path becomes clearly defined.Here,this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches,with an emphasis on practical considerations in stem cell biology.
