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

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

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

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

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