To develop the methodology to purify and culture expand mesenchymal stem cells (MSC) from human bone marrow and investigate the optimal system for MSC differentiation into chondrocytes mononuclear cells were harvested by gradient centrifugation on Percoll at density of 1 073g/ml and seeded in low glucose DMEM containing 10% fetal calf serum. The purity of cells was evaluated by flow cytometric technique. MSC of 2 passages thereafter were absorbed into a biomaterial of gelatin sponge and induced to differentiate in to chondrocytes for one week under the influence of TGF ? and other inductive agents. The feature of chondrocytes in engineered tissues was identified by toluidine blue staining at various time points. The results showed that human mesenchymal stem cells culture expanded were positive for CD166, CD29, and CD44, but negative for CD34, CD45, and HLA DR. Furthermore, when treated cells absorbed into the biomaterial were implanted subcutaneously into BALB c nude mice, they formed cartilage like tissues after 4 weeks.Our conclusions is that cultured marrow MSC have unique biological features and the capacity to differentiate into chondrocytes in vivo , so they are useful for cartilage engineering by serving as the seed cells.

Objective To investigate the heterogeneity of immunomodulatory function of exosomes secreted from human umbilical cord-derived mesenchymal stem cells(hUC-MSC).Methods Five different hUC-MSCs lines were isolated and cultured.Exosomes were isolated from the supernatant.The expression of specific surface markers CD9,CD63,CD81 and CD44 was detected by flow cytometry.The protein concentration of hUC-MSCs exosomes(hUC-MSCs-ex)was evaluated by the BCA assay.Concanavalin A and rhIL-2 stimulated umbilical cord blood mononuclear cells (UBMCs) from healthy donor were co-cultured at different concentrations of hUC-MSCs-ex from different cell lines for 72 h.The growth rate of UBMCs was detected by MTT assay.ELISA was used to test the levels of IFN-γ and TNF-α of the supernatants.The UBMCs co-cultured with hUC-MSCs were co-cultured with K562 cells at the ratio of 5∶1.The cytotoxic activity was calculated by MTT assay.Results hUC-MSCs-ex expressed CD9,CD63,CD81and CD44.Different hUC-MSCs lines had different regulating activities on the proliferation,secretion and killing ability of UBMCs.Conclusion hUC-MSCs-ex has heterogeneity of immunomodulatory function on UBMCs.

BACKGROUND: Using bone marrow mesenchymal stem cells (MSCs) infected by an adenoviral vector cen-ying human hepatocyte growth factor (HGF) gene, which was independently developed by our laboratory and has been approved by SFDA in clinical trial to repair bone defects via cell therapy.OBJECTIVE: To investigate the bone restoration effect of mssenchymal stem cell and hepatocyte growth factor in the pathophysiological course of femoral head osteonecresis defects.DESIGN, TIME AND SETTING: An in vivo experiment of ceUular-rnatedal science. The experiment was performed at the Beijing Institute of Radiation Medicine and the 66400 Orthopaedic Hospital of Chinese PLA from September to December 2007. MATERIALS: Eighteen New Zealand rabbits with clean grade, aged 26-28 weeks, were supplied by Beijing Kaiyuan rabbit livestock farm. Bone matrix gelatin was purchased from Shanghai Xiaobo Technological Development Limited Co., Ltd. METHODS: MSCs from New Zealand white rabbit were isolated and culture-expanded by adhesion method and their in vitro ostengenesis and adipogenesis were identified. Eighteen New Zealand white rabbits were created femoral heads defect models and randomly divided into 3 groups, with 6 animals in each group. Animals in each group were treated with scaffold of bone matrix gelatin (27 mm3 per defect) only, scaffolds seeded with MSCs (1×107 per defect) or MSCs infected by an adenoviral vector carrying human hepatocyte growth factor gene (MSC/HGF, 1×107 per defect), respectively. Histological examination was conducted at 3 months post operation. MSCs or MSC/HGF were labeled with carboxyfluorescein diacetate succinmidyl ester dye, treated with cobalt chloride for 72 hours and their proliferative status was evaluated and compared by flow cytomatric techniques. MAIN OUTCOME MEASURES: The differentiation of MSCs, therapeutic effect of MSCs in treating femoral head necrosis, and potent ability of MSCs/HGF against hypoxia.RESULTS: The adherent cells could differentiate into osteoblasts and adipocytes under in vitro inductive condition. Histological examination revealed that the bone defects from both control and MSCs-treated groups were fille:d with fibrous tissue, though blood vessels were evident in MSCs-treated group, whereas new bony tissues were obvious in MSC/HGF group. The result was further confirmed by Lane-Sandhu scaling, which indicated that new bone formation was more evident in MSC/HGF-treated group compared with MSCs-treated or control group (P< 0.01). Flow cytometry analysis showed that the proportion of MSC/HGF-treated group that had experienced cell division was significantly higher than that of MSCs-treated group after cobalt treatment (P < 0.001).CONCLUSION: MSC/HGF exhibit greater osteogeneeis in vivo in this model compared their counterparts, which might be attributed to their resistance to hypoxic injury. The results here suggest that HGF gene modification might be an optional strategy for the application of MSCs in the management of avascular osteonecrosis.

To explore the effect of icariside ? on osteoprotegerin(OPG) expression in osteoblasts, primary osteoblasts were cultured with different concentration of icariside ? and 17?estradiol. As a major metabolite of icarrin in vivo, icariside ? exerted a better effect on OPG expression which plays a key role in osteoporosis. 20 ng/dl icariside ? also exerted better effect on OPG secretion than that of positive control group. Consequently, icariside ?could be a candidate for osteoporosis treatment.

In this study, the underlying antileukemic mechanisms of homoharringtonine (HHT) were investigated. K562 cell line was used to observe the effects of HHT on the induction of apoptosis and on the expression of the specific chimeric protein P210(bcr/abl), as evaluated by flow cytometric annexin V-PI dual labeling technique and Western blot. The results showed that HHT induced K562 cells to apoptotic death at the concentrations of 5 - 20 ng/ml, and some of the cells became necrotic when exposed to a higher concentration. The amount of P210(bcr/abl) oncoprotein was decreased by approximately 70% when the cells were exposed to HHT for 48 hours, however, that of its partner P145(c-abl) proto-oncoprotein was not affected. It is clear from the study that HHT is an inhibitor of P210(bcr/abl) oncoprotein and therefore promotes the apoptosis of CML cells. It could be promising that HHT be used extensively in the chemotherapy of patients with CML.

BACKGROUND:Recently,application of stem cells and growth factor to promoting lung regeneration in repair of emphysema lesion has been a hot focus in study.Thus,it is worth to pay attention on whether stem cells carrying relevant foreign growth factor gene can repair emphysema lesion.OBJECTIVE:To evaluate the effidency of adenovirus vector mediated green fluorescence protein(Ad-GFP)transfecting bone marrow mesenchymal stem cells(BMSCs)and its effect on the cell proliferation,to explore oriented migration of intravenously administrated BMSCs transfected with Ad-GFP in the lung tissues of pulmonary emphysema rats.METHODS:MSCs were separated and purified from the bone marrow of rats by density gradient centrifugation and by adherence.At different multiplicity of infection(MOI),transfection efficiency was observed by laser confocal microscopy.At 48 hours of transfection,MTT method was used to evaluate the proliferation of MSCs.A total of 16 Wistar rats were randomly divided into emphysema model group and control group(n=8).Model rats were established by exposure to cigarette smoke.MSCs,transfected with Ad-GFP,were grafted into the body of rats via tail vein.Lungs derived at 24 hours after implantation,and frozen sections were made.Migration and survival of MSCs in the lung tissues were observed by fluorescence microscopy.RESULTS AND CONCLUSION:MSCs from Wistar rats were successfully cultured,grew well and infected by Ad-GFP.The highest transfection effincincy(88.42 %)could be achieved at MOI of 200.Green fluorescent protein labeling had little effect on proliferation of MSCs by different MOI(P＞0.05).At 24 hours posttransplantation,the green fluorescence-positive tissue was Found in the lung tissues of emphysema model group and control group.Compared with control group,the expression of GFP in lung tissues was higher in emphysema model group(P＜0.05).These suggested that introduction of target gene cannot affect proliferation and homing property of BMSCs.

The generation of large quantities of novel human T cell clones ex vivo would make a wide range of gene-and immuno-therapies for tumor and AIDS possibly. Although it is well established that T cells are derived from CD34(+) cells, the involvement of thymic fragments from either human or murine fetus makes the in vitro T cell perliferation very cumbersome. In this report, cord blood mononuclear cells were used as accessory cells to support the differentiation of CD34(+) cells into naive T cells stimulated with SCF and IL-2. CD4(+) and CD8(+) T cells, under the cultural conditions, were continuously produced in vitro at least over a period of 3 weeks and their ratios changed gradually. CD4/CD8 double positive T cells and RAG-2 gene were existed, and RAG-2 gene, reponsible for TCR rearrangement, was expressed during the cell proliferation. Our study presents a simple culture system in vitro to acquire large quantities of naive T cell clones.

Transfer of drug resistance genes into hematopoietic cells is an attractive approach to protect hematopoietic system from the toxic effects by chemotherapeutic agents in cancer patients. In this study, transduction of mdr-1 in combination with dihydrofolate reductase (dhfr) gene was performed, and the expression of exogenous genes and chemoprotection capacity in mouse bone marrow cells were observed. The results showed that approximately 15% of bone marrow cells transfected with the retroviral vector expressed mdr-1 as assayed by flow cytometry. Gene transfer resulted in about 0.9 - 13 fold and 0.5 - 2.6 fold increase in the resistance of CFU-GM to taxol and methotrexate in vitro, respectively (P < 0.05). Moreover, seven months after transplantation to syngeneic mice with mdr-1 and dhfr-transfected bone marrow cells, peripheral blood cells in recipients were still positive for gp170 as evaluated by FACS as well as for mdr-1 and dhfr by PCR amplification. These results indicate that hematopoietic progenitors can be transfected by retrovirus containing mdr-1 and dhfr genes, and that functional drug resistance accompanies their expressions. Furthermore, genetic chimerism might exist in hematopoietic stem cells. In conclusion, transfer and expression of mdr-1 and dhfr genes in bone marrow cells might be applicable in gene therapy research in cancer patients.

Objective This study aimed to establish a reliable primary culture protocol for preparing murine spleen-derived mesenchymal stem cells ( MSCs) by tissue explant culture.Methods Healthy mouse spleens were crushed by syringe handle to harvest spleen mesenchymal tissues.Then the tiny pieces of spleen tissue were digested by collagenase II before seeded into culture flasks.The morphological characteristics of spleen tissue-derived cells were observed under the inverted microscope.Further, the surface antigen profile of the cells was analyzed by flow cytometry (FACS).The cells were induced to differentiate into osteoblasts and adipocytes.Results The murine spleen-derived MSCs exhibited a spindle-shaped appearance.The FACS results showed that the spleen-derived MSCs highly expressed CD29, CD44, CD105 and Sca-1, but weakly expressed CD11b, CD34, CD45 and Ia. In addition, the spleen-derived MSCs steadily differentiated into osteoblasts and adipocytes in the induction medium.Conclusions A method of primary culture of murine spleen-derived MSCs by explant culture is successfully established.The harvested MSCs exhibit high purity and cell proliferation ability, and provide a reliable cell model for related researches.

BACKGROUND:Spinocerebel ar ataxia is a inherited neurodegenerative disease with progressive cerebel ar masonic movement disorders as the main clinical manifestation. So far, no drug is available to control the disease progression. OBJECTIVE:To observe the clinical effect of umbilical cord mesenchymal stem cells in treating spinocerebel ar ataxia by intrathecal injection. METHODS:Thirty-eight cases of spinocerebel ar ataxia were given umbilical cord mesenchymal stem cells by intrathecal injection, 1×106/kg once a week, four times as a course. These 38 cases received 52 courses. Among them, 27 cases received 1 course, 8 cases received 2 courses and 3 cases received 3 courses. International Cooperative Ataxia Rating Scale (ICARS) and Activity of Daily Living Scale (ADL) were used to evaluate patients’ neural functions (the greater scores, the more severe damage) and ability of daily living (the lower score, the stronger the ability of daily living). After treatment, al patients were subjected to fol ow-up visit. RESULTS AND CONCLUSION:The total effective rate of 52 courses of treatment was 84.62%. ICARS and ADL scores were significantly decreased at 1 month after treatment (P<0.01). In most of effective patients, unstable walking and standing, slow movement, upper limb fine motor disorder, writing difficulties, dysarthria, eye movement disorders were improved. After treatment, common adverse effects were dizziness (1 case), low back pain (2 cases), headache (1 case), and fever (2 cases). Al these symptoms disappeared within 1-3 days. No treatment-related adverse events happened in the median fol ow-up of 39 months (11-59 months). The il ness of effective patients had been stable for 1-19 months, average (5.95±4.84) months. Intrathecal injection of umbilical cord mesenchymal stem cells is safe to ameliorate clinical symptoms to some extent within a certain time. It may delay the progression of spinocerebel ar ataxia. Multiple courses of treatment can help to further improve neurological function in most patients.