OBJECTIVETo study the expression of matrix metalloproteinases (MMPs ) in the decidual stromal cells (DSCs) and extravillous cytotrophoblasts (EVCT) in human early pregnancy and explore the change of MMPs in endometrial stromal cell (ESC) decidualization and its impact on implantation and placentation.

METHODSThe decidua and villi from 5 women with early pregnancy and mid-secretory endometrium from 5 normal women were collected and cultured in vitro, and the supernatants of the culture media were collected after 48 hours of incubation. The expression of the MMPs in the ESCs, DSCs and EVCTs was detected using Luminex xMAP system simultaneously and the difference in MMPs expression and their correlations were analyzed with SPSS10.0 software.

RESULTSThe MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, and MMP-9) were expressed in ESCs, DSCs and EVCTs, while MMP-12 was not found in ESCs and MMP-13 not in DSCs. Expressions of MMP-8, MMP-12, and MMP-13 were lowered. Compared with the ESCs, DSCs and EVCTs showed significantly lowered expressions of MMP-1, MMP-3, and MMP-7 (P<0.05), whereas expression of MMP-2 and MMP-9 increased significantly, and the high expressions of MMP-1, MMP-3, and MMP-7 was especially obvious in the DSCs. The expressions of MMP-1, MMP-3, and MMP-7, however, were significantly decreased in the EVCTs in comparison with the DSCs. Significant correlations were noted between MMP-1, MMP-3, and MMP-7, and MMP-2 was closely correlated with MMP-9. MMP-8 was significantly lower and MMP-12 and MMP-13 showed no obvious variation in the cell culture.

CONCLUSIONMMPs are secreted by ESCs, DSCs and EVCTs. Diverse MMPs play an important role in proliferation and differentiation of the ESC to affect embryo implantation and placentation. All MMPs establish a balance to co-regulate the process of pregnancy.

Adult ; Cells, Cultured ; Decidua ; cytology ; enzymology ; Endometrium ; cytology ; enzymology ; Female ; Humans ; Isoenzymes ; metabolism ; Matrix Metalloproteinases ; metabolism ; Pregnancy ; Pregnancy Trimester, First ; Stromal Cells ; cytology ; enzymology ; Trophoblasts ; cytology ; enzymology

OBJECTIVETo investigate telomerase activity in rabbit bone marrow stromal cells (BMSCs) during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor (GDNF) on the expression of telomerase.

METHODSBMSCs were acquired from rabbit marrow and divided into control group, GDNF (10 ng/ml) group. Cytokine.NSCs medium (prepared by our lab, Patent No. ZL02134314. 4) supplemented with 10 percent fetal bovine serum (FBS) was used to induce BMSCs differentiation along neural pathway. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neuron-specific endase (NSE), and gial fibrillary acidic protein (GFAP). The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activities were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA).

RESULTSBMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomerase activities were undetectable in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomerase expression were observed in the two groups.

CONCLUSIONSRabbit BMSCs do not display telomerase activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomerase activity of BMSCs.

Animals ; Bone Marrow Cells ; enzymology ; Cell Differentiation ; Glial Cell Line-Derived Neurotrophic Factor ; Immunohistochemistry ; Rabbits ; Stromal Cells ; enzymology ; Telomerase ; metabolism

OBJECTIVETo investigate the telomerase activity in mesenchymal stem cells (hMSCs) from human bone marrow after their in vitro committed differentiation into adipocytes and cryopreservation.

METHODShMSCs were isolated from human bone marrow. The isolated hMSCs were induced to differentiate into adipocytes in vitro or cryopreserved. TRAP assay (telomerase repeat amplification protocol assay) was employed to detect telomerase activity in those hMSCs.

RESULTSTelomerase activity (RTA) in hMSCs (n=19) was (1.46 +/-0.67)%, while that in hMSCs-derived adipocytes (n=3) was (11.80 +/-2.52)% (P<0.001). RTA of hMSCs-passage 1.3 (n=10) was (1.46+/-0.83)%, and that of hMSCs-passage 4-7(n=9) was (1.46 +/-0.47)% (P=0.99). Cryopreservation did not affect the telomerase activity in hMSCs, RTA of fresh hMSCs (n=13) was (1.41 +/-0.44)%, RTA of frozen hMSCs (n=6) was (1.57 +/-1.07)% (P=0.64).

CONCLUSIONhMSCs are telomerase-negative, but telomerase activity in hMSCs-derived adipocytes is upregulated.

Adipocytes ; cytology ; enzymology ; Bone Marrow Cells ; cytology ; enzymology ; Cell Differentiation ; Cells, Cultured ; Cryopreservation ; Humans ; Mesenchymal Stromal Cells ; cytology ; enzymology ; Telomerase ; metabolism

BACKGROUNDRecent studies on bone have shown an endocrine role of the skeleton, which could be impaired in various human diseases, including osteoporosis, obesity, and diabetes-associated bone diseases. As a sensor and regulator of energy metabolism, AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism. The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.

METHODSReverse transcription-polymerase chain reaction (PCR) for relative quantification, real-time PCR for absolute quantification, and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types, including primary human mesenchymal stem cells (hMSCs) and hFOB, Saos-2, C3H/10T1/2, MC3T3-E1, 3T3-L1, and C2C12 cells.

RESULTSAMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types. AMPKγ1 mRNA was abundantly expressed in C3H/10T1/2, MC3T3-E1, 3T3-L1, and C2C12 but not detected in human-derived cell types. AMPKγ2 mRNA was mildly expressed in all cell types. AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells. AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2, in which AMPKβ2 protein overwhelmed AMPKβ1 expression. AMPKγ1 and AMPKγ2 proteins were expressed in C3H/10T1/2, MC3T3-E1, 3T3-L1, and C2C12 cells and only AMPKγ2 protein was expressed in hMSCs, hFOB and Saos-2 cells. AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.

CONCLUSIONThe combination of AMPK α, β, and γ subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.

AMP-Activated Protein Kinases ; genetics ; metabolism ; Animals ; Cell Line ; Humans ; Mesenchymal Stromal Cells ; enzymology ; Mice ; Phosphorylation

The aim of this study was to investigate the expression and localization of cyclooxygenase-1 and -2 (COX-1 and COX-2) in synovial tissues from patients with rheumatoid arthritis (RA). Synovial tissues from 9 patients with RA and 5 patients with osteoarthritis (OA) were examined for COX-1 and COX-2 expressions by immunohistochemical staining using 2 polydonal COX-1 and COX-2 antibodies. In RA synovia, synovial lining cells showed intense immunostaining for COX-1, whereas slight to moderate staining was observed in inflammatory cells, stromal fibroblast-like cells and vascular endothelial cells. There was no significant difference in COX-1 expression between RA and OA synovia. The localization of COX-2 expression dearly differed from that of COX-1 expression, being most intense in inflammatory cells. However, there was no difference in COX-1 and COX-2 expressions between RA and OA synovial tissues. Our observations support that inflammatory mechanisms modulated by COX-1 and COX-2 in chronic RA synovium might be similar to those in chronic OA synovium.
Adult ; Aged ; Arthritis, Rheumatoid/pathology ; Arthritis, Rheumatoid/enzymology* ; Cell Division ; Female ; Fibrin/metabolism ; Human ; Isoenzymes/metabolism ; Isoenzymes/biosynthesis* ; Male ; Middle Age ; Neutrophil Infiltration ; Osteoarthritis/enzymology ; Prostaglandin-Endoperoxide Synthase/metabolism ; Prostaglandin-Endoperoxide Synthase/biosynthesis* ; Stromal Cells/pathology ; Stromal Cells/enzymology ; Synovial Membrane/pathology ; Synovial Membrane/enzymology*

BACKGROUNDThis study characterized the cardiac telocyte (TC) population both in vivo and in vitro, and investigated its telomerase activity related to mitosis.

METHODSUsing transmission electron microscopy and a phase contrast microscope, the typical morphological features of cardiac TCs were observed; by targeting the cell surface proteins CD117 and CD34, CD117 + CD34 + cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture. Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8. Under this conditioned medium, the process of cell division was captured, and the telomerase activity of CD117 + CD34 + cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs), cardiac fibroblasts (CFBs), cardiomyocytes (CMs).

RESULTSCardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms). In addition, 64% of the primary cultured cardiac TCs were composed of CD117 + CD34 + cardiac TCs; which was verified by immunofluorescence. In a live cell imaging system, CD117 + CD34 + cardiac TCs were observed to enter into cell division in a short time, followed by an significant invagination forming across the middle of the cell body. Using a real-time quantitative telomeric-repeat amplification assay, the telomerase concentration in CD117 + CD34 + cardiac TCs was obviously lower than in BMSCs and CFBs, and significantly higher than in CMs.

CONCLUSIONSCardiac TCs represent a unique cell population and CD117 + CD34 + cardiac TCs have relative low telomerase activity that differs from BMSCs, CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis.

Animals ; Antigens, CD34 ; metabolism ; Fibroblasts ; enzymology ; ultrastructure ; Flow Cytometry ; Mesenchymal Stromal Cells ; enzymology ; ultrastructure ; Mice ; Mice, Inbred C57BL ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Microscopy, Phase-Contrast ; Myocytes, Cardiac ; enzymology ; ultrastructure ; Proto-Oncogene Proteins c-kit ; metabolism ; Telomerase ; metabolism ; Vimentin ; metabolism

BACKGROUNDMesenchymal stem cells (MSCs) transplantation may partially restore heart function in the treatment of acute myocardial infarction (AMI). The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 (HO-1) on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells.

METHODSIn vitro, MSCs were divided into four groups: (1) non-transfected MSCs (MSCs group), (2) MSCs transfected with the pcDNA3.1-Lacz plasmid (Lacz-MSCs group), (3) MSCs transfected with pcDNA3.1-hHO-1 (HO-1-MSCs group), and (4) MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin (SnPP) (HO-1-MSCs + SnPP group). Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at < 1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor (TGF-β) and fibroblast growth factor (FGF-2) in the supernatant. In vivo, 28 Chinese mini-pigs were randomly allocated to the following treatment groups: (1) control group (saline), (2) Lacz-MSCs group, (3) HO-1-MSCs group, and (4) HO-1-MSCs + SnPP group. About 1 × 10(7) of autologous stem cells or an identical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging (MRI) assay and postmortem analysis were assessed four weeks after stem cell transplantation.

RESULTSPost hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs ((874.88 ± 68.23) pg/ml) compared with Lacz-MSCs ((687.81 ± 57.64) pg/ml, P < 0.001). FGF-2 was also significantly increased in the HO-1-MSCs ((1106.48 ± 107.06) pg/ml) compared with the Lacz-MSCs ((853.85 ± 74.44) pg/ml, P < 0.001). In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area compared with the Lacz-MSCs group (14.24 ± 1.66/HPFs vs. 11.51 ± 1.34/HPFs, P < 0.001). Arteriolar density was also significantly higher in HO-1-MSCs group than in the Lacz-MSCs group (7.86 ± 2.00/HPFs vs. 6.45 ± 1.74/HPFs, P = 0.001). At the same time, the cardiac function was significantly improved in the HO-1-MSCs group compared with the Lacz-MSCs group ((53.17 ± 3.55)% vs. (48.82 ± 2.98)%, P < 0.05). However, all these effects were significantly abrogated by SnPP.

CONCLUSIONMSCs provided a beneficial effect on cardiac function after ischemia/reperfusion by the induction of therapeutic angiogenesis, and this effect was amplified by HO-1 overexpression.

Animals ; Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Heme Oxygenase-1 ; genetics ; metabolism ; Magnetic Resonance Imaging ; Mesenchymal Stromal Cells ; cytology ; enzymology ; metabolism ; Myocardial Reperfusion Injury ; enzymology ; metabolism ; Swine ; Swine, Miniature

OBJECTIVETo investigate the expression dynamics and significance of matrix metalloproteinase-2 (MMP-2) membrane type-matrix metalloproteinase-2 (MT-MMP-2) in hepatic fibrosis and its reversal counterpart.

METHODSAn experimental CCl4 induced hepatic fibrosis rat model was established by intraperitoneal administration of carbon tetrachloride for 2, 4, 6, 8, 10 weeks, and normal rats were used as a control group. The immunohistochemical methods and in situ hybridization were used to detect MMP-2,MT-MMP-2 mRNA and related antigens in the liver.

RESULTSMMP-2,MT-MMP-2 mRNA and related antigens were expressed in mesenchymal cells and parts of hepatocytes besides active pathological changes, especially in the fibrous septum and portal area. Expression of MMP-2,MT-MMP-2 mRNA and related antigens were increased in hepatic fibrosis and decreased gradually in its reversal counterpart.

CONCLUSIONThis study suggested that mesenchymal cells are the main cellular origins of MMPs. The levels of MMP-2 and MT-MMP-2 antigens and gene expression were closely related to hepatic fibrosis. MMP-2 and MT-MMP-2 may play important roles in hepatic fibrosis and its reversal counterpart.

Animals ; Carbon Tetrachloride Poisoning ; Gene Expression Regulation, Enzymologic ; Hepatocytes ; enzymology ; Liver ; enzymology ; pathology ; Liver Cirrhosis, Experimental ; enzymology ; etiology ; pathology ; Male ; Matrix Metalloproteinase 2 ; biosynthesis ; genetics ; Matrix Metalloproteinases ; biosynthesis ; genetics ; Matrix Metalloproteinases, Membrane-Associated ; Mesenchymal Stromal Cells ; enzymology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo study the compatibility between human adipose-derived mesenchymal stem cells and porcine bone scaffolds.

METHODSPorcine bone tissues were co-cultured with adipose-derived mesenchymal stem cells, and the complex was observed under scanning electron microscope. The viability and alkaline phosphatase (ALP) activity of the cells were examined with the cells co-cultured with human bone scaffold as the control.

RESULTSAt 4 and 10 days after the co-culture, the adipose-derived mesenchymal stem cells were observed to extend pseudopodia to adhere to the two scaffold materials. MTT assay showed that the cell proliferation on both of the materials increased with time, and the two cell complexes exhibited similar pattern of changes in ALP activity.

CONCLUSIONAs the seed cells, human adipose-derived mesenchymal stem cells exhibit good comparability with porcine bone scaffold, suggesting their potential of constructing tissue-engineered bone graft.

Adipose Tissue ; cytology ; Adult ; Alkaline Phosphatase ; metabolism ; Animals ; Bone and Bones ; cytology ; metabolism ; Cell Proliferation ; Coculture Techniques ; Humans ; Materials Testing ; Mesenchymal Stromal Cells ; cytology ; enzymology ; metabolism ; ultrastructure ; Microscopy, Electron, Scanning ; Swine ; Time Factors ; Tissue Scaffolds

OBJECTIVETo determine the effect of aspirin on cell proliferation, alkaline phosphatase (ALP) activity, cell cycle and apoptosis in murine bone marrow stromal cells, so as to explore an appropriate dose range to improve bone regeneration in periodontal treatment.

METHODSST2 cells were stimulated with aspirin (concentrations of 1, 10, 100 and 1 000 μmol/L) for 1, 2, 3, 5 and 7 d. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. After ST2 cells were treated for 1, 3 and 7 d, ALP activity was measured by ALP kit, cell cycle and apoptosis were measured by flow cytometry (FCM) after treated for 48 h.

RESULTSMTT assays showed that various doses of aspirin have different effects on the cell growth. Briefly, lower concentrations (1, 10 μmol/L) of aspirin promoted the cell growth, the A value of 0, 1 and 10 μmol/L aspirin 7-day-treated cells were 0.313±0.012, 0.413±0.010 and 0.387±0.017 respectively (P <0.01 vs control), and so did the ALP level ([4.3±0.9], [6.0±0.3] and [7.7±0.4] μmol·min(-1)·g(-1), P <0.05 vs control), while higher concentrations, especially 1000 μmol/L of aspirin might inhibit the cell growth with time going, A value and ALP level were 0.267±0.016, (4.3±1.3) μmol·min(-1)·g(-1) respectively (P <0.05 vs control). Cell cycle analysis revealed no changes in comparison to control cells after treatment with 1 or 10 μmol/L aspirin, but it was observed that cell mitosis from S phase to G2/M phase proceeded at higher concentrations of 100 μmol/L aspirin, and the cell cycle in phase G0/G1 arrested at 1000 μmol/L. Parallel apoptosis/necrosis studies showed that the percentage of cells in apoptosis decreased dramatically at all doses of aspirin, the apoptosis rates of ST2 cells responded to 0, 1, 10, 100 and 1000 μmol/L aspirin were (11.50±0.90)%, (5.30±0.10)%, (5.50±0.10)%, (4.90±0.90)% and (7.95±0.25)% respectively (P<0.05 vs control).

CONCLUSIONSThis study demonstrated that lower dosage of aspirin can promote ST2 cells growth, osteogenic activity and inhibit its apoptosis. Aspirin maybe used for the bone reconstruction with a proper concentration.

Alkaline Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Aspirin ; administration & dosage ; pharmacology ; Bone Regeneration ; Cell Cycle ; drug effects ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Formazans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; enzymology ; Mice ; Periodontics ; Tetrazolium Salts ; Time Factors