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

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 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

OBJECTIVETo research the effect of icariin (ICA) on the proliferation and differentiation of bone mesenchymal stem cells (BMSCs) and to study its influence on the expressions of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) in the progress of BMSCs differentiating into osteoblast, for providing an experimental evidence to explain the mechanism of ICA, also for exploring the feasibility of establishing a platform upon TGF-beta, and BMP-2 to screen out the medicine in preventing and treating osteoporosis.

METHODSAfter the most effective concentration of ICA for promoting the differentiation of BMSCs into osteoblast was judged with the indices like alkaline phosphatase (ALP), etc., a grouped experiment was conducted for the sake of studying the effect and mechanism of ICA in its process of inducing BMSCs differentiation into osteoblast through detecting expression of ALP and calcium nodes, as well as the expressions of TGF-beta1, and BMP-2 with ELISA.

RESULTSThe most effective concentration of the ICA on the BMSCs differentiation was judged as 1 x 10(-9) mol/L, ICA of that concentration showed effects in enhancing the expressions of osteoblast-indices and increasing the secretion of TGF-beta1, and BMP-2. Besides, the increase of TGF-beta1, and BMP-2 was revealed in all the groups, in which ICA showed its influence visibly.

CONCLUSIONICA could promote the differentiation of BMSCs into osteoblast; the up-regulation of TGF-beta1, and BMP-2 expressions is possibly one of the action mechanisms of various interventional drugs in their differentiation promoting progress.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Flavonoids ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Osteoblasts ; cytology ; Rabbits ; Transforming Growth Factor beta1 ; metabolism ; Up-Regulation

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

OBJECTIVETo explore the role of bone morphogenetic protein-7 (BMP-7) in strontium ranelate (Sr)-induced osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

METHODSBMSCs were isolated from 4-week-old rats and cultured in vitro. The third or fourth passages of BMSCs were examined using alkaline phosphatase kit for changes in ALP activity in response to treatment with different concentrations of Sr. Calcium nodules in the induced cells were detected using alizarin red staining, and the cellular BMP-7 expression was detected by Western blotting.

RESULTSWithin the concentration range of 0.1-3.0 mmol/L, Sr dose-dependently increased ALP activity in rat BMSCs. ALP activity reached the highest level after treatment with 3 mmol/L Sr, which also significantly promoted the formation of calcium nodules. Within the range of 0.1-3.0 mmol/L, Sr dose-dependently enhanced the expression of BMP-7, and its peak expression occurred following 3 mmol/L Sr treatment. Noggin (100 ng/ml), an inhibitor of BMP-7, obviously suppressed Sr-induced over-expression of BMP-7 and reduced ALP activity and calcium nodule formation in the BMSCs.

CONCLUSIONSr promotes osteogenic differentiation of rat BMSCs by increasing the expression of BMP-7.

Animals ; Bone Density Conservation Agents ; pharmacology ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Organometallic Compounds ; pharmacology ; Osteoblasts ; cytology ; Osteogenesis ; drug effects ; Rats ; Thiophenes ; pharmacology

OBJECTIVETo explore the role of the basic fibroblast growth factor (bFGF) in the directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into Leydig cells.

METHODSAfter purification and identification, we inoculated the third-generation BMSCs of SD rats onto a six-orifice board and then randomly divided them into groups A (normal saline control), B (human chorionic gonadotropin [hCG] + platelet-derived growth factor [PDGF] induction), C (hCG + PDGF + 5.0 ng/ml bFGF induction), D (hCG + PDGF + 10.0 ng/ml bFGF induction), and E (hCG + PDGF + 20.0 ng/ml bFGF induction). On the 7th, 14th and 21st day of induction, we observed the morphological changes of the cells and measured the level of testosterone (T) and expression of 3 beta hydroxy steroid dehydrogenase (3β-HSD) in the supernatant by immunofluorescence staining.

RESULTSAfter induction, the BMSCs of groups B, C, D, and E exhibited microscopic features of enlarged size, inter-connection, long-shuttle or irregular shape, adherent growth, and large round nuclei, all characteristic of Leydig cells. With the prolonging of time and enhanced concentration of bFGF, gradual increases were observed in the T level and the count of 3β-HSD-positive BMSCs in the four induction groups, with statistically significant differences between group B and groups C, D, and E (P < 0.05), as well as between group C and groups D and E (P < 0.05), but not between D and E (P > 0.05).

CONCLUSIONThe bFGF has an obvious promoting effect in the in vitro induced differentiation of rat BMSCs into Leydig cells.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; Cells, Cultured ; Chorionic Gonadotropin ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Leydig Cells ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism

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

We hypothesized that the formation and differentialtion of osteoclasts are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow in the early stage of stroke. We randomly divided white female Sprague-Dawley (SD) rats (n = 30) into two groups, stroke (n = 15) and sham group (n = 15). On the 7th day after stroke, after cutting away the epiphyses of the femurs and tibias, diaphyseal channels were flushed using alpha-minimum essential medium (alpha-MEM) and bone marrow cells were collected. Bone marrow stem cells, which were extracted from the femur and tibia, were cultured on the 7th day after middle cerebral artery occlusion. We then estimated the ratio of non-adherent cells to total bone marrow cells that included osteoclast precursor cells. After culturing these cells separately, cells that tested positive on the tartrate resistant acid phosphatase (TRAP) were counted and bone resorption was evaluated by using the OAAS(TM) plate. In comparison to the control group, the stroke group showed a higher increase of non-adherent cells in the hemiplegic side bone marrow. In addition, after the primary culture, the stroke group showed an increased number of TRAP positive cells and a higher degree of bone resorption estimated by OAAS(TM) plate. As a result, osteoclastogenesis and osteoclast differentiation are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow and these changes are detected as early as within the first week after middle cerebral artery occlusion in SD rats.
Animals ; Bone Marrow Cells/cytology/drug effects ; Bone Resorption/*physiopathology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Female ; Femur/cytology ; Osteoclasts/cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells/cytology/metabolism ; Stroke/*metabolism/pathology ; Tartrates/pharmacology ; Tibia/cytology