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

OBJECTIVETo explore the role of CXCR4/STAT3 in mesenchymal stromal cell (MSC)-mediated drug resistance of AML cells.

METHODSAML cell lines U937 and KG1a and primary AML cells were co-cultured with MSC from bone marrow of healthy donors. The AML cell lines cultured alone were used as control. Apoptosis induced by mitoxantrone was measured by flow cytometry. Expression of CXCR4 and STAT3 protein were detected by Western blot. After incubated with STAT3 inhibitor Cucurbitacin I or CXCR4 antagonist AMD3100, the apoptosis of AML cells induced by mitoxantrone was evaluated.

RESULTSApoptosis of AML cells (U937 and KG1a) and primary AML cells induced by mitoxantrone significantly decreased in cocultured group than that of control group [U937 cells: (20.08±1.53)% vs (45.33 ± 1.03)% , P=0.004; KG1a cells: (25.60 ± 1.82)% vs (40.33 ± 3.29)% , P=0.020]. Expression of phosphorylated STAT3 and CXCR4 protein in AML cells were upregulated in cocultured group. After addition of Cucurbitacin I into the co-culture system, the apoptosis rate of primary AML cells significantly increased. Similar results of the apoptosis rates were also detected when the inhibitor of CXCR4 AMD3100 was added to overcome the stromal cell-mediated drug resistance. Besides, the expression of p-STAT3 in AML cells after incubated with AMD3100 decreased significantly.

CONCLUSIONSAML cells cocultured with MSC leads to the up-regulation of phosphorylated STAT3 and CXCR4 proteins, which resulted in AML cells resistance to chemotherapeutic drugs. Therefore targeting STAT3 or CXCR4 could be a new therapeutic strategy of AML.

Acute Disease ; Apoptosis ; Coculture Techniques ; Drug Resistance, Neoplasm ; Flow Cytometry ; Gene Expression Regulation, Leukemic ; Heterocyclic Compounds ; Humans ; Leukemia ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Receptors, CXCR4 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; U937 Cells ; Up-Regulation

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

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.
Animals ; Blood Cells/*cytology ; Bone Morphogenetic Protein 2/genetics ; *Cell- and Tissue-Based Therapy ; Femur Head Necrosis/metabolism/*pathology/*therapy ; Gene Expression Regulation ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology ; Osteonecrosis/*pathology/*therapy ; PPAR gamma/genetics ; Rabbits ; Transplantation, Homologous

The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell (hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential.
Bone and Bones ; Cell Differentiation ; Humans ; Mesenchymal Stromal Cells ; Stem Cells ; cytology ; Tissue Engineering ; Urine ; 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