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 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

OBJECTIVETo investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on VP16-induced apoptosis of Nalm-6 cells.

METHODShUC-MSC were isolated and identified using morphological observation and flow cytometry, then Nalm-6 cells were treated with hUC-MSC with or without VP16. Apoptosis and cell cycle were assayed by FACS. The mRNA levels of apoptosis-related genes BCL-2, BAX and caspase-3 were detected by quantitative RT-PCR, and the protein levels of BCL-2, BAX and caspase-3 were examined by Western blot.

RESULTSFACS showed that hUC-MSC inhibited the proliferation and decreased apoptosis of Nalm-6 cells resulted from VP16. The quantitative RT-PCR showed that hUC-MSC increased the mRNA expression level of BCL-2 and decreased the expression level of BAX and caspase-3 (P < 0.05). Western blot showed that the protein expression level of BCL-2 increased, and expression level of BAX and caspase-3 decreased in Nalm-6 cells after co-culture with hUC-MSC (P < 0.05).

CONCLUSIONhUC-MSC may protect Nalm-6 cells from apoptosis induced by VP16 through regulation of BCL-2, BAX and caspase-3.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Etoposide ; adverse effects ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Umbilical Cord ; cytology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the effect of total flavonoids of Herba Epimedium (FHE) on BMP-2/RunX2/OSX signaling pathway in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

METHODSPassage 3 BMSCs were randomly divided into the control group, the experimental group, and the inhibitor group. BMSCs in the control group were cultured in 0.2% dimethyl sulfoxide + Osteogenuxic Supplement (OS) fluid + DMEM/F12 culture media. BMSCs in the experimental group were intervened by 20 microg/mL FHE. BMSCs in the inhibitor group were intervened by 20 microg/mL FHE and 1 microg/mL NOGGIN recombinant protein. At day 9 alkaline phosphatase (ALP) activity was measured. Calcium nodules were stained by alizarin red staining and the density was observed. The transcription expression of osteogenic differentiation-related proteins (type I collagen, osteocalcin, and osteopontin) and related factors of BMP-2/RunX2/OSX signaling pathway was assayed by RT-PCR.

RESULTSCompared with the control group, ALP activities were enhanced and the density of calcium nodules significantly increased; type I collagen, osteocalcin, and osteopontin expression levels were increased in the experimental group. The expression of osteogenesis-related transcription factor was also increased in the experimental group. Noggin recombinant protein inhibited FHE promoting BMSCs osteogenesis in the inhibitor group. Compared with the experimental group, ALP activity decreased (P < 0.05), the density of calcium nodules was lowered, expression levels of type I collagen, osteocalcin, osteopontin significantly decreased (P < 0.05) in the inhibitor group.

CONCLUSION20 microg/mL FHE promoted osteogenic differentiation process of BMSCs by BMP-2/RunX2/OSX signaling pathway.

Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Osteocalcin ; metabolism ; Osteogenesis ; drug effects ; Osteopontin ; metabolism ; Signal Transduction ; Sp7 Transcription Factor ; Transcription Factors ; 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 evaluate the application of tendon-derived stem cells (TDSC) and bone marrow-derived mesenchymal stem cells (BMSC) for patellar tendon injury repair in rat model.

METHODSTDSCs and BMSCs were isolated from patellar tendons or bone marrow of healthy SD rats. The patellar tendon injury model was induced in 60 SD rats, then the animals were divided into 3 groups with 20 in each group: rats in TDSC group received transplantation of TDSC with fibrin glue in defected patellar tendon, rats in BMSC group received BMSC with fibrin glue for transplantation and those in control group received fibrin glue only. The gross morphology, histology and biomechanics of the patellar tendon were examined at 1, 2, 4, 6 and 8 weeks after the treatment.

RESULTSGross observation showed that the tendon defects in TDSC group and BMSC group almost disappeared in week 8, while the boundary of tendon defects in control group was still visible. Histology examination showed that the neo-tendon formation in TDSC group and BMSC group was observed at week 8, while there was no neo-tendon formation in control group. Biomechanics study showed that the ultimate stress and Young Modulus, relative ultimate stress and relative Young Modulus increased with the time going in all groups(all P<0.05); the ultimate stress and Young Modulus, relative ultimate stress and relative Young Modulus of TDSC and BMSC groups were significantly higher than those in control group at week 4, 6 and 8(all P<0.05). There was no difference in ultimate stress and Young Modulus between TDSC group and BMSC group(P>0.05), however, the relative Young Modulus of TDSC group was significantly higher than that in BMSC group at week 8(P<0.05).

CONCLUSIONAllogeneic TDSC and BMSC transplantation facilitates the repair of tendon injury and improves the biomechanics of tendon. TDSC is more suitable for in vivo tendon regeneration than BMSC.

Animals ; Bone Marrow ; Elastic Modulus ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Tendon Injuries ; therapy ; Tendons ; cytology ; Wound Healing

Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.
Cell Differentiation ; Consensus ; Human Embryonic Stem Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Tendons ; cytology ; Tissue Engineering