Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.
Humans ; Forkhead Box Protein M1/genetics* ; Female ; Macrophages/cytology* ; Endometriosis/genetics* ; Ubiquitin Thiolesterase/genetics* ; Cells, Cultured ; Endometrium/metabolism* ; Ubiquitination ; Adult ; Interleukin-10/metabolism* ; Interleukin-6/metabolism* ; Protein Stability ; Stromal Cells/metabolism*

BackgroundCorneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.

MethodsADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.

Results:ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40-200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = -82.742, P < 0.01; collagen II: t = -72.818, P < 0.01; collagen III: t = -104.452, P < 0.01; collagen IV: t = -133.426, P < 0.01, and collagen V: t = -294.019, P < 0.01; and fibronectin: t = -92.491, P < 0.01, respectively).

Conclusion:The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

Adipose Tissue ; cytology ; Animals ; Cell Proliferation ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Exosomes ; metabolism ; Extracellular Matrix ; metabolism ; Fibroblasts ; cytology ; metabolism ; Matrix Metalloproteinases ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rabbits

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.
Acute Lung Injury/chemically induced/*therapy ; Angiopoietin-1/*genetics ; Animals ; Bronchoalveolar Lavage Fluid ; Cytokines/metabolism ; Endotoxins ; Genetic Therapy ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung/metabolism ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/metabolism ; Neutrophils/metabolism ; Rats ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/*cytology

OBJECTIVETo investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells.

METHODSThe co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b.

RESULTSUC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells.

CONCLUSIONUC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.

Cell Differentiation ; Cell Line, Tumor ; Humans ; Interleukin-6 ; metabolism ; Leukemia, Promyelocytic, Acute ; pathology ; Mesenchymal Stromal Cells ; metabolism ; Tretinoin ; pharmacology ; Umbilical Cord ; cytology

BACKGROUNDThe functional improvement following bone marrow stromal cells (BMSCs) transplantation after stroke is directly related to the number of engrafted cells and neurogenesis in the injured brain. Here, we tried to evaluate whether 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), a free radical scavenger, might influence BMSCs migration to ischemic brain, which could promote neurogenesis and thereby enhance treatment effects after stroke.

METHODSRat transient middle cerebral artery occlusion (MCAO) model was established. Two separate MCAO groups were administered with either MCI-186 or phosphate-buffered saline (PBS) solution to evaluate the expression of stromal cell-derived factor-1 (SDF-1) in ischemic brain, and compared to that in sham group (n = 5/ group/time point[at 1, 3, and 7 days after operation]). The content of chemokine receptor-4 (CXCR4, a main receptor of SDF-1) at 7 days after operation was also observed on cultured BMSCs. Another four MCAO groups were intravenously administered with either PBS, MCI-186, BMSCs (2 × 106), or a combination of MCI-186 and BMSCs (n = 10/group). 5-bromo-2-deoxyuridine (BrdU) and Nestin double-immunofluorescence staining was performed to identify the engrafted BMSCs and neuronal differentiation. Adhesive-removal test and foot-fault evaluation were used to test the neurological outcome.

RESULTSMCI-186 upregulated the expression of SDF-1 in ischemic brain and CXCR4 content in BMSCs was enhanced after hypoxic stimulation. When MCAO rats were treated with either MCI-186, BMSCs, or a combination of MCI-186 and BMSCs, the neurologic function was obviously recovered as compared to PBS control group (P < 0.01 or 0.05, respectively). Combination therapy represented a further restoration, increased the number of BMSCs and Nestin+ cells in ischemic brain as compared with BMSCs monotherapy (P < 0.01). The number of engrafted-BMSCs was correlated with the density of neuronal cells in ischemic brain (r = 0.72 , P < 0.01) and the improvement of foot-fault (r = 0.70, P < 0.01).

CONCLUSIONMCI-186 might promote BMSCs migration to the ischemic brain, amplify the neurogenesis, and improve the effects of cell therapy.

Animals ; Antipyrine ; analogs & derivatives ; therapeutic use ; Bone Marrow Cells ; cytology ; physiology ; Brain Ischemia ; drug therapy ; metabolism ; therapy ; Chemokine CXCL12 ; metabolism ; Disease Models, Animal ; Infarction, Middle Cerebral Artery ; drug therapy ; metabolism ; therapy ; Male ; Mesenchymal Stromal Cells ; physiology ; Neurogenesis ; physiology ; Rats ; Rats, Sprague-Dawley ; Stroke ; drug therapy ; metabolism ; therapy

BACKGROUNDMesenchymal stem cells (MSCs) are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/β-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs (DKKs) are considered as an antagonist to block Wnt/β-catenin signaling pathway by binding the receptor of receptor-related protein (LRP5/6). DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/β-catenin signaling pathway.

METHODSStable MSCs were randomly divided into four groups: MSCs control, MSCs + transforming growth factor-β (TGF-β), MSCs + DKK1, and MSCs + TGF-β + DKK1. Flow cytometry was used to identify MSCs. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test. Immunofluorescence was used to detect protein expression in the Wnt/β-catenin signaling pathways. Western blotting analysis was employed to test expression of fibroblast surface markers and, finally, real-time reverse transcription polymerase chain reaction was employed to test mRNA expression of fibroblast surface markers and Wnt/β-catenin signaling proteins.

RESULTSCultivated MSCs were found to conform to the characteristics of standard MSCs: expression of cluster of differentiation (CD) 73, 90, and 105, not expression of 34, 45, and 79. We found that DKK1 could maintain the normal cell morphology of MSCs. Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs + TGF-β. However, the expression was lower in the MSCs + TGF-β + DKK1. Immunofluorescence showed high expression of all Wnt/β-catnin molecules in the MSCs + TGF-β group but expressed in lower quantities in MSCs + TGF-β + DKK1 group. Finally, mRNA expression of fibroblast markers vimentin, α-smooth muscle actin and Wnt/β-catenin signaling proteins β-catenin, T-cell factor, and glycogen synthase kinase-3β was significantly increased in MSCs + TGF-β group compared to control (P < 0.05). Expression of the same fibroblast markers and Wnt/β-catenin was decreased to regular quantities in the MSCs + TGF-β + DKK1 group.

CONCLUSIONSDKK1, Wnt/β-catenin inhibitors, blocks the Wnt/β-catenin signaling pathway to inhibit the process of MSCs fibrosis. It might provide some new ways for clinical treatment of certain diseases.

Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fibroblasts ; cytology ; metabolism ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Rats ; Transforming Growth Factor beta ; genetics ; metabolism

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

ObjectiveTo explore the feasibility of inducing human umbilical cord mesenchymal stem cells (HUMSCs) to differentiate into Leydig cells in the interstitial tissue of the rat testis.

METHODSHUMSCs were obtained by tissue blocks culture attachment and their purity and multi-lineage differentiation ability were verified by flow cytometry and chondrogenic/adipogenic/osteogenic differentiation. Then the HUMSCs were marked by CM-Dil and transplanted into the interstitial tissue of the rat testis. At 4 and 8 weeks after transplantation, the survival and differentiation status of the HUMSCs were observed by immunofluorescence staining and flow cytometry. The suspension of the rat Leydig cells was obtained at 8 weeks for determining the expression of the Leydig cell marker 3β-HSD in the HUMSCs, the cells labeled with CM-Dil were sorted and cultured, and the medium collected after 3 days of culture for measurement of the testosterone level.

RESULTSThe expression of the Leydig cell marker CYPllal was not observed in the HUMSCs at 4 weeks but found at 8 weeks after transplantation and the differentiation rate of 3β-HSD was about 14.5% at 8 weeks. CM-Dil labeled cells survived after sorting and testosterone was detected in the medium.

CONCLUSIONSHUMSCs are likely to differentiate into Leydig cells in the interstitium of the rat testis.

Animals ; Biomarkers ; metabolism ; Carbocyanines ; Cell Differentiation ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Feasibility Studies ; Humans ; Leydig Cells ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Testis ; cytology ; Time Factors ; Umbilical Cord ; cytology

OBJECTIVETo compare the biological characteristics and immunosuppressive activity between human amniotic mesenchymal stem cells (hAMSC) and human bone marrow mesenchymal stem cells (hBMMSC).

METHODSMSC from human amnion and bone marrow were isolated using enzymatic digestion and Ficoll-Hypaque density gradients, respectively. Their biological characteristics were compared by morphology, cell growth curves, cell cycle profile analysis, immunophenotype and immunofluorescence assay. Their immunosuppressive activities were studied on total activated T-cells with phytohemagglutinin (PHA-PBMSC). An in vitro co-culture was performed to compared the lymphocyte proliferation and the supernatant level of IFN-γ were measured by CCK-8 method and ELISA, respectively.

RESULTSBoth hAMSC and hBMMSC demonstrated fibroblast-like morphology. The hAMSC were able to be amplified for at least 15 passages, while the hBMMSC only for 6-7 passages. There was no significant difference in the proportion of G2/M phase cells of the 2 cells types (P>0.05). By FACS analysis for immunophenotype, both MSC were shown to be positive for CD105, CD90, CD73 and negative for CD34, CD45, CD11b, CD19, HLA-DR, but hAMSC were positive for Oct-3/4, which was in contrast to hBMMSC. Both of them expressed vimentin. Both the cells exhibited a inhibitory role on the lymphocyte proliferation induced by PHA in co-culture conditions, that was increased with the increase MSC proportion and both the suppressing effecs were enhanced. The supernatant IFN-γ levels of hAMSC co-cultured with lymphocyte at a ratio of 1:1 after 72 hours were measured by ELISA, and the level of IFN-γ was significantly lower than that in the same co-culture system of hBMMSC. In contrast to the IFN-γ in the PHA-stimulated group, the IFN-γ level in both co-culture groups was significantly lower.

CONCLUSIONMSC from amnion displayed a higher proliferative capacity and stem cell properties, compared with hBMMSC. Both MSC can inhibit lymphocyte proliferation and suppress IFN-γ secretion induced by PHA in vitro.

Amnion ; cytology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunophenotyping ; Immunosuppression ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes ; cytology

OBJECTIVETo investigate the effect of microRNA-382 (miR-382) on the biological properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSC).

METHODSThe mimics and inhibitor of miR-382 were transfected into hUC-MSC with lipo2000. Inverted microscopy was used to observe the morphology change of hUC-MSC. The proliferation of hUC-MSC was detected by CCK-8. Oil red O and alizarin red staining were applied to assess the adipogenic and osteogenic differentiation of hUC-MSC. Cetylpyridinium chloride was used to the quantitative analysis of osteogenic differentiation. The expression of Runx2 and some cytokines were detected by RT-PCR.

RESULTSmiR-382 did not influence the morphology, proliferation and adipogenic differentiation of hUC-MSC miR-382 inhibited the expression of Runx2, thus could inhibit the osteogenesis of hUC-MSC, being confirmed by alizarin red stain; miR-382 could influence the expression of key cytokines secreted from hUC-MSC, such as IL-6, IDO1, G-CSF, M-CSF, GM-CSF.

CONCLUSIONmiR-382 decreases the expression of Runx2 and inhibites the osteogenesis of hUC-MSC. In addition, it also affects the expression of some key cytokines secreted from hUC-MSC.

Cell Differentiation ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Granulocyte Colony-Stimulating Factor ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Interleukin-6 ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; metabolism ; Osteogenesis ; Transfection ; Umbilical Cord ; cytology