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 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 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 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 the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts.

METHODS(1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, and t test.

RESULTS(1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (with P values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (with P values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%, t=6.710, P<0.01].

CONCLUSIONShAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.

Amnion ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; metabolism ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Male ; Mesenchymal Stromal Cells ; chemistry ; Pregnancy ; Vascular Endothelial Growth Factor A ; metabolism