Bone marrow mesenchymal stem cells (MSCs) are somatic stem cells that can differentiate into progenies of multiple lineages. They play an important role in hematopoiesis and stem cell therapy due to their multi-lineage potentials and immunomodulatory properties. Oxidative stress is a disturbed redox state caused by accumulation of reactive oxygen species. It can induce the senescence and apoptosis of MSCs via phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and p53 pathways, and inhibit the proliferation and differentiation of MSCs through apurinic/apyrimidinic endonuclease/redox factor 1 (APE/REF-1) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, using anti-stress medication and hypoxic preconditioning, the functions of MSCs can be further enhanced. Accordingly, further studies on the effect of oxidative stress on MSCs and its signaling pathways may be meaningful for the treatment of hematologic diseases and for improving stem cell therapy.
Bone Marrow Cells ; cytology ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Oxidative Stress ; Signal Transduction

This study was to establish an iron overload bone marrow (BM) model by co-culturing the mononuclear cells from BM with iron, and investigate its hematopoiesis changes. The iron overload model was set up by adding different concentration of ferric citrate (FAC) into the mononuclear cells from BM and culturing for different time, and the model was confirmed by detecting labile iron pool (LIP). Then the apoptosis of hematopoietic cells, ability of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) and percentage of the CD34(+) cells of the BM cells all were determined. The changes of these indexes were tested after the iron-overloaded BM was treated with deferasirox (DFO). The results showed that after BM cells were cultured with FAC at different concentrations for different time, the LIP increased in time-and concentration-dependent manners. The intracellular LIP reached maximum level when cultured at 400 µmol/L of FAC for 24 hours. The detection of BM cell hematopoietic function found that the apoptotic rate of the FAC-treated cells (24.8 ± 2.99%) increased significantly, as compared with normal control (8.9 ± 0.96%)(p < 0.01). The ability of hematopoietic colony forming in FAC-treated cells decreased markedly, as compared with normal control (p < 0.05). The percentage of CD34(+) cells of FAC-treated cells (0.39 ± 0.07%) also decreased significantly, as compared with normal control (0.91 ± 0.12%)(p < 0.01). And these changes could be alleviated by adding DFO. It is concluded that the iron-overloaded model has been set by adding iron into the mononuclear cells from BM in vitro, and the hematopoietic function of iron-overloaded BM is deficient. These changes can be alleviated by removing the excess iron from the BM cells through treating with DFO. These findings would be helpful to further study the mechanism of iron-overload on the hematopoiesis of BM and also useful to find the way to treat iron-overload patients with hematopoietic disorders.
Bone Marrow Cells ; cytology ; Cells, Cultured ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Humans ; Iron ; metabolism ; Iron Overload

The release of microvesicles(MV) is one of the critical mechanisms underlying the angiogenesis-promoting activity of mesenchymal stem cells(MSC). This study was aimed to explore the appropriate condition under which MSC releases MV. Bone marrow samples from 5 healthy adults were collected, and MSC were isolated, culture-expanded and identified. MSC at passage 5 were suspended in medium without or medium with 10% fetal(FCS) calf serum and seeded into culture dishes. The culture was separately maintained in hypoxia (1% oxygen) or normoxia (around 20% oxygen), and 20 dishes of cells (2×10(6)/dish) were used for each group. The supernatants were collected for MV harvesting. The cell number was counted with trypan blue exclusion test and the protein contents in the MV were determined. MV were identified by observation under an electron microscope. The surface markers on MV were analyzed by flow cytometry. MTT test was performed to observe the pro-proliferative activity of MV that were added into the culture of human umbilical cord vein endothelial cells at a concentration of 10 µg/ml. The results showed that the majority of MV released by MSC were with diameters of less than 100 nm, and MV took the featured membrane-like structure with a hypodense center. They expressed CD29, CD44, CD73 and CD105, while they were negative for CD31 and CD45. The increase multiples of the adherent trypan blue-resistant cells cultured in normoxia with serum, in normoxia without serum, in hypoxia with serum and hypoxia in the absence of serum were 4.05 ± 0.73, 1.77 ± 0.48, 5.80 ± 0.65 and 3.69 ± 0.85 respectively, and the estimated protein contents per 10(8) cells were 463.48 ± 138.74 µg, 1604.07 ± 445.28 µg, 2389.64 ± 476.75 µg and 3141.18 ± 353.01 µg. MTT test showed that MV collected from MSC in hypoxia seemed to promote the growth of endothelial cells more efficiently than those from cells in normoxia. It is concluded that hypoxia can enhance the release of microvesicles from MSC, and cultivation of MSC in hypoxia and medium without serum may provide an appropriate condition for MV harvesting.
Bone Marrow Cells ; cytology ; metabolism ; Caveolae ; metabolism ; Cell-Derived Microparticles ; metabolism ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism

In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cells in vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHA in vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced into neural stem cells, and then differentiated into neuron-like cells in vitro.
Bone Marrow Cells/*cytology ; *Cell Differentiation ; Cells, Cultured ; Glial Fibrillary Acidic Protein/metabolism ; Neurons/*cytology ; Rats, Wistar ; Stromal Cells/cytology

AIMTo observe expressions and changes of Tau protein, pSer202 and Tau protein's contents during the differentiation process of bone-marrow mesenchymal stem cells (MSCs) into neural cells, and discuss Tau's effects on it.

METHODSEGF and bFGF were combined for the induction of 4th, 8th, and 12th-MSCs into neural cells. Expressions of Tau protein and pSer202 were tested by immunocytochemistry. ELISA assay was applied for testing Tau protein's contents during differentiation process.

RESULTSPositive rates of Tau protein in uninduced MSCs of 4th, 8th, and 12th-MSCs were under < 6%; After 14-day induction, the cellular morphologic characteristics in different passages were very similar to neurons, positive rates of Tau protein had no significant differences between passages (P > 0.05), but had differences with their uninduced groups (P < 0.05). There hadn't had expression of pSer202 in uninduced and induced groups of passages. ELISA assay indicated that there was an upward tendency in Tau protein's contents during the 14-day induction process, those in the 14th day had no significant differences between passages too (P > 0.05).

CONCLUSIONThe increase in Tau protein's expressions and its non-phosphorylated state may make for MSCs differentiating into normal neural cells and formation of neuronal processes.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Guinea Pigs ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; tau Proteins ; metabolism

OBJECTIVETo investigate the role of mesenchymal stem cells (BMSCs) in multiple myeloma (MM) bone marrow (BM) microenrivonment and their effect on myeloma cells survival and bortezomib induced apoptosis.

METHODSBMSCs were derived from BM of untreated myeloma patients (MM-BMSCs) and healthy donors (HD-BMSCs), respectively. The phenotype, proliferation time and cytokine secretion of MM-BMSCs were detected and compared with HD-BMSCs. Then BMSCs were co-cultured with myeloma cell line NCI-H929 and bortezomib in vitro. The NCI-H929 cells proliferation and bortezomib induced cell apoptosis were investigated.

RESULTSMM-BMSCs and HD-BMSCs were isolated successfully. The phenotype of MM-BMSCs was similar to that of HD-BMSCs. Expressions of CD73, CD105, CD44 and CD29 were positive, but those of CD31, CD34, CD45 and HLA-DR (< 1%) negative. The proliferation time of MM-BMSCs was longer than that of HD-BMSCs (82 h vs 62 h, P < 0.05). Moreover, over-expressions of IL-6 and VEGF in MM-BMSCs culture supernatant were detected as compared with that in HD-BMSCs [(188.8 ± 9.4) pg/ml vs (115.0 ± 15.1) pg/ml and (1497.2 ± 39.7) pg/ml vs (1329.0 ± 21.1) pg/ml, respectively]. MM- BMSCs supported survival of the myeloma cells NCI-H929 and protected them from bortezomib induced cell apoptosis.

CONCLUSIONSMM-BMSCs is benefit for myeloma cells proliferation and against cell apoptosis induced by bortezomib. Over-expression of IL-6 and VEGF maybe play a critical role in these effects.

Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Bortezomib ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Multiple Myeloma ; metabolism

The study was aimed to explore the influence of co-culture ex vivo of CD34+ cells from two units of cord blood (CB) on the homing-related adherent molecule expression of each other. Mesenchymal stem cells (MSCs) were obtained from human bone marrow. Two units of CB CD34+ cells were co-cultured on 12 Gy gamma-ray irradiated MSC layer. Their adherent molecule expressions were assessed by flow cytometry. The results showed that the purity of the isolated CD34+ cells was (98.25+/-0.93)%. After co-culture on MSC layer for 6 days, the proportion of CD34+ cells of each unit was dropped to (60.4+/-6.32)% and (60.2+/-5.12)% respectively, but there was no significant difference from the control groups. The expressions of CD44, CD62L, CD184 and CD26 on CD34+ cells of each unit remained unaffected. The expression of CD162 was downregulated and CD54 was first increased but then dropped to the level before co-culture. But there was no significant difference between the experimental and control groups. In conclusion, co-culture of CD34+ cells from two units of CB may have no effects on the adherent molecule expressions of each other.
Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion Molecules ; metabolism ; Coculture Techniques ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology

Bone Marrow Cells ; cytology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; Humans ; Myocardium ; pathology

OBJECTIVETo investigate the possibility of differentiation of human mesenchymal stem cells (hMSC) into epidemic cells in vitro.

METHODShMSCs were segregated from normal adult human bone marrow by Percoll solution (1.073 g/ml) , and were cultured, purified, and amplified to 3th passage in vitro. Then the hMSCs were randomly divided into control group ( with treatment of normal L-DMEM medium) and experimental group (with treatment of L-DMEM medium containing epidermal growth factor,insulin,tretinoin, calcium chloride). After 7 days of culture, the morphologic changes of hMSCs in the 2 groups were observed with inverted phase contrast microscope. The expressions of P63 and PCK of hMSCs were assessed with immunohistochemical methods.

RESULTSThe shape of hMSCs in experimental group became irregular or oblong in shape, while that in control group were still in spindle shape. Immunohistochemical results showed that hMSCs were P63 and PCK positive in the experimental group, while those in control group were negative.

CONCLUSIONHuman mesenchymal stem cells can differentiate into epidemic cell in vitro.

Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; Humans ; Keratins ; metabolism ; Membrane Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo screen and identify differentially expressed proteins of mesenchymal stem cells (MSC) during endothelial differentiation.

METHODSMSCs were induced to endothelial differentiation with vascular endothelial growth factor (VEGF) and epithelial growth factor (EGF) mixture. The whole cell proteins were extracted and isolated by two-dimensional gel electrophoresis. After gel was analyzed by Imagemaster 5.0 software, differentially expressed proteins were partially selected and identified by MALDI-TOF-MS.

RESULTSThe differentiated MSC highly expressed endothelial cells related markers, CD31, CD34 and FVIIIAg were 56.8%, 38.8% and 14.5% respectively by flow cytometer. Compared with the primary cultured MSC, the differentiated cells differentially expressed 91 proteins. Among the 19 identified proteins, 11 up-regulated and 8 down-regulated, which include cytoskeletal proteins, such as myosin, filamin, vimentin and vinculin; cell metabolism enzymes, such as ORP-150, ERO1-α, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, protein disulfide-isomerase A3, FAS and enolase 3; nuclear factors, such as TAR DNA binding protein, guanine nucleotide binding protein and hypoxia up-regulated protein 1; VEGF receptors, such as KDR and so on.

CONCLUSIONSCytoskeletal proteins, metabolism enzymes and KDR were all involved in endothelial differentiation of MSC. These proteins may be the regulatory targets for endothelial differentiation of MSC.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Proteome ; analysis ; Proteomics ; Rats ; Rats, Wistar