Immunogenic potential of human bone marrow mesenchymal stromal cells is enhanced by hyperthermia

Background aims

Bone marrow–derived mesenchymal stromal cells (MSCs) have been reported to suppress T-cell proliferation and used to alleviate the symptoms of graft-versus-host disease (GVHD). MSCs are a mixed cell population and at this time there are no tools to isolate the cells responsible for the T-cell suppression. We wanted to find a way to enhance the immune-modulatory actions of MSCs and tried varying the temperature at which they were cultured.

Effects of age and gender on WNT gene expression in human bone marrow stromal cells

WNT signaling pathways play important roles in the behavior of human bone marrow stromal cells. Although WNT expression has been examined in human bone marrow stromal cells (hMSCs) with limited numbers of subjects or from commercial sources, there are conflicting results on WNT gene expression in hMSCs. Furthermore, the effects of age and gender on WNT expression in hMSCs are largely unknown. In this study, we evaluated RNA expression of all the WNT genes in hMSCs from 19 subjects, 12 women and 7 men, aged from 36 to 85 years. Analysis of WNT gene expression in young and old groups indicated that WNT7B and 14 were expressed significantly higher in the young group. WNT2 and WNT13 showed a trend of higher expression in young group. WNT7B, 13, and 14 were inversely correlated with age. Further analysis for gender‐specific difference indicated that WNT16 was expressed significantly higher in men than in women. WNT11 showed a trend of higher expression in hMSCs from women. For the hMSCs from women, WNT13 was inversely correlated with age and WNT4 was positively correlated with age. For the hMSCs from men, WNT7B and WNT14 were inversely correlated with age. These data indicated that most of the age‐related WNT genes belong to the canonical WNT signaling pathway. Further, there are gender‐specific differences in the expression of WNT4, 7B, 13, 14, and 16 in hMSCs. Age and gender account for many of the sample‐to‐sample variations in WNT gene expression in human marrow stromal cells. J. Cell. Biochem. 106: 337–343, 2009. © 2008 Wiley‐Liss, Inc.     

Curcumin inhibits osteoclastogenesis by decreasing receptor activator of nuclear factor-kappaB ligand (RANKL) in bone marrow stromal cells

Curcumin (diferuloylmethane), a pigment derived from turmeric, has anti-oxidant and anti-inflammatory activities. Accumulating evidence points to a biochemical link between increased oxidative stress and reduced bone density. Osteoclast formation was evaluated in co-cultures of bone marrow stromal cells (BMSC) and whole bone marrow cells (BMC). Expression of receptor activator of nuclear factor-kappaB ligand (RANKL) was analyzed at the mRNA and protein levels. Exposure to curcumin led to dose-dependent suppression of osteoclastogenesis in the coculture system, and to reduced expression of RANKL in IL-1alpha-stimulated BMSCs. Addition of RANKL abolished the inhibition of osteoclastogenesis by curcumin, whereas the addition of prostaglandin E2(PGE2) did not. The decreased osteoclastogenesis induced by curcumin may reduce bone loss and be of potential benefit in preventing and/or attenuating osteoporosis.     

RANK ligand expression in heat shock factor-2 deficient mouse bone marrow stromal/preosteoblast cells

Heat Shock Proteins (HSP) are molecular chaperones activated upon cellular stress/stimuli. HSP gene expression is regulated by Heat Shock Factors (HSF). We have recently demonstrated a functional role for heat shock factor-2 (HSF-2) in fibroblast growth factor-2 (FGF-2)-induced RANK ligand (RANKL), a critical osteoclastogenic factor expression on stromal/preosteoblast cells. In the present study, we show that FGF-2 treatment did not induce RANKL expression in HSF-2-/-stromal/preosteoblast cells. Interestingly, HSF-2 deficiency resulted in rapid induction of alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in these cells. Furthermore, FGF-2 did not induce osteoclast formation in co-culture of normal mouse spleen cells and HSF-2-/-stromal/preosteoblast cells. Electron microscopy analysis demonstrated that osteoclasts from HSF-2-/-mice have poorly developed ruffled borders. These data further confirm that HSF-2 plays an important role in FGF-2-induced RANKL expression in stromal/preosteoblast cells. HSF-2 deficiency has pleotropic effects on gene expression during osteoblast differentiation and osteoclastogenesis in the bone microenvironment. Novel therapeutic agents that modulate HSF-2 activation may have therapeutic utility against increased levels of FGF-2 and bone destruction associated with pathologic conditions.     

MiR‐9 promotes multiple myeloma progression by regulating TRIM56/NF‐κB pathway

miR‐9 has been reported to play a pivotal role in multiple human cancers by acting as an oncogene or tumor suppressor. In this study, we explored the possible role and molecular mechanism of miR‐9 in multiple myeloma (MM). The miR‐9 expression was examined by quantitative real‐time polymerase chain reaction assay. Transfection with miR‐9‐mimics, miR‐9‐inhibitor, pcDNA‐TRIM56, or si‐TRIM56 into cells was used to change the expression levels of miR‐9 and TRIM56. Western blot analysis was used to detect the expression of TRIM56, p65, p‐p65, IκBα, and p‐IκBα. The potential target of miR‐9 was confirmed by luciferase reporter assay. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium (MTT) assay, colony formation assay, and flow cytometry were used to assess the abilities of cell proliferation and apoptosis. miR‐9 was upregulated in MM patients and cell lines, and miR‐9 overexpression promoted proliferation and repressed apoptosis in MM cell lines. TRIM56 was confirmed as a target of miR‐9. Moreover, TRIM56 reversed miR‐9‐mediated pro‐proliferation and anti‐apoptosis effect on MM cell lines. Furthermore, nuclear factor‐κB (NF‐κB) pathway was involved in miR‐9/TRIM56‐mediated regulation on MM cell lines. miR‐9 promoted the development and progression of MM by regulating TRIM56/NF‐κB pathway, thereby providing a potential microRNA‐based target for MM therapy.     

Flesh‐eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor‐κB ligand

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock‐like syndrome. Human CD46‐expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast‐mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10⁵ colony‐forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor‐κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro‐computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4⁺ and CD8⁺ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte‐derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast‐like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

Gamma irradiation preserves immunosuppressive potential and inhibits clonogenic capacity of human bone marrow‐derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are promising candidates for the treatment of graft‐versus‐host and autoimmune diseases. Here, by virtue of their immunosuppressive effects, they are discussed to exhibit inhibitory actions on various immune effector cells, including T lymphocytes that promote the underlying pathology. While it becomes apparent that MSCs exhibit their therapeutic effect in a transient manner, they are usually transplanted from third party donors into heavily immunocompromised patients. However, little is known about potential late complications of persisting third party MSCs in these patients. We therefore analysed the effect of gamma irradiation on the potency and proliferation of MSCs to elucidate an irradiation dose, which would allow inhibition of MSC proliferation while at the same time preserving their immunosuppressive function. Bone marrow‐derived MSCs (BM‐MSCs) were gamma‐irradiated at increasing doses of 5, 10 and 30 Gy and subsequently assessed by colony formation unit (CFU)‐assay, Annexin V‐staining and in a mixed lymphocyte reaction, to assess colony growth, apoptosis and the immunosuppressive capacity, respectively. Complete loss of proliferative capacity measured by colony formation was observed after irradiation with a dose equal to or greater than 10 Gy. No significant decrease of viable cells was detected, as compared to non‐irradiated BM‐MSCs. Notably, irradiated BM‐MSCs remained highly immunosuppressive in vitro for at least 5 days after irradiation. Gamma irradiation does not impair the immunosuppressive capacity of BM‐MSCs in vitro and thus might increase the safety of MSC‐based cell products in clinical applications.     

Osteogenic differentiation of multipotent mesenchymal stromal cells isolated from human bone marrow and subcutaneous adipose tissue

Cellular populations with phenotypes similar to multipotent mesenchymal stromal cells were isolated from two different sources, including human bone marrow (BM) and subcutaneous adipose tissue (SAT). Comparative analysis of the efficiency of differentiation in the direction of osteogenesis has revealed morphological changes confirmed by staining with Alizarin red and von Kossa in bone marrow cells at the 14th day and in adipose tissue cells at the 28th day of cultivation in the medium with inductors. Analysis of expression of the osteopontin, osteocalcin, and bone sialoprotein genes in RT-PCR reactions has detected essential differences in the potential of these cells to differentiate into bone tissue cells. Cells isolated from BM of both the control and experimental groups were positive for octeopontin (OP) on the 14th day. Unlike these cells, in cells isolated from SAT in medium without an inductor, no product of OP gene expression was identified. In the cells subjected to differentiation, OP appeared at day 14. In the BM cells, octeocalcin (OC) was found at the 14th day, while the bone sialoprotein (BS) was found at the 21st day of cultivation in induction medium. In cells isolated from SAT, OC, and BS were not detected, even at the 28th day after the beginning of induction.     

CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200^pos cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up‐regulated in CD200^pos cells compared to CD200^neg fraction. At the functional level, CD200^pos cells were prone to mineralize the extra‐cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200^pos cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down‐regulated. As dexamethasone has anti‐inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro‐inflammatory cytokines interleukin‐1β and tumour necrosis factor‐α increased CD200 membrane expression but down‐regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro‐inflammatory or pro‐osteogenic, CD200 expression was down‐regulated when nuclear‐factor (NF)‐κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro‐inflammatory cytokines through the same pathway: NF‐κB.     

Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis

Using an in vitro osteogenic culture system, we carried out a restriction fragment differential display (RFDD-PCR) to identify genes expressed by these cells in their undifferentiated stage and not expressed, or expressed at a lower level, in a closely related but distinct cell type: bone marrow stromal cells (BMSC)-derived osteoblasts (BDO). Forty-seven candidate regulated genes, selected by RFDD, were analyzed by RT-PCR analysis in three cell clones and in primary cultures from seven different donors. A subset of three genes were confirmed as upregulated in BMSC relative to BDO in every primary culture and cloned population examined: betaIG-h3, IGFbp3, and LOXL2. Their differential expression was confirmed by Northern analysis and the corresponding proteins were detected by immunolocalization in BMSC.