OBJECTIVETo observe the proliferation state of transplanted cells in acute myocardial infarction (AMI) rats, and the endothelial progenitor cells (EPCs) preconditioned by salvianolic acid B in different ratios with the bone mesenchymal stem cells (BMSCs).

METHODSThe cultivation and purification of EPCs were performed by density-gradient centrifugation and plastic adherence method. Two types of cells were identified by immunocytochemical method (CD34, CD133, and CD44). The rat model of AMI was prepared by ligation of left anterior descending artery. The EPCs were pre-treated with the optimal concentration of salvianolic acid B (8 microg/ mL). They were mixed with BMSCs in different proportions (EPCs/BMSCs in the ratio of 1:1, 2:1, 4:1, and 8:1, respectively). BMSCs and EPCs were injected into the myocardial infarction area. The infarcted area was determined by the N-BT staining and hematoxylin-eosin staining. The expression of Ki-67 was detected by immunohistochemical assay.

RESULTSCompared with the model group (19.60% +/- 3.23%), the myocardial infarction area of each implanted group obviously decreased (P < 0.05). Of them, the decrease was most obvious in the 4:1 group (11.37% +/- 2.18%) and the 8:1 group (9.23% +/- 2.35%, P < 0.05). Compared with the model group (cell/high magnification, 5.17 +/- 2.31), the Ki-67 positive cell number of each implanted groups significantly increased (P < 0.05). Of them, the Ki-67 positive cell number was obviously higher in the 8:1 group (15.00 +/- 3.16, P < 0.05).

CONCLUSIONSEPCs pretreated by salvianolic acid B combined with BMSCs could reduce the myocardial infarcted area, improve the proliferation of BMSCs in the peripheral infarction and local ischemia. Besides, along with the increase of the implant proportion of EPCs, the infarct area was gradually reduced, and the proliferative expression was gradually enhanced.

Animals ; Benzofurans ; pharmacology ; Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; Endothelial Cells ; cytology ; drug effects ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Myocardial Infarction ; metabolism ; pathology ; Rats ; Rats, Wistar ; Transplantation Conditioning

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.
Animals ; Ascorbic Acid ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line ; Cellular Senescence ; drug effects ; DNA Damage ; DNA Repair ; drug effects ; DNA Replication ; drug effects ; Disease Models, Animal ; Heterochromatin ; metabolism ; pathology ; Humans ; Mesenchymal Stem Cells ; metabolism ; pathology ; Mice ; Nuclear Lamina ; metabolism ; pathology ; Reactive Oxygen Species ; metabolism ; Telomere Homeostasis ; drug effects ; Werner Syndrome ; drug therapy ; genetics ; metabolism

To isolate bone marrow mesenchymal stem cells (BM-MSCs) and establish the model of chronic kidney disease (CKD) of Wuzhishan (WZS) mini-pig, and to study the repairment effect of BM-MSCs on CKD-induced renal fibrosis in vitro.
 Methods: Density gradient method was used to isolate and culture BM-MSCs. The cells were verified by morphology, phenotype, differentiation and so on. The left partial ureteral obstruction (LPUUO) was used to establish the CKD model, which was evaluated by B-ultrasound, single-photon emission computed tomography (SPECT), HE and Masson staining. The cells were divided into 3 groups, the tissue plus BM-MSCs group, the tissue group, and the BM-MSCs group, respectively. Seven days later, the supernatants were collected to observe the changes of hepatocyte growth factor (HGF) cumulative release. HE and Masson staining was used to observe the changes of renal tissue.
 Results: The isolated BM-MSCs possessed the features as follow: fibroblast-like adherent growth; positive in CD29 and CD90 expression while negative in CD45 expression; osteogenic induction and alizarin red staining were positive; alcian blue staining were positive after chondrogenic induction. Twelve weeks after the operation of LPUUO, B-ultrasound showed the thin renal cortical with pelvis effusion; SPETCT showed the left kidney delayed filling and renal impairment. The accumulation of HGF in the tissue plus BM-MSCs group was significantly higher than that in the tissue alone group at the 1st, 5th, 6th, 7th day, respectively (P<0.05). HE staining showed the different degree of renal lesions between the tissue plus BM-MSCs+CKD group and the tissue alone group, which was aggravated with the time going. Masson staining showed that the cumulative optical density of blue-stained collagen fibers in tissue plus BM-MSCs group was significantly lower than that in the tissue group at the 5th to 7th day (P<0.05).
 Conclusion: BM-MSCs from WZS mini-pig can inhibit or delay the progress of CKD-induced renal fibrosis through autocrine HGF in vitro.
Animals ; Autocrine Communication ; physiology ; Bone Marrow Cells ; Cells, Cultured ; Fibrosis ; physiopathology ; prevention & control ; Hepatocyte Growth Factor ; metabolism ; Kidney ; drug effects ; pathology ; physiopathology ; Mesenchymal Stem Cells ; drug effects ; Renal Insufficiency, Chronic ; complications ; physiopathology ; Swine ; Swine, Miniature ; Ureteral Obstruction ; complications

OBJECTIVETo investigate the effects of sodium copper chlorophyllin (SCC) on the proliferation, differentiation and immunomodulatory function of mesenchymal stem cells (MSCs) from mice with aplastic anemia.

METHODSA mouse model of aplastic anemia was established by exposure of BALB/c mice to sublethal doses of 5.0 Gy Co60 γ radiation, followed by transplantation of 2×10(6) lymph node cells from DBA/2 donor mice within 4 h after radiation. Aplastic anemic BALB/c mice were randomly divided into six groups: the treated groups, which received 25, 50, or 100 mg/kg/day SCC, respectively; a positive control group treated with cyclosporine A (CsA); and an untreated model control group (model group); while, the non-irradiated mice as the normal control group. SCC or CsA were administered by gastrogavage for 20 days, starting on day 4 after irradiation. Peripheral blood cells were counted and colony-forming fibroblasts (CFU-F) in the bone marrow were assayed. The ability of MSCs to form calcium nodes after culture in osteoinductive medium was also observed. The immunosuppressive effect of MSCs on T lymphocytes was analyzed by enzyme-linked immunosorbent assay and flow cytometry, to evaluate the efficacy of SCC in mice with aplastic anemia.

RESULTSPeripheral blood white cell and platelet counts were increased by medium and high SCC doses, compared with the untreated control. CFU-Fs were also increased compared with the untreated control, and the numbers of calcium nodes in MSCs in osteoinductive medium were elevated in response to SCC treatment. The percentage of Forkhead box protein 3 (FOXP3(+)) T cells was increased in T cell-MSC cocultures, and the cytokine transforming growth factor β1 was up-regulated in SCC-treated groups.

CONCLUSIONThe results of this study suggest that SCC not only promotes the proliferation and differentiation of MSCs, but also improves their immunoregulatory capacity in mice with aplastic anemia.

Anemia, Aplastic ; blood ; pathology ; therapy ; Animals ; Anthraquinones ; metabolism ; Biomarkers ; metabolism ; Bone Marrow Cells ; drug effects ; pathology ; Calcium ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Chlorophyllides ; pharmacology ; Colony-Forming Units Assay ; Female ; Immunosuppression ; Leukocyte Count ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred DBA ; Osteoblasts ; drug effects ; pathology ; Platelet Count ; T-Lymphocytes ; drug effects

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.
Adolescent ; Animals ; Cell Death/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Transformation, Neoplastic/drug effects/pathology ; Child ; Cytosine Deaminase/*genetics/therapeutic use ; Fluorouracil/pharmacology ; Genetic Therapy ; Genomic Instability/drug effects ; Humans ; Karyotype ; Mesenchymal Stromal Cells/*cytology/drug effects/metabolism ; Mice ; Multipotent Stem Cells/cytology/drug effects/metabolism ; Neoplasms/therapy ; Retroviridae/*metabolism ; Time Factors ; *Transduction, Genetic

OBJECTIVETo evaluate whether human mesenchymal stem cells (hMSCs) administration alter the clinical course of hyperoxia-induced lung injury.

METHODShMSCs were obtained from bone marrow aspirates from healthy donors after informed consent was signed, hMSCs were separated, cultured, amplified, identified and labeled with BrdU. For BrdU labeling, a sterile stock solution was added to the culture medium 48 h before the end of culture, at a final concentration of 10 micromol/L. Thirty-two 3-day old SD rats from four litters were randomly divided into four groups, as hyperoxia exposed + hMSC group (A), air-exposed + hMSC group (B), hyperoxia exposed group (C), and air-exposed group (D). The rats from the group A and the group C were placed in a sealed Plexiglas chamber with a minimal in- and outflow, providing six to seven exchanges per hour of the chamber volume and maintaining O2 levels above 95%, while the rats in the group B and the group D were only exposed to room air. Seven days later, all of them were taken out of the chamber, rats in the group A and B were injected intraperitoneally with hMSCs (1 x 10(5) in 50 microl of PBS) immediately, while the rats in the group C and D were only treated with 50 microl of PBS 3 days later. All the animals were sacrificed by an injection of sodium pentobarbital (120 mg/kg), perfused with cold 0.9% NaCl, and the left lungs were removed, the upper lobes of which were ground as tissue homogenates and used for ELISA, while the inferior lobes were stored at -70 degrees C until use for RT-PCR. The right lungs were fixed in situ for 2 h by the intratracheal instillation with 10% neutral formalin and then postfixed for 24 h. Sagittal sections (4-microm) of paraffin-embedded middle lobe and upper lobe of the right lung were used for immunohistochemistry and histology, respectively.

RESULTS(1) There was a significant difference in the value of RAC (raditive alveoli coant) among the 4 groups (11.145 +/- 1.331, 13.941 +/- 0.985, 9.595 +/- 0.672, 14.819 +/- 1.080, F = 43.234, P = 0.000). RAC in group A and C were significantly reduced compared with subjects in group D (P < 0.05, P < 0.05); and there was also a significant difference between group A and group C (P < 0.05), but not between group B and D subjects (P > 0.05). (2) There were significant differences in the levels of both TNFalpha and TGFbeta(1) in the homogenate of lungs among the 4 groups (142.933 +/- 24.017, 79.033 +/- 11.573, 224.088 +/- 41.915, 76.500 +/- 10.373, F = 59.970, P = 0.000; 1726.484 +/- 91.086, 1530.359 +/- 173.441, 2047.717 +/- 152.057, 1515.777 +/- 131.049, F = 24.977, P = 0.000). The levels of TNFalpha and TGFbeta1 were significantly elevated in both group A and group C when compared with subjects in group D (P < 0.05 for both). Concentrations of TNFalpha and TGFbeta1 were both significantly decreased in group A versus group C (P < 0.05 for both). There was no significant difference between group B and D subjects in the fields of TNFalpha and TGFbeta(1) (P > 0.05 for both). (3) BrdU-labelled cells were observed at alveolar wall and bronchioles in both group A and group B, and there was a significant difference in BrdU-labeled cells between two groups (0.230 +/- 0.026, 0.190 +/- 0.015; t = 3.769, P = 0.002), but none was found in group C and group D. Electrophoresis of the PCR products showed a 224 bp band, specific for Alu mRNA, in 7 of 8 rats of group A and 5 of 8 rats of group B, respectively, but no such band was found in group C and group D.

CONCLUSIONhMSCs administered by intraperitoneal injection could be implanted in the lungs of newborn rats, and they could effectively protect the rats against damage to the lungs caused by hyperoxia.

Animals ; Animals, Newborn ; Bone Marrow Cells ; drug effects ; Bromodeoxyuridine ; pharmacology ; Cell Communication ; Cell Differentiation ; drug effects ; Cells, Cultured ; Hematopoietic Stem Cells ; Humans ; Hyperoxia ; metabolism ; Infant, Newborn ; Lung ; pathology ; Lung Injury ; pathology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Oxygen ; metabolism ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta ; analysis ; Tumor Necrosis Factor-alpha ; analysis

Pulmonary arterial hypertension (PAH) causes right ventricular failure due to a gradual increase in pulmonary vascular resistance. The purposes of this study were to confirm the engraftment of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) placed in the correct place in the lung and research on changes of hemodynamics, pulmonary pathology, immunomodulation and several gene expressions in monocrotaline (MCT)-induced PAH rat models after hUCB-MSCs transfusion. The rats were grouped as follows: the control (C) group; the M group (MCT 60 mg/kg); the U group (hUCB-MSCs transfusion). They received transfusions via the external jugular vein a week after MCT injection. The mean right ventricular pressure (RVP) was significantly reduced in the U group after the 2 week. The indicators of RV hypertrophy were significantly reduced in the U group at week 4. Reduced medial wall thickness in the pulmonary arteriole was noted in the U group at week 4. Reduced number of intra-acinar muscular pulmonary arteries was observed in the U group after 2 week. Protein expressions such as endothelin (ET)-1, endothelin receptor A (ERA), endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 significantly decreased at week 4. The decreased levels of ERA, eNOS and MMP-2 immunoreactivity were noted by immnohistochemical staining. After hUCB-MSCs were administered, there were the improvement of RVH and mean RVP. Reductions in several protein expressions and immunomodulation were also detected. It is suggested that hUCB-MSCs may be a promising therapeutic option for PAH.
Animals ; Cytokines/metabolism ; Disease Models, Animal ; Endothelin-1/metabolism ; Fetal Blood/*cytology ; Gene Expression Regulation/drug effects ; Hemodynamics ; Humans ; Hypertension, Pulmonary/chemically induced/*therapy ; Hypertrophy, Right Ventricular/physiopathology ; Immunohistochemistry ; Lung/metabolism/pathology ; Male ; Matrix Metalloproteinase 2/metabolism ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology/metabolism ; Monocrotaline/toxicity ; Nitric Oxide Synthase Type III/metabolism ; Pulmonary Artery/pathology ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/metabolism

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti-cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno-ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10AEMT). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10AEMT. Stem-like cells of MCF7 and MDA-MB-231, and MCF10AEMT cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10AEMT cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.
ATP-Binding Cassette Transporters/*genetics/metabolism ; Adenine Nucleotide Translocator 2/antagonists & inhibitors/genetics ; Adenoviridae/*genetics ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects/genetics ; Breast Neoplasms ; Cadherins/antagonists & inhibitors/genetics ; Cell Line, Tumor ; Cell Survival/drug effects/genetics ; Cell Transdifferentiation/drug effects ; Doxorubicin/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epithelial-Mesenchymal Transition/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Humans ; Neoplasm Proteins/*genetics/metabolism ; Neoplastic Stem Cells/drug effects/*metabolism/pathology ; RNA, Small Interfering/*genetics ; Signal Transduction/drug effects

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
Adipogenesis/drug effects ; Alkaline Phosphatase/biosynthesis/genetics ; Antigens, Differentiation/biosynthesis/genetics ; Bone Marrow/pathology ; Cell Differentiation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/biosynthesis/genetics ; Culture Media ; Gene Expression Regulation ; Humans ; Lipoprotein Lipase/biosynthesis/genetics ; Mesenchymal Stem Cells/*drug effects/metabolism/pathology ; Osteoblasts/drug effects/*metabolism/pathology ; Osteogenesis/drug effects ; PPAR gamma/biosynthesis/genetics ; Parathyroid Hormone/*pharmacology ; Peptide Fragments/*pharmacology ; Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors

AIMTo study the effects of bone marrow MSCs transplantation on the apoptosis of alveolar wall cells and the expression of Bcl-2 and Bax of lung tissue in papain and Co60-induced pulmonary emphysema rats.

METHODSFemale Lewis rats were randomly divided into three groups: control group, emphysema group, emphysema + MSCs transplantation group. Rats were sacrificed at days 14 and 28 after treatment. Morphologic analysis of the lung tissue was performed. The apoptosis of the lung cells was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of Bcl-2 and Bax were determined by immunohistochemical staining.

RESULTSEmphysematous changes of the lung tissue were observed in emphysema group and emphysema + MSCs transplantation group. However, the emphysematous change in emphysema + MSCs transplantation group was improved compared with the emphysema group. There was significant difference in the number of alveolar counted per unit area (MAN), mean alveoli area (MAA) and mean linear interval(MLI) between emphysema group and emphysema + MSCs transplantation group. The apoptotic index of the alveolar wall cells in emphysema + MSCs transplantation group was less than that in the emphysema group. The percentage of Bcl-2 positive cells in emphysema + MSCs transplantation group was significantly higher than that in the emphysema group. The percentage of Bax positive cells in emphysema + MSCs transplantation group was significantly lower than that in the emphysema group. The ratio of Bcl-2/Bax of emphysema + MSCs transplantation group was significantly higher than that in the emphysema group.

CONCLUSIONBone marrow MSCs transplantation inhibits the apoptosis of alveolar wall cells, upregulates the expression of Bcl-2 and downregulates the expression of Bax. This may be part of the reason that bone marrow MSCs transplantation improves the papain and Co60-induced pulmonary emphysema.

Animals ; Apoptosis ; Bone Marrow Transplantation ; Cells, Cultured ; Cobalt Isotopes ; adverse effects ; Female ; Lung ; cytology ; Mesenchymal Stem Cell Transplantation ; Papain ; adverse effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pulmonary Alveoli ; drug effects ; pathology ; radiation effects ; Pulmonary Emphysema ; chemically induced ; metabolism ; pathology ; surgery ; Rats ; bcl-2-Associated X Protein ; metabolism