Co-culture Of Bone Marrow Stromal Cells Research Articles

Bone Marrow Stromal Cells Act as Feeder Cells for Tendon Fibroblasts through Soluble Factors

Feeder effects of bone marrow stromal cells (BMSCs) on tendon fibroblasts were investigated using a co-culture method for the application of ligament or tendon tissue engineering and cell therapy. BMSCs had significant effects on enhancing cell proliferation, the ability of cells to migrate, and cell adhesivity but little effect on the extracellular matrix (ECM) synthesis of tendon fibroblasts without cell-cell contact. Furthermore, the conditioned medium from BMSCs, despite the existence of fibroblasts, significantly increased the number of fibroblasts. Based on these results, the mechanism of the feeder effects is considered to be a certain signal of soluble factors from BMSCs to the fibroblasts. Comparative proteome analysis of the conditioned medium from co-culture of fibroblasts and BMSCs revealed less expression of plasminogen, which showed inhibitory effects on fibroblast proliferation. With regard to the relationships between plasminogen and BMSCs in the co-culture system, we speculate that BMSCs allow resolution of plasminogen or its cleavage activity in the medium via some mechanism.

CNTO328 Abrogates Glucocorticoid (GC) Resistance Conferred by Interleukin (IL)-6 and the Bone Marrow Microenvironment in Multiple Myeloma (MM).

Given the critical role that IL-6 plays in MM cell proliferation, survival, and resistance to GCs, we evaluated the ability of CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, to overcome GC resistance in cell line models of human MM. In the presence of IL-6, the MM cell lines ANBL-6 and KAS-6 were resistant to the cytotoxic activity of dexamethasone (Dex) as assessed by cell viability assays both in suspension culture and in the context of patient-derived stromal cells. Resistance to dexamethasone was readily reversed by CNTO328, but not an isotype control antibody, in suspension culture. For example, in the case of the ANBL-6 model, viability was reduced by 12% with CNTO328 alone, 8% with Dex, but 74% with the combination, consistent with a synergistic interaction Given the ability of other growth factors in the bone marrow microenvironment to confer GC resistance in preclinical models of MM, we evaluated the activity of the CNTO328 and Dex combination in ANBL-6 and KAS-6 cells using a physiologically-relevant MM cell/patient-derived bone marrow stromal cell co-culture system. Importantly, bone marrow stromal cells rendered ANBL-6 and KAS-6 cells resistant to Dex in cell viability assays, and CNTO328 was able to reestablish Dex sensitivity, thus confirming a central role of IL-6 in bone marrow stroma-mediated GC resistance. Furthermore, treatment of ANBL-6 and KAS-6 cells with Dex alone did not induce apoptosis in this co-culture system, whereas the combination of CNTO328 and Dex led to a synergistic induction of apoptosis. In KAS-6 cells, IL-6-mediated Dex resistance was not overcome using pharmacologic inhibitors to p38, PI-3 kinase, mTor or MEK, suggesting that other IL-6 signaling pathways are likely involved. In contrast, the mTor inhibitor rapamycin was capable of sensitizing ANBL-6 cells to Dex in the presence of IL-6, suggesting that this pathway may be relevant to IL-6-mediated GC resistance in these cells. Induction of the pro-apoptotic Bcl-2 family member, Bim, has been shown to play an important role in GC-mediated cell death in lymphocytes as well as preclinical lymphoma and acute lymphoblastic leukemia models. Interestingly, although treatment of ANBL-6 cells in the presence of IL-6 with either CNTO328 or dexamethasone did not lead to induction of Bim, the combination led to a 3.3-fold increase in its expression. Taken together, the above data demonstrate that inhibition of IL-6 signaling with CNTO328 can effectively overcome IL-6-mediated GC resistance even in the presence of bone marrow stroma, and provide a compelling rationale for translation of this combination into clinical trials for patients suffering from MM. Furthermore, we show that the ability of CNTO328 to overcome GC resistance may be mediated in part by its ability to reverse IL-6-mediated repression of GC-induced Bim expression. Studies evaluating the relevance of Bim modulation in IL-6-mediated GC resistance and the molecular pathways that mediate this effect are on-going.

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38α MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFα-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFα-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFα-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1α as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

The anti-tumoral effect of PI3K inhibitor and MEK inhibitor combined with STI571 on chronic myeloid leukemia cells in a bone marrow stromal cell co-culture system

The anti-tumoral effect of PI3K inhibitor and MEK inhibitor combined with STI571 on chronic myeloid leukemia cells in a bone marrow stromal cell co-culture system

Bone marrow stromal cells increase astrocyte survival via upregulation of phosphoinositide 3-kinase/threonine protein kinase and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways and stimulate astrocyte trophic factor gene expression after anaerobic insult

Transplantation of bone marrow stromal cells improves animal neurological functional recovery after stroke. Astrocytes are known to provide structural, trophic and metabolic support for neurons. Thus astrocytes are critical for neural survival during post-ischemia. However, information on the effects of bone marrow stromal cells on astrocytic survival post-ischemia is unavailable. We investigated the influence of rat bone marrow stromal cells on rat astrocytic apoptosis and survival post-ischemia employing an anaerobic chamber. Our data indicate that rat bone marrow stromal cells reduce cell death and apoptosis, and increase the DNA proliferation rate in astrocytes post-ischemia. Mitogen-activated protein kinase kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/threonine protein kinase pathways are involved in cell survival. Western blot showed that rat bone marrow stromal cells activate these two pathways in astrocytes post-ischemia, and upregulate total extracellular signal regulated kinase 1/2 and threonine protein kinase. Since astrocytes produce various neurotrophic factors, we performed reverse transcription polymerase chain reaction to investigate rat bone marrow stromal cells’ effect on astrocyte growth factor gene expression post-ischemia. We observed that brain-derived neurotrophic factor, vascular endothelial growth factor and basic fibroblast growth factor gene expression was enhanced by rat bone marrow stromal cell coculture. These data suggest that bone marrow stromal cells increase astrocytic survival post-ischemic injury. This protective function might involve the activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/threonine protein kinase pathways. Upregulation of brain-derived neurotrophic factor, vascular endothelial growth factor and basic fibroblast growth factor may also contribute to astrocyte survival.

Experimental study of in vitro chondrogenesis by co-culture of bone marrow stromal cells and chondrocytes

Chondrogenic microenvironments play a very important role in chondrogenesis of bone marrow stromal cells (BMSC). This study explored the feasibility of in vitro chondrogenesis by co-culture of BMSC and chondrocytes so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSC and thus promote in vitro chondrogenesis of BMSC. Porcine BMSC and auricular chondrocytes were in vitro expanded respectively and then were mixed at a ratio of 8:2 (BMSC:chondrocyte). 200 microl mixed cells(5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 9 mm in diameter and 3 mm in thickness, as co-culture group. Chondrocytes and BMSC with the same cell number were seeded respectively onto the scaffolds as positive control (chondrocyte group) and negative control (BMSC group). 200 microl chondrocytes (1.0 x 10(7)/ml, equal to the chondrocyte number of co-culture group) alone were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results. Cells in all groups had fine adhesion to the scaffolds and could secrete extracellular matrix. In both co-culture group and positive control group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture and formed homogenous mature cartilage after 8 weeks of in vitro culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features, and abundant type II collagen was also detected by immunohistochemistry in both groups. The average wet weight and GAG content of co-culture group were both more than 80% of those of positive control group. In negative control group, however, the constructs shrunk gradually during in vitro culture and cartilage-like tissue could only be observed at the edge area of the construct. In low concentration chondrocyte group, the constructs also shrunk gradually during in vitro culture and the average wet weight was below 40% of that of the positive control group although histology showed a small amount cartilage formation. Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of BMSC and thus promote the in vitro chondrogenesis of BMSC.

The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague–Dawley rats

There are numerous parallels between the heamatolymphopoietic and nervous systems in terms of the mechanisms regulating their development. We proposed that neural stem cells (NSCs) may respond to the microenvironmental signals provided by bone marrow stromal cells (BMSCs) which regulate the differentiation and maturation of hematolymphopoietic stem cells. First, we isolated and proliferated BMSCs from the femur and tibia, and NSCs from the midbrain of Sprague–Dawley (SD) rats, and then investigated the effects of BMSCs on the differentiation of NSCs into neurons, astrocytes and oligodendrocytes by directly plating neurospheres on BMSC monolayers in serum-free conditions. The results confirmed that BMSCs induced NSCs to differentiate selectively into neurons. The percentage of neurons significantly increased in 7 days in vitro co-cultures of NSCs and BMSCs as compared to NSCs cultures alone. When the duration of the cultures was extended to 12 days in vitro, BMSCs enhanced the survival of neurons derived from these NSCs; our investigation then focused on the underlying mechanism for this effect of BMSCs. NSCs were cultured with BMSC conditioned-medium and co-cultured with membrane fragments of live BMSCs or paraformaldehyde fixed BMSCs, the inducing activity of BMSCs was solely detectable in BMSC conditioned-medium, indicating that soluble factors secreted by BMSCs were responsible for its effect on the neuronal differentiation of NSCs. Therefore, BMSCs may provide a powerful tool for therapeutic neurological applications.

Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.