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

OBJECTIVEWe explored the expressions of the Notch and Wnt signaling pathways and their significance in the repair process of alveolar bone defects by establishing animal models with a composite of autologous bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) to repair bone defects in the extraction sockets of rabbits.

METHODSA total of 36 two-month-old male New Zealand white rabbits were randomly divided into four groups, and the left mandibular incisors of all the rabbits were subjected to minimally invasive removalunder general anesthesia. BMSC-PRF compounds, single PRF, and single BMSC were implanted in Groups A, B, and C. No material was implanted in Group D (blank control). The animals were sacrificed at 4, 8 and 12 weeks after surgery, the bone defect was immediately drawn, and the bone specimens underwent surgery after four, eight, and twelve weeks, with three rabbits per time point. The expressions of Notch1 and Wnt3a in the repair process of the bone defect were measured via immunohistochemical and immunofluorescence detection.

RESULTSImmunohistochemistry showed that the expressions of Notch1 and Wnt3a in Groups A, B, and C were higher than that in Group D at the fourth and eighth week after operation (P<0.05). By contrast, the expressions of Notch1 and Wnt3a in Group D were higher than those in Groups A, B, and C at the twelfth week (P<0.05). Immunofluorescence showed that the expressions of both Notch1 and Wnt3a reached their peaks in the new bone cells of the bone defect after four weeks following surgery and gradually disappeared when the bone was repaired completely.

CONCLUSIONNotch1 and Wnt3a signaling molecules are expressed in the process of repairing bone defects using BMSC-PRF composites and can accelerate the healing by regulating the proliferation and differentiation of BMSCs. Moreover, the expressions of Notch and Wnt are similar, and a crosstalk between them may exist it.

Alveolar Bone Grafting ; methods ; Animals ; Blood Platelets ; Bone Marrow Cells ; cytology ; Bone Transplantation ; methods ; Bone and Bones ; abnormalities ; Cell Differentiation ; Fibrin ; administration & dosage ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma ; Rabbits ; Random Allocation ; Receptor, Notch1 ; metabolism ; Tissue Engineering ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; Wound Healing

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 investigate effects of retinol on the expressions of epidermal growth factor (EGF), stem cell factor (SCF), colony-stimulating factor 1 (CSF1) and leukemia inhibitory factor (LIF) in cultured human umbilical-derived mesenchymal stem cells (UCMSCs).

METHODSHuman UCMSCs were isolated from human umbilical cord and identified for immunophenotypes. The cells were then cultured in DMEM/F12 media supplemented with 12% fetal bovine serum (FBS), 12% FBS+1 µmol/L retinol, 15% knockout serum replacement (KSR) and 15% KSR+ 1 µmol/L retinol. The expressions of the cytokines EGF, SCF, CSF1 and LIF in the cells were detected using RT-PCR and ELISA.

RESULTSThe isolated cells exhibited characteristic immunophenotypes of human UCMSCs and expressed EGF, CSF1 and SCF at both mRNA and protein levels but not LIF protein. Retinol (1 µmol/L) significantly promoted the expressions of SCF and CSF1 at both mRNA and protein levels but did not result in changes of EGF and LIF expressions in human UCMSCs.

CONCLUSIONRetinol at the concentration of 1 µmol/L can promote expression of SCF and CSF1 in human UCMSCs in vitro.

Cell Differentiation ; Cells, Cultured ; EGF Family of Proteins ; metabolism ; Humans ; Immunophenotyping ; Leukemia Inhibitory Factor ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Stem Cell Factor ; metabolism ; Umbilical Cord ; cytology ; Vitamin A ; pharmacology

OBJECTIVETo screen the differentially expressed miRNAs and their target genes in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) to better understand the mechanism for regulating the balance between osteoblast and adipocyte differentiation.

METHODSCultured hMSCs were induced for adipogenic differentiation, and at 0, 7, 14, and 21 days of induction, the cells were examined for miRNA and mRNA expression profiles using miRNA chip and transcriptome sequencing (RNA-seq) techniques. Correlation analysis was carried out for the miRNAs and mRNAs of potential interest. The databases including TargetScan, PicTar and miRanda were used to predict the target genes of the differentially expressed miRNA.

RESULTSThe expression of miR-140-5p was down-regulated and leukemia inhibitory factor receptor (LIFR) expression increased progressively during adipogenic differentiation of hMSCs, showing a negative correlation between them. Target gene prediction using the 3 databases identified LIFR as the target gene of miR-140-5p.

CONCLUSIONmiRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.

Adipocytes ; cytology ; Adipogenesis ; Cell Differentiation ; Cells, Cultured ; Down-Regulation ; Humans ; Leukemia Inhibitory Factor Receptor alpha Subunit ; metabolism ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; genetics ; Oligonucleotide Array Sequence Analysis ; Osteoblasts ; cytology ; RNA, Messenger ; Transcriptome

OBJECTIVETo observe the effect of Jianpi Bushen Qingchang Huashi Recipe (JBQHR) on proliferation and migration of bone marrow mesenchymal stem cells (BMSCs).

METHODSBMSCs were isolated and cultured in vitro with adherence screening method to prepare cell suspension. No drug intervention was given to BMSCs in the vehicle control group. JBQHR at 0.39, 0.78, 1.56 µg/mL was added in BMSCs of low, mid, and high dose JBQHR groups for co-incubation. Its effect on the proliferation of BMSCs was detected by CCK-8. BMSCs migration and chemotactic ability was detected using Transwell method. Each dose JBQHR combined ERK kinase inhibitor U0126 was set up as control. The phosphorylation of extracellular regulated protein kinase (ERK) and CAMP responsive element-binding protein (CREB) were detected by Western blot.

RESULTSCompared with the vehicle control group, the proliferation of BMSCs and BMSCs migration number could be promoted in the 3 JBQHR groups (P < 0.05). Besides, the proliferation of BMSCs was better in mid and high dose JBQHR groups than in the low dose JBQHR group (P < 0.05). Compared with the vehicle control group, the phosphorylation of ERK and CREB could be elevated in the 3 JBQHR groups (P < 0.05), and could be inhibited by U0126 (P < 0.01). Compared with the low dose JBQHR group, the phosphorylation of ERK increased in mid and high dose JBQHR groups with statistical difference (P < 0.05).

CONCLUSIONJBQHR could promote the proliferation and migration of BMSCs, and its mechanism might be related to ERK/CREB signaling pathway

Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; MAP Kinase Signaling System ; Mesenchymal Stromal Cells ; cytology ; drug effects