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

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

OBJECTIVETo explore the effect of MicroRNA-146a (miR-146a) on the ability of BM-MSC to differentiate into adipocytes and osteoblasts.

METHODSBM-MSC were isolated from the bone marrow of healthy donors. The differentiation of BM-MSC into adipocytes and osteoblasts cells were done in vitro. After transfection with miR-146a inhibitor or mimics, the expression of miR-146a in BM-MSC was detected by real time quantitative PCR. The effect of MicroRNA-146a on the differentiation potential of BM-MSC was evaluated after transfection.

RESULTSBM-MSC possessed the ability to differentiate into adipocytes and osteoblasts cells when cultured in the induction medium. The expression of miR-146a was correspondingly down-regulated and up-regulated in BM-MSC after transfection. Compared with the control group, the expression of miR-146a was down-regulated (P < 0.01) after transfection with miR-146a inhibitor, while after transfection with miR-146a mimics it was significantly up-regulated. This study proved that the transfection with miR-146a inhibitor can inhibit BM-MSC differentiate into adipocytes (P < 0.01), while transfection with miR-146a mimics can promote differentiation of BM-MSC into adipocytes (P < 0.01). No effect of miR-146a inhibitor or miR-146a mimics on osteogenic differentiation of BM-MSC was observed (P > 0.05).

CONCLUSIONBM-MSC possess the ability to differentiate into adipocytes and osteoblasts. The miR-146a can promote BM-MSC to differentiate into adipocytes.

Adipocytes ; cytology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; metabolism ; Osteoblasts ; cytology ; Osteogenesis ; Transfection

OBJECTIVETo study the regulation of SIRT1 by transcription factor SREBP-1 in adipogeneic differentiation of bone marrow mesenchymal stem cells (BMMSC).

METHODSOil red O staining was used to identify the adipogenic differentiation of BMMSC; the mRNA transcription levels of AP2, LPL, SREBF-1, SIRT1 gene were detected by RT-PCR; the expession level of SREBP-1 was determined by Western-blot. The chromatin immunoprecipitation (ChIP) assay was used to investigate the binding of SREBP-1 to SIRT1 promoter.

RESULTSBMMSC exposed to adipogenesis inducing medium become mature adipocytes at day 14; the mRNA transcription levels of AP2, LPL, SREBF-1, SIRT1 genes were up-regulated in adipocyte differentiation of BMMSC; the protein level of SREBP-1 was higher obviously; SIRT1 gene sequences was succesfully amplified from the genomic DNA immunoprecipitated by SREBP-1 antibody.

CONCLUSIONSREBP-1 can bind to the promoter region of the SIRT1 gene in adipogenesis of BMMSC, and may be involved in the transcriptional regulation of the SIRT1 gene.

Adipocytes ; cytology ; Adipogenesis ; Cell Differentiation ; Cells, Cultured ; Chromatin Immunoprecipitation ; Gene Expression Regulation ; Humans ; Mesenchymal Stromal Cells ; cytology ; Promoter Regions, Genetic ; Sirtuin 1 ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Up-Regulation

OBJECTIVETo investigate the modulatory effect of the MSC derived from low attaching culture systems (suspending MSC) on T lymphocytes and the related mechanisms.

METHODSThe suspending MSC were generated from mouse compact bones by using low attaching plates and adherent cell culture flasks, respectively. The morphology of suspending MSC was observed under the inverted microscope and the cells were induced to differentiate into osteoblasts and adipocytes. Further, the surface antigen profile of MSC was analyzed with flow cytometry. In addition, the culture medium (CM) of suspending MSC and adherent MSC was collected and added into the activated T cell cultures before detection of the proliferation by CFSE assay. Moreover, the modulaory effects of the CM on the T cell-derived cytokines were detected by quantitative PCR. Also, the mRNA expression of cytokines of MSC was detected.

RESULTSThe suspending MSC grew in floating cell spheres and differentiated into osteoblasts and adipocytes in the induction medium. Furthermore, the suspending MSC shared the typical immuno-phenotype with their adherent counterparts. In addition, the results of CFSE assay demonstrated that suspending MSC derived CM suppressed ConA induced T cell proliferation. The results of quantitative PCR revealed that suspending MSC expressed transforming factor β1 and interleukin-6 at a higher level and suppressed the T cell expressing interferon γ and interleukine-17A.

CONCLUSIONThe suspending MSC exerted an unique modulatoy effect on T cells, which is quite different to adherent MSC.

Adipocytes ; cytology ; Animals ; Cell Adhesion ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Culture Media, Conditioned ; Flow Cytometry ; Immunophenotyping ; Interleukin-6 ; metabolism ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Osteoblasts ; cytology ; T-Lymphocytes ; cytology ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo construct a co-culture system for bone marrow mesenchymal stem cells (BMMSC) and multiple myeloma (MM) cells, and to investigate the effects of co-cultured BMMSC on the migrating and homing of multiple myeloma cells.

METHODSThe BMMSC from the transgenic mice with green fluorescent protein (GFP) fetal bone were cultured by adherent screening. A co-culture system of BMMSC and MM cell line XG-7 cells was constracted, the proliferation and apoptosis of cells were determined by trypan blue exclusion and Annexin V/PI, respectively, MDC staining was employed to detect the autophagy. The moving direction distribution of molecule in BMMSC and XG-7 cells labeled with PE-CD138 in co-culture process were observed dinamically by confocal microscopy.

RESULTSAfter co-culture with GFP-BMMSC, the resistance of XG-7 cells to apoptosis and autophagy were enhanced; at the same time, their proliferation increased. Apoptosis rates of XG-7 cells directly and indirectly co-cultured with BMMSC were (6.23 ± 0.12)% and (6.97 ± 0.03)% respectively, which were lower than that of XG-7 cells cultured alone (17.90 ± 1.46)% (P < 0. 01). There was low level of autophagy in XG-7 cells co-cultured with BMMSC. XG-7 cells are highly polarized and contained a specialized membrane domain with specific protein and lipid components to contact with BMMSC under confocal microscope. After methyl-β-cyclodextrin treatment, the molecules were normally enriched in the specialized domain.

CONCLUSIONBMMSC can protect XG-7 cells from apoptosis and autophagy, and obviously promote the proliferation of XG-7 cells, and can influence the migrating and homing of multiple myeloma cells.

Animals ; Apoptosis ; Autophagy ; Bone Marrow Cells ; cytology ; Cell Line, Tumor ; Cell Movement ; Coculture Techniques ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Transgenic ; Multiple Myeloma ; pathology

OBJECTIVETo investigate the effects of intercellular adhesion molecule-1(ICAM-1) on the adherence between mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC).

METHODSMSC and EPC were isolated, cultured and expanded from the 6-8 weeks aged C57BL/6 murine bone marrow by in vitro. Immuno-fluorescence was used to detect the expression of ICAM-1 in MSC group, EPC group and co-cultured MSC and EPC group. The mRNA and protein levels of ICAM-1 were detected by RT-PCR and Western blot respectively, then, the ICAM-1 adherence between MSC and EPC was observed by adding different concentration of neutralizing antibody.

RESULTSThe expression of ICAM-1 on surface of MSC and EPC could be detected by cell immunofluorescence method. According to results of the semiquantitative fluorescene detection, the fluorescence strength of MSC+EPC co-cultured group (89.02 ± 24.52) was higher than that of MSC group (31.25 ± 2.95) and EPC group (34.32 ± 5.02), and there was statistical difference between them (P < 0.01), but there was no obvious difference between MSC group and EPC group (P > 0.05). RT-PCR detection showed that the expression levels of ICAM-1 in MSC+EPC co-cultured group were higher than that in MSC group and that in EPC group (P < 0.01), and expression level of ICAM in EPC group was higher than that in MSC group (P < 0.01). Western blot detection showed that the expression level of ICAM-1 protein in MSC+EPC co-cultured group (0.33 ± 0.4) was higher than that in MSC group (0.11 ± 0.01) (P < 0.05) and than that in EPC group (0.19+0.02) (P < 0.05), However, the expression level of ICAM-1 protein in EPC group was higher than that in MSC group (P < 0.05). The test of different concentrations against neutralizing antibody showed that with the increasing of concentration of ICAM-1 neutralizing antibody, the adhesion capability of MSC and EPC was gradually decreasing.

CONCLUSIONThe ICAM-1 can mediate the adherence process between MSC and EPC.

Animals ; Bone Marrow ; Cell Adhesion ; Coculture Techniques ; Endothelial Progenitor Cells ; cytology ; Intercellular Adhesion Molecule-1 ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL

OBJECTIVETo study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.

METHODSExperiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.

RESULTSThe cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.

CONCLUSIONLIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.

Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Genes, Homeobox ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Octamer Transcription Factor-3 ; metabolism ; Organic Chemicals ; Proliferating Cell Nuclear Antigen ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Umbilical Cord ; cytology