OBJECTIVETo construct a co-culture system for bone marrow mesenchymal stem cells (BMMSC) and multiple myeloma (MM) cells, and to investigate the effects of co-cultured BMMSC on the migrating and homing of multiple myeloma cells.

METHODSThe BMMSC from the transgenic mice with green fluorescent protein (GFP) fetal bone were cultured by adherent screening. A co-culture system of BMMSC and MM cell line XG-7 cells was constracted, the proliferation and apoptosis of cells were determined by trypan blue exclusion and Annexin V/PI, respectively, MDC staining was employed to detect the autophagy. The moving direction distribution of molecule in BMMSC and XG-7 cells labeled with PE-CD138 in co-culture process were observed dinamically by confocal microscopy.

RESULTSAfter co-culture with GFP-BMMSC, the resistance of XG-7 cells to apoptosis and autophagy were enhanced; at the same time, their proliferation increased. Apoptosis rates of XG-7 cells directly and indirectly co-cultured with BMMSC were (6.23 ± 0.12)% and (6.97 ± 0.03)% respectively, which were lower than that of XG-7 cells cultured alone (17.90 ± 1.46)% (P < 0. 01). There was low level of autophagy in XG-7 cells co-cultured with BMMSC. XG-7 cells are highly polarized and contained a specialized membrane domain with specific protein and lipid components to contact with BMMSC under confocal microscope. After methyl-β-cyclodextrin treatment, the molecules were normally enriched in the specialized domain.

CONCLUSIONBMMSC can protect XG-7 cells from apoptosis and autophagy, and obviously promote the proliferation of XG-7 cells, and can influence the migrating and homing of multiple myeloma cells.

Animals ; Apoptosis ; Autophagy ; Bone Marrow Cells ; cytology ; Cell Line, Tumor ; Cell Movement ; Coculture Techniques ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Transgenic ; Multiple Myeloma ; pathology

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 investigate whether plasma from patients with systemic lupus erythematosus (SLE) inhibits the suppressive effects of mesenchymal stem cells (MSCs) on lupus B lymphocytes.

METHODSMSCs isolated and expanded from the bone marrow of healthy donors were co-cultured with B cells purified from the peripheral blood of SLE patients in the presence of fetal bovine serum or pooled plasma from SLE patients, and the proliferation and maturation of the B lymphocytes were analyzed.

RESULTSs Co-culture with normal MSCs obviously inhibited the proliferation of lupus B cells and suppressed the maturation of B lymphocytes, which showed lowered expressions of CD27 and CD38. The pooled plasma from SLE patients significantly inhibited the suppressive effects of normal MSCs on B cell proliferation and maturation.

CONCLUSIONPlasma from SLE patients negatively modulates the effects of normal MSCs in suppressing lupus B cell proliferation and maturation to affect the therapeutic effect of MSC transplantation for treatment of SLE. Double filtration plasmapheresis may therefore prove beneficial to enhance the therapeutic effects of MSC transplantation for SLE.

B-Lymphocytes ; pathology ; Cell Proliferation ; Coculture Techniques ; Humans ; Lupus Erythematosus, Systemic ; blood ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; Plasma

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 simulate the chemical microenvironment of injured brain tissue, and to explore the effect of this chemical microenvironment on temperature sensitive umbilical cord mesenchymal stem cells (tsUC).

METHODSRat models of traumatic brain injury (TBI) were made by fluid percussion injury, and then the brain tissue extracts of the injured regions were acquired. Human umbilical cord mesenchymal stem cells (UC) were isolated and cultured, and the tsUC were obtained through the infection of temperature-sensitive Simian 40 Large T- antigen (ts-SV40LT) retrovirus. After that, both the two kinds of cells were cultured on the polyacrylamide gels which mimicking the elastic modulus of brain. Four groups were included: UC cultured under normal temperature (UC group), UC cultured added brain tissue extract under normal temperature (UC plus extract group), tsUC cultured under mild hypothermia (tsUC group), and tsUC added brain tissue extract under mild hypothermia for 3 days, then normal temperature for 4 days (tsUC plus extract group). After 24 hours, the apoptosis level was checked. Cell growth and morphological changes in each group were given dynamic observation. Seven days later, cell immunofluorescences were implemented for examining neural differentiation level.

RESULTSCompared with UC plus extract group, the apoptosis and proliferation in UC plus extract group were significantly reduced (P < 0.01) and increased (P < 0.01) respectively. Cell immunofluorescence showed that the both GFAP and Neuron positive cells were significantly enhanced in UC plus extract group than those in tsUC plus extract group.

CONCLUSIONtsUC combining with mild hypothermia could significantly reverse injury induced cell apoptosis, improve cell proliferation and neural differentiation under chemical microenvironment after brain injury, which confirmed the adaptation and resistance of tsUC under mild hypothermia after TBI.

Animals ; Apoptosis ; Brain ; cytology ; pathology ; Brain Injuries ; pathology ; Cell Proliferation ; Humans ; Mesenchymal Stromal Cells ; chemistry ; Neurons ; cytology ; Rats ; Temperature ; Umbilical Cord ; cytology

Proangiogenic cells (PACs) display surface markers and secrete angiogenic factors similar to those used by myelomonocytic cells, but, unlike myelomonocytic cells, PACs enhance neovascularization activity in experimental ischemic diseases. This study was performed to reveal the differential neovascularization activities of PACs compared with those of myelomonocytic cells. We cultured PACs and CD14+-derived macrophages (Macs) for 7 days. Most of the surface markers and cytokines in the two cell types were alike; the exceptions were KDR, beta8 integrin, interleukin-8 and monocyte chemotactic protein-1. Unlike Macs, PACs significantly enhanced mesenchymal stem cell (MSC) transmigration. PACs and Macs increased neovascularization activity in an in vitro co-culture of human umbilical vein endothelial cells and MSCs and in an in vivo cotransplantation in Matrigel. However, the use of Macs resulted in inappropriately dilated and leaky vessels, whereas the use of PACs did not. We induced critical hindlimb ischemia in nude mice, and then transplanted PACs, Macs or vehicle into the mice. We obtained laser Doppler perfusion images weekly. At 2 weeks, mice treated with PACs showed significantly enhanced perfusion recovery in contrast to those treated with Macs. After day 7, when cells were depleted using a suicidal gene, viral thymidine kinase, to induce apoptosis of the cells in vivo by ganciclovir administration, we found that the improved perfusion was significantly abrogated in the PAC-treated group, whereas perfusion was not changed in the Mac-treated group. PACs caused an increase in healthy new vessels in in vitro and in vivo models of angiogenesis and enhanced long-term functional neovascularization activity in the hindlimb ischemia model, whereas Macs did not. Nevertheless, the angiogenic potential and long-term functional results for a specific cell type should be validated to confirm effectiveness and safety of the cell type for use in therapeutic angiogenesis procedures.
Animals ; Bone Marrow Cells/cytology ; Cells, Cultured ; Cytokines/analysis ; Human Umbilical Vein Endothelial Cells ; Humans ; Ischemia/*pathology ; Macrophages/*cytology/pathology ; Male ; Mesenchymal Stromal Cells/*cytology/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neovascularization, Pathologic/*pathology ; *Neovascularization, Physiologic

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

Bone marrow mesenchymal stem cells (MSCs) transplantation could repair injury tissue, but no study confirms whether MSCs can promote the proliferation of endogenous lung stem cells to repair alveolar epithelial cells of mice with chronic obstructive pulmonary disease (COPD). This study was designed to investigate the effect of MSCs on the proliferation of endogenous lung stem cells in COPD mice to confirm the repair mechanism of MSCs. The mice were divided into control group, COPD group, and COPD+MSCs group. The following indexes were detected: HE staining of lung tissue, the mean linear intercept (MLI) and alveolar destructive index (DI), the total cell number in bronchoalveolar lavage fluid (BALF), pulmonary function, alveolar wall apoptosis index (AI) and proliferation index (PI), the number of CD45(-)/CD31(-)/Sca-1(+) cells by flow cytometry (FCM), and the number of bronchoalveolar stem cells (BASCs) in bronchoalveolar duct junction (BADJ) by immunofluorescence. As compared with control group, the number of inflammatory cells in lung tissue was increased, alveolar septa was destroyed and the emphysema-like changes were seen, and the changes of lung function were in line with COPD in COPD group; AI of alveolar wall was significantly increased and PI significantly decreased in COPD group. There was no significant difference in the number of CD45(-)/CD31(-)/Sca-1(+) cells and BASCs between control group and COPD group. As compared with COPD group, the number of inflammatory cells in BALF was decreased, the number of CD45(-)/CD31(-)/Sca-1(+) cells and BASCs was increased, AI of alveolar wall was decreased and PI was increased, and emphysema-like changes were relieved in COPD+MSCs group. These findings suggested that MSCs transplantation can relieve lung injury by promoting proliferation of endogenous lung stem cells in the cigarette smoke-induced COPD mice.
Animals ; Cell Proliferation ; Lung ; pathology ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Pulmonary Disease, Chronic Obstructive ; pathology ; therapy

This study was aimed to explore the regulation of arsenic trioxide (As₂O₃) on imbalance between adipogenic and osteogenic differentiation of BM-MSC from patients with aplastic anemia(AA). The BM-MSC from AA patients were separated and purified, placed into the adipogenic and osteogenic differentiation culture system, then added the As₂O₃, CsA, As₂O₃combined with CsA were added to corresponding differentiation culture system, the concentration of As₂O₃and CsA were set at 0.001 µmol/ml and 2.5 mmol/ml respectively, the cells were divided into As₂O₃group, the CsA group, combined group and control group (no drug). The cell morphological observation, oil red 'O' staining, Von-Kossa staining, and RT-PCR were used to detect corresponding differentiation marks. The results indicated that in respect to adipogenic differentiation, cellular morphology observing and oil red 'O' staining showed that the rate of adipocyte differentiation in As₂O₃group was (18.3 ± 1.9)%, which was lower than the (91.8 ± 2.7)% in the CsA group and (92.1 ± 1.2)% in control group (P < 0.05), there was no significant difference in comparison with (8.3 ± 1.9)% in the combined group (P > 0.05), but the rate of differentiation in CsA group was higher than that in combined group (P < 0.05), and there was no significant difference in comparison wtih control group. RT-PCR showed that the LPL-mRNA expression level in As₂O₃group were significantly lower than that in the CsA group and the control group (P < 0.05), no difference was observed while compared with the combined group (P > 0.05), but the LPL-mRNA expression level in CsA group was significantly higher than that in the combined group (P < 0.05). In terms of osteogenetic differentiation, the calcium deposition in As₂O₃group and combined group was obviously observed while rarely in the CsA group and the control group when detected by the Von-Kossa staining. OST-mRNA expression level in As₂O₃group were higher than that in CsA group and the control group (P < 0.05), while compared with the combined group, there was no significant difference (P > 0.05), but the OST-mRNA expression level in the CsA group was lower than that in the combined group (P < 0.05). It is concluded that As₂O₃can significantly enhance the ability of BM-MSC from AA patients to differentiate into osteoblast, also can inhibit the adipogenic differentiation, in contrast, the CsA can not promote the osteoblast differentiation of BM-MSC from AA patients.
Adipocytes ; cytology ; drug effects ; Anemia, Aplastic ; pathology ; Arsenicals ; pharmacology ; Bone Marrow ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Osteoblasts ; Osteogenesis ; drug effects ; Oxides ; pharmacology

The purpose of this study was to investigate the effect of bone marrow mesenchymal stem cells (BMMSC) from patients with chronic myeloid leukemia (CML) in blastic phase (Bp) on K562 cells and the primary CML-Bp cells, and to explore its potential mechanisms. K562 cells and primary CML-Bp cells were co-cultured with BMMSC of different groups; the cell proliferation was detected by MTT method, the cell apoptosis rate and mitochondrial membrane potential were measured by flow cytometry, the expression levels of Caspase-8, Caspase-9, and activated Caspase-3 in cells were measured by Western blot. The results showed that the CML-Bp BMMSC could enhance the survival rate of K562 cells treated with adviamycin (ADM) and display protective effect on K562 cells and primary CML-Bp mononuctear cells, inhibited ADM-induced leukimia cell apoptosis (P < 0.05); as compared with CML-chronic phase (CML-Cp) BMMSC and normal BMMSC, the CML-Bp BMMSC showed the highest protective effect on leukemic cells, the mitochondrial membrane potential of co-cultured cells slightly droped (P < 0.05). In the CML-Bp BMMSC cultured with K562 cells, the expression level of caspase-3 was more down-regulated than that in K562 alone plus ADM group, while the expression of caspase-9 significantly increased (P < 0.05). It is concluded that the CML-Bp BMMSC down-regulates ADM-induced leukemia cell appoptosis, its mechanism may relate with the inhibition of mitochondrial membrane potential drop, the stabilization of unactive expression of caspase-9 and down-regulation of caspase-3 expression.
Apoptosis ; Bone Marrow ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Mesenchymal Stromal Cells ; cytology ; metabolism