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

OBJECTIVETo establish an immortalized marrow mesenchymal stem cell line to facilitate advances in cartilage engineering research.

METHODSHuman telomerase reverse transcriptase (hTERT) cDNA was transferred into primary human marrow mesenchymal stem cells (hMSC) by retroviral vector pLEGFP-C1-hTERT. Subsequently G418 resistant cell clone was screened and expanded for further studies. hMSC biomarkers and hTERT expression were confirmed by examination. Transfected hMSC was induced to differentiate into chondrocyte using TGF-P1 and dexamethasone.

RESULTSUp-regulated hTERT expression was detected in transfected hMSC. hMSC-hTERT cells could be induced to differentiate into chondrocyte. Higher telomerase activity in transfected cells was maintained for 50 population doublings so far. Collagen II could be detected in induced transfected hMSC by immunocytochemical and hybridization in situ.

CONCLUSIONSEctopic expression of hTERT can effectively immortalize hMSC in vitro. Immortalized hMSC can be induced to differentiate into chondrocyte under certain condition. It may be an ideal target of further studies in cartilage engineering.

Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Transformed ; Chondrocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVETo review the recent developments in the mechanisms of glucocorticoids induced osteonecrosis of femoral head (ONFH) and introduce a new theory of ONFH.

DATA SOURCESBoth Chinese- and English-language literatures were searched using MEDLINE (1997 - 2011), Pubmed (1997 - 2011) and the Index of Chinese-language Literature (1997 - 2011).

STUDY SELECTIONData from published articles about mechanisms of glucocorticoids induced ONFH in recent domestic and foreign literature were selected. Data extraction Data were mainly extracted from 61 articles which are listed in the reference section of this review.

RESULTSGlucocorticoids are steroid hormones secreted by the adrenal cortex that play a pivotal role in the regulation of a variety of developmental, metabolic and immune functions. However, high dose of exogenous glucocorticoids usage is the most common non-traumatic cause of ONFH. Glucocorticoids can affect the metabolisms of osteoblasts, osteoclasts, bone marrow stromal cells and adipocytes which decrease osteoblasts formation but increase adipocytes formation and cause ONFH finally.

CONCLUSIONSGlucocorticoids affect the differentiation of mesenchymal stem cells, through activating or inhibiting the related transcript regulators of osteogenesis and adipogenesis. At last, the size and volume of mesenchymal stem cells derived adipocytes will increase amazingly, but the osteoblasts will be decreased obviously. In the meantime, the activity of the osteoclasts will be activated. So, these mechanisms work together and lead to ONFH.

Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Femur Head ; cytology ; Glucocorticoids ; metabolism ; pharmacology ; Humans ; Osteonecrosis ; etiology ; metabolism ; Stromal Cells ; drug effects ; metabolism

Hepatocyte growth factor (HGF) is one of major growth factors in the bone marrow microenvironments with which the proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells were closely contacted. However, its roles in the regulation of proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells remain unclear. This study was aimed to investigate the effect of HGF on biological characteristics of bone marrow-derived mesenchymal stem cells. Expression of c-Met, the receptor for HGF was detected by immunohistochemistry assay, cell proliferation was determined by MTT, activity of ALP was quantitatively assayed, cell migration and anoikis-induced MSC apoptosis were analyzed. The results showed that HGF not influenced the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Treatment of bone marrow-derived mesenchymal stem cells with recombinant human hepatocyte growth factor resulted in inhibition of anoikis-induced apoptosis. HGF significantly stimulated the migration of bone marrow-derived mesenchymal stem cells. Both PI-3 kinase and MAPK kinase were proved to be involved in HGF-induced migration. It is concluded that HGF/c-Met signal regulates the apoptosis and migration of bone marrow-derived mesenchymal stem cells.
Anoikis ; drug effects ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Proto-Oncogene Proteins c-met ; biosynthesis

UNLABELLEDIn this study, the roles of hematopoietic inhibitors elaborated by bone marrow endothelial cells in the proliferation and differentiation of hematopoietic progenitors were investigated. Murine bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and the components with > 10 kD and < 10 kD were obtained by centrifugal ultrafiltration. The effect of BMEC-CM and its components on proliferation of hematopoietic progenitors was evaluated by CFU-GM and HPP-CFC assay and antibody neutralization test. The expression of the inhibitors in BMEC and BMEC-CM was detected by RT-PCR and Western blot, and change of proliferation and differentiation-related genes during expansion of hematopoietic progenitors was examined by membrane hybridization technique.

THE RESULTS(1) When BME C-CM and its components directly were added to CFU-GM and HPP-CFC culture system, BMEC-CM had no effect on colony formation, > 10 kD component enhanced and < 10 kD component inhibited the formation of CFU-GM and HPP-CFC. (2) When BMEC-C M and its components were added to liquid culture system of marrow cells, after 24 hours incubation, CFU-GM decreased and HPP-CFC increased significantly in B MEC-CM group, CFU-GM increased and HPP-CFC had no significant change in > 10 kD component group; and both CFU-GM and HPP-CFC reduced in < 10 kD group. (3) MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha, IFN-gamma and T beta 4 were expressed in murine marrow endothelial cells, and MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha and T beta 4 were existed in BMEC-CM. (4) Antibody neutralization test results demonstrated that TGF-beta, MSP, MIP-1 alpha, IFN-gamma and T beta 4 existed in BMEC-CM had significant suppressive effects on CFU-GM and HPP-CFC. (5) T beta 4 combined with 5 hematopoietic cytokines (SCF, IL-3, IL-6, GM-CSF and EPO) added to CD34(+) cells expansion culture system, HPP-CFC significantly increased compared with 5 cytokines group. T beta 4 could downregulated the expression of proliferation and differentiation-related genes and signal transduction-related genes. It is concluded that BM EC-CM promotes the proliferation of early hematopoietic progenitor cells, and this effect is related with the inhibitors existed in BMEC-CM and it could be executed via influencing cell proliferation and differentiation-related genes and signal-related genes.

Animals ; Bone Marrow Cells ; metabolism ; Cell Division ; drug effects ; Cytokines ; genetics ; pharmacology ; Endothelium ; cytology ; metabolism ; Hematopoietic Stem Cells ; drug effects ; physiology ; Mice ; RNA, Messenger ; analysis ; Thymosin ; pharmacology

This study was aimed to investigate the growth-promoting activity of thrombin on mesenchymal stem cells (MSC) and its mechanisms. Human bone marrow MSC were cultured in serum-free medium supplemented with graded concentrations of thrombin, and the proliferation status of MSC was detected by MTT test. The expression levels of protease-activated receptors (PAR) and c-MYC gene were detected by PCR. Activated Akt signaling pathway was revealed by Western blot, and specific inhibitors of the signaling pathways were used to confirm the effects. The results showed that thrombin stimulated MSC proliferation in a dose-dependent manner; the minimal concentration of thrombin for stimulating MSC growth was 0.5 U/ml, and the promoting effect reached its maximum when thrombin at a dose of 8 U/ml was employed. PCR results showed that MSC expressed the two types of PAR1 and PAR2. After PAR1 was blocked with a specific inhibitor SCH79797, the growth-promoting effect of thrombin was inhibited, while this phenomenon was not observed when MSC were exposed to FSLLRY-NH2, a specific inhibitor for PAR2. Further experiments showed that after exposure to thrombin, the AKT signaling pathway in MSC was promptly activated, and c-MYC expression was greatly up-regulated. Meanwhile, when LY294002, a specific AKT inhibitor, was added into the culture medium, the up-regulation of c-MYC expression was reduced, accompanied by the low rate of MSC growth. It is concluded that thrombin can stimulate MSC proliferation by eliciting PAR1-mediated AKT activation and subsequent up-regulation of c-MYC expression.
Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Receptors, Thrombin ; metabolism ; Signal Transduction ; drug effects ; Thrombin ; pharmacology

OBJECTIVETo investigate the inhibition effect of leukemic bone marrow stromal cells (BMSCs) on daunorubicin (DNR) induced apoptosis of human Jurkat cell line, and analyze the differentially expressed genes between Jurkat cells cocultured with leukemic BMSCs or without.

METHODSSuppression subtractive hybridization (SSH) was employed to establish subtracted cDNA library of differentially expressed genes in Jurkat cells cocultured with leukemic BMSCs and DNR. The cDNA fragments were sequenced and analyzed.

RESULTSThe differentially expressed gene cDNA library was successfully developed. Primary screening was done by reverse Northern hybridization. Thirty up-regulated and 22 down-regulated cDNA fragments were isolated and sequenced. Analysis and comparison were performed in GenBank using BLAST. These genes are related to cell cycle regulation, cell apoptosis and energy metabolism.

CONCLUSIONLeukemic BMSCs influence gene expression of Jurkat cells. The resulting differentially expressed genes might be associated with the protection of leukemic cells by BMSCs from injury.

Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; metabolism ; pathology ; Cells, Cultured ; Coculture Techniques ; Daunorubicin ; pharmacology ; Gene Expression Profiling ; Gene Expression Regulation, Leukemic ; drug effects ; Gene Library ; Humans ; Jurkat Cells ; Stromal Cells ; drug effects ; metabolism ; pathology

OBJECTIVETo explore the effect of Chinese herbs for Shen invigorating and blood activating (CHSIBA) on the number of endothelial progenitor cells (EPCs) in the bone marrow and the peripheral blood and the signaling pathway of bone marrow matrix metalloproteinase 9 (MMP-9) of the myocardial infarction (MI) model rats.

METHODSThe MI rat model was established by ligation. Thirty successfully modeled rats were randomly divided into the high dose CHSIBA group, the low dose CHSIBA group, and the model group, 10 in each group. Besides, another 10 normal rats were recruited as the blank group. Rats in the high dose CHSIBA group and the low dose CHSIBA group were administered with CHSIBA at 3 g/kg and 1.5 g/kg body weight by gastrogavage (by adding them in 4 mL physiological saline), once daily. Rats in the model group and the blank group were administered with 4 mL physiological saline once daily. The EPCs were collected from the bone marrow and the peripheral blood 4 weeks later. Seven days later the CD34/CD133 phenotype was identified in collected sticking wall cells using flow cytometry. The MMP-9 and water soluble Kit ligand (sKitL) were detected using Western blot. The expressions of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1alpha (SDF-1alpha) were detected using ELISA.

RESULTSThe CD34/CD133 positive rate and the EPC quantity in the bone marrow and the peripheral blood were higher in the high dose CHSIBA group and the low dose CHSIBA group than in the model group (P < 0.05, P < 0.01). Besides, the expressions of VEGF, SDF-1alpha, MMP-9, and sKitL in the bone marrow and the peripheral blood were also higher in the high dose CHSIBA group and the low dose CHSIBA group than in the model group (P < 0.05, P < 0.01).

CONCLUSIONCHSIBA could activate MMP-9 signaling pathway, increase its upstream and downstream signal expression levels, and mobilize EPCs in the bone marrow to enter the blood circulation.

Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Myocardial Infarction ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stem Cells ; drug effects ; metabolism

Transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) are abundant proteins in the bone matrix. However, their interaction in controlling osteoblast differentiation is not clearly understood. In this study, HBMSCs were cultured in collagen gel matrix with different condition of exogenous rhBMP-2 and TGF-beta1 in order to determine the interaction of BMP-2 and TGF-beta1 on human bone marrow stromal cells (HBMSCs) differentiation. The cultured cells were analyzed for cell proliferation, alkaline phophatase (ALP) activity and mineralization stainning with Von-Kossa. The cells treated with TGF-beta1 exhibited a higher rate of cell growth than those without. However, the cells cultured in collagen gel matrix showed a lower rate of cell growth than the cells cultured in a monolayer. To investigate the effects of both cytokines on osteoblast differentiation, the cells were treated with 0, 1, 5, 10 ng/ml of TGF-beta1 for 2 days. This was followed by culturing with 0, 1, 5, and 10 ng/ml of TGF-beta1 and 100 ng/ml of rhBMP-2 together for 3 days with the alkaline phosphatase (ALP) activity measured. The cells treated with 1 ng/ml of TGF-beta1 responded efficiently to rhBMP-2 and expressed ALP activity with a level equivalent to that exhibited by cells that were not treated with TGF-beta1. The cells treated with 5 and 10 ng/ml of TGF-beta1 showed a dramatic decrease in ALP activity. The cells treated with 10ng/ml of TGF-beta1 followed by rhBMP-2 alone exhibited an intermediate ALP activity. The cells treated with 100 ng/ml of rhBMP-2 demonstrated Von-Kossa positive solid deposits after 3 weeks, while there were few Von-Kossa positive solid deposits when the cells treated with 10 ng/ml of TGF-beta1. These results show that TGF-beta1 inhibits the effects of rhBMP-2 on the osteoblast differentiation of HBMSCs in a dose dependant manner. Furthermore, the effects of TGF-beta1 on HBMSCs are reversible. This suggest that TGF-beta1 and rhBMP-2 are coordinately controlled during the osteoblast differentiation of HMBSCs.
Alkaline Phosphatase/metabolism ; Bone Marrow Cells/*drug effects ; Bone Morphogenetic Proteins/*pharmacology ; Cell Differentiation/drug effects ; Cell Division/drug effects ; Cells, Cultured ; Collagen/*pharmacology ; Human ; Recombinant Proteins/pharmacology ; Stromal Cells/*drug effects ; Transforming Growth Factor beta/*pharmacology

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.
Anemia, Aplastic ; metabolism ; Apoptosis ; drug effects ; Autophagy ; Bone Marrow Cells ; cytology ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Signal Transduction ; Sirolimus ; pharmacology