Objective To investigate the feasibility of imaging of bone marrow mesenchymal stem cells (BMMSCs) labeled with superparamagnetic iron oxide(SPIO) transplanted into coronary artery in vivo using magnetic resonance imaging (MRI), and the redistribution of the cells into other organs. Methods BMMSCs were isolated, cultured from bone marrow of Chinese mini swine, and double labeled with SPIO and CMDiI(Cell TrackerTM C-7001). The labeled cells were injected into left anterior descending coronary artery through a catheter. The injected cells were detected by using MRI at 1 week,3weeks after transplantation. And different organs were harvested and evaluated the redistribution of transplanted cells through pathology. Results The SPIO labeled BMMSCs injected into coronary artery could be detected through MRI and confirmed by pathology and maintained more than 3 weeks. The SPIO labeled cells could be clearly imaged as signal void lesions in the related artery. The pathology showed that the injected cells could be distributed into the area of related artery, and the cells injected into coronary artery could be found in the lung, spleen, kidney, but scarcely in the liver, the structures of these organs remained normal. Conclusion The SPIO labeled BMMSCs injected into coronary artery can be detected by using MRI, the transplanted cells can be redistributed into the non-targeted organs.
Objective The bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into insul in-producing cells (IPCs) in vitro. However, low differentiation efficiency and poor maturity are the main obstacles. To investigate the feasibil ity of BMSCs differentiation into IPCs in diabetic pancreatic microenvironment of pigs. Methods BMSCs were isolated and purified from the bone marrow of a 4-week-old male pig. Fifteen female pigs (aged 8 to 10 weeks, weighing 8 to 10 kg) were randomly divided into 3 groups: normal control group (group A, n=5), diabetic control group (group B, n=5), and BMSCs transplanted group (group C, n=5). The pigs of groups B and C were treated by auris vein injections of styeptozocin and alloxan for 3 days to induce diabetes mell itus (DM) model, whose blood glucose level 2 days all greater than 17 mmol/L was successful DM model. A total of 1.1 mL of the 3rd passage BMSCs labeled with enhanced green fluorescent protein (EGFP), with cell density of 5 × 107/ mL, were injected into subcapsular pancreas of group C at multi ple points, normal saline at the same dosage into those of groups A and B. After 30 days of monitoring blood glucose, the histological analysis of islet number and size were done; the immunofluorescence staining was used to detect the protein expression of insul in in the new-formed islets. The EGFP+ cells were collected from the sections using laser-capture microdissection; RT-PCR was used to detect insulin mRNA and pancreatic and duodenal homeobox factor 1 (PDX1) mRNA expressions from EGFP+ cells, and the insul in and sexdetermining region of the Y chromosome (SRY) genes were detected by fluorescence in situ hybridization (FISH). Results The blood glucose level decreased significantly in group C when compared with that in group B from 18 days and gradually decreased with time (P lt; 0.05). The histological observation showed that the number of islets was increased significantly in group C when compared with that in group B (10.9 ± 2.2 vs. 4.6 ± 1.4, P lt; 0.05), and there was no significant difference when compared with that in group A (10.9 ± 2.2 vs.12.6 ± 2.6, P gt; 0.05). The size of new-formed islets in group C was significantly smaller than that in group A [(47.2 ± 19.6) μm vs. (119.6 ± 27.7) μm, P lt; 0.05]. The immunofluorescence staining showed that new-formed islets of group C expressed insulin protein. RT-PCR showed that the microdissected EGFP+ cells of group C expressed insulin mRNA and PDX-1 mRNA. FISH showed that the new-formed islet cells of group C contained SRY gene in Y chromosome and insulin double positive cells. Conclusion BMSCs can differentiate into IPCs in diabetic pancreatic microenvironment of pigs.
Objective To review the research progress of the current methods of inducing bone marrow mesenchymal stem cells (BMSCs) to chondrogenic differentiation in vitro so as to provide references for researches in cartilage tissue engineering. Methods Various methods of inducing BMSCs differentiation into the chondrogenic l ineage in vitro inrecent years were extensively reviewed and analyzed. Results Adding exogenous growth factors is still the mainly methodof inducing BMSCs differentiation into the chondrogenic l ineage; among the members, transforming growth factor β (TGF-β) family is recognized as the most important chondrogenic induction factor. Other important inducing factors include various chemical factors, physical factors, transgenic methods, and the microenvironmental induction. But the problems of low inducing efficiency and unstable inducing effects still exist. Conclusion The progress of chondrogenic induction of BMSCs promotes its util ization in cartilage tissue engineering. Further researches are needed for establ ishing more efficient, simpler, and safer inducing methods.
Objective To observe the characteristics and related gene expression of osteoblastic differentiation in porcine bone marrow mesenchymal stem cells (MSCs) during. Methods Bone marrow from 6 landrace pigs, 3-month-old about 50 kg, was aspirated from the medullary cavity of the proximal tibia. The MSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The MSCs from passage 1 were cultivated in DMEM with 1×10-8mmol/L dexamethasone (Dex), 10 mmol/L β-glycerophosphate (β-GP), 82 μg/ml ascorbic acid (Asc) and 10% inactivated fetal bovine serum (FBS) up to 21 days. The MSCs were cultivated in basic DMEM as a control. Cell morphology was observed by microscope. Cell proliferation was tested by using the fluorescent dye SYBR green I measurement. Osteoblastic differentiation was evaluated by alkaline phosphatase (ALP) histochemical staining, quantitative calcium deposit, and real-time PCR technology. Results Characterization of primary MSCs: At day 1, most cells depicted round and floating hematopoietic cells. Colonies consisting of fibroblastlike cells were observed at day 3 after removal of nonadherent cells, colonies grew to various sizes at day 7. Thirteen population doublings took place in primary culture. Osteoblastic differentiation: During osteogenic stimulation, cellular morphology of MSCs changed from a fibroblastic shape to a cubical form. Cell proliferation had no impact in osteogenic medium compared to basic medium (Pgt;0.05). At day 14, ALP staining presented b positive. Calcium deposit pronouncedly increased at day 21 (Plt;001). Furthermore, the mRNA levels of core binding factor α1 (Cbfα1), osterix, ALP, collagen Ⅰ(ColⅠ), osteonectin (ON) and osteocalcin (OC) increased gradually. Cbfα1, ON and ALP genes increased at early stage of osteoblastic differentiation. Osterix and OC at day 21 were significantly increased when compared with that at day 7 (Plt;0.05). ColⅠ was increased at day 14 (Plt;0.05). Conclusion Porcine MSCs harvested from bone marrow by density gradient centrifugation are capable of osteoblastic differentiation in vitro. The potential of osteoblastic differentiation relies upon upregulation of genes specific to this lineage under the osteogenic conditions.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) embedded in fibrin glue around chemical extracted acellular nerve allograft (CEANA) on the peripheral nerve regeneration. Methods Twenty-oneadult male C57 mice (weighing 25-30 g) and 15 adult male Balb/c mice (weighing 25-30 g) were selected. The sciatic nerves were harvested from the Balb/c mice to prepare CEANA. The BMSCs were isolated from 3 C57 mice and were cultured; BMSCs embedded in fibrin glue were cultured for 3, 7, 14, and 21 days. Then the supernatant was obtained and co-cultured with PC12 cells for 2 days to observe the PC12 cell growth in vitro. The other 18 C57 mice were used to establ ish the left sciatic nerve defect models of 10 mm and divided into 3 groups: autogenous nerve graft with fibrin glue (group A, n=6), CEANA graft with BMSCs (5 × 106) embedded in fibrin glue (group B, n=6), and CEANA graft with fibrin glue (group C, n=6). The right sciatic nerves were exposed as the controls. At 2, 4, 6, and 8 weeks, the mouse static sciatic index (SSI) was measured. The histomorphometric assessment of triceps surae muscles and nerve grafts were evaluated by Masson staining, toluidine blue staining, and transmission electron microscope (TEM) observationat 8 weeks after operation. Results BMSCs were uniform distribution in fibrin glue, which were spherical in shape, and the cells began to grow apophysis at 3 days. PC12 cells differentiated into neuron-l ike cells after addition supernatant co-cultured after 2 days. Incisions healed well in each group. At 2, 4, 6, and 8 weeks, the SSI increased gradually in 3 groups. SSI in group A was higher than that in groups B and C at 4, 6, and 8 weeks after operation (P lt; 0.05). SSI in group B was sl ightly higher than that in group C, but had no significant difference (P gt; 0.05). At 8 weeks, the wet weight recovery rate of triceps surae muscles and fibers number of myel inated nerve were better in group B than in group C, but worse in group B than in group A, showing significant differences (P lt; 0.05). The triceps surae muscle fibers area and myel in sheath thickness had significant differences between group B and group C (P lt; 0.01), but there was no significant difference between group A and group B (P gt; 0.05). Conclusion BMSCs embedded in fibrin glue around CEANA can improve functional recovery of peripheral nerve injury.
Objective To explore the paracrine effect of bone marrow mesenchymal stem cells (BMSCs) on dexamethasone-induced inhibition of osteoblast function in vitro. Methods The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups: the control group (group A), dexamethasone group (group B), dexamethasone+BMSCs conditioned medium (1:1) group (group C), and BMSCs conditioned medium group (group D). After 24 hours of culture, the alkaline phosphatase (ALP) content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1 (COL1A1), RUNX2, ALP, and Osteocalcin were detected by real-time fluorescence quantitative PCR (RT-qPCR); alizarin red staining was used to observe calcium nodules formation at 21 days. Results After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D (P < 0.05), but no significant difference was found between groups C and D (P > 0.05). The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D (P < 0.05), but difference was not significant between groups A, C, and D (P > 0.05). The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D (P < 0.05), but difference had no significance between groups A and C (P > 0.05). The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A (P < 0.05); the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B (P < 0.05). The gene expression of COL1A1 was significantly higher in group D than group B (P < 0.05), but difference was not significant between groups B and C, and between groups C and D (P > 0.05). The cells of group A all died at 6 days after culture; at 21 days, the calcium no dule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D. Conclusion BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
Objective To explore the role and possible mechanisms of bone marrow mesenchymal stem cell (BMSC) in the lipopolysaccharide (LPS)-induced inflammatory response involving alveolar macrophages through the inflammatory pathways. Methods ptges and ptges shRNA were transfected into BMSC by lentivirus, and stable ptges overexpression BMSC (BMSC-PGE2(+)) and PTGEs silencing BMSC (BMSC-PGE2(-)) were established. Macrophages were divided into control group, LPS group, LPS+BMSC group, LPS+BMSC-PGE2(+) group and LPS+BMSC-PGE2(-) group. The expression levels of nucleotide-bound oligomerized domain-like receptor 3 (NLRP3), precursor cysteinyl aspartate specific proteinase 1 (pro-caspase-1), caspase-1 and pro-IL-1β proteins were detected by Western blot. The mRNA expression levels of NLRP3 and caspase-1 were determined by RT-PCR. The expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, IL-18 and prostaglandin E2 (PGE2) in cell supernatant were detected by ELISA. Results The intervention of LPS significantly increased the expression of NLRP3, pro-caspase-1, caspase-1 and pro-IL-1β in macrophages. After co-culture with BMSC, the expression of each protein decreased significantly. After the overexpression of PGE2, the difference of protein expression further decreased. The expression of NLRP3 and caspase-1 mRNA in LPS group increased significantly, but decreased significantly after co-culture with BMSC. Overexpression of PGE2 could increase this difference, but there was no significant change in PGE2 silent group. The results of ELISA showed that the contents of TNF-α, IL-1β and IL-18 in cell supernatant were the highest in LPS group. Adding BMSC and overexpressing PGE2 could decrease the related inflammatory factors. The levels of IL-10 and PGE2 in LPS group were higher than those in control group, and further increased in LPS+BMSC group and LPS+BMSC-PGE2(+) group with significant differences. Conclusions When inflammation is induced by LPS, BMSC can significantly mitigate the inflammatory response within macrophages. This process is likely mediated through the overexpression of PGE2, which inhibits the NLRP3-mediated pyroptosis pathway.
ObjectiveTo study the effect of Schwann cells (SCs) promoting the function of nitric oxide (NO) secretion of bone marrow mesenchymal stem cells (BMSCs) derived endothelial cells so as to lay the experimental foundation for research of the effect of nerves on vessels during the process of tissue engineering bone formation. MethodsSCs were collected from 1-day-old Sprague Dawley (SD) rats,and identified through S100 immunohistochemistry (IHC).BMSCs were collected from 2-week-old SD rats and induced into endothelial cells (IECs),which were identified through von Willebrand factor (vWF) and CD31 immunofluorescence (IF).Transwell system was used for co-culture of SCs and IECs without contact as the experimental group,and simple culture of IECs served as the control group.The NO concentration in the medium was measured at 1,3,5,and 7 days after culture; the mRNA expressions of nitric oxide synthetase 2 (NOS2) and NOS3 were detected by real-time fluorescence quantitative PCR (RT-qPCR) at 1,3,7,and 10 days. ResultsSCs and IECs were identified through morphology and immunology indexes of S100 IHC,vWF and CD31 IF.Significant differences were found in the NO concentration among different time points in 2 groups (P<0.05); the NO concentration of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 3 days.NOS2 mRNA expression of the experimental group was significantly higher than that of the control group (P<0.05); difference was significant in the NOS2 mRNA expression among different time points in 2 groups (P<0.05).NOS3 mRNA expression of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 10 days.No significant difference was found in NOS3 mRNA expression among different time points in the experimental group (F=6.673,P=0.062),but it showed significant differences in the control group (F=36.581,P=0.000). ConclusionSCs can promote NO secretion of BMSCs derived endothelial cells,which is due to promoting the activity of NOS.