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-1ß 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.

Regulation and function of immunosuppressive molecule human leukocyte antigen G5 in human bone tissue.

Bone-marrow mesenchymal stem cells (MSCs) are the origin of bone-forming cells with immunomodulation potential. HLA-G5 is among the generated immunosuppressive molecules. HLA-G proteins play a crucial role in promoting the acceptance of allografts. However, the mechanisms regulating the expression of HLA-G5 in human MSCs are unknown. We induced differentiation of MSCs and found that HLA-G5 was greatly up-regulated only in osteoblastic cells (+63% for mRNA). Growth plates and bone callus postfracture in adults showed that only bone-lining cells and mesenchymal progenitors were positive for HLA-G5. Use of gene silencing and dominant-negative factors revealed that HLA-G5 depends on the expression and function of the skeletogenesis master genes RUNX2 and DLX5. In addition, HLA-G5 could directly inhibit osteoclastogenesis by acting on monocytes through SHP1. However, in mature osteoblasts, the expression of HLA-G5 protein was greatly suppressed whereas the proosteoclastogenic factor, RANKL, was concomitantly increased. Down-regulation of HLA-G5 expression during the maturation of osteoblasts was due to binding of the repressor GLI3, a signal transducer of the Hedgehog pathway, to the GLI binding element within the HLA-G promoter. Our findings show that mesenchymal progenitors and osteoblastic cells specifically express HLA-G5 during osteogenesis, with a key role in bone homeostasis.

Specific lineage-priming of bone marrow mesenchymal stem cells provides the molecular framework for their plasticity.

Lineage-priming is a molecular model of stem cell (SC) differentiation in which proliferating SCs express a subset of genes associated to the differentiation pathways to which they can commit. This concept has been developed for hematopoietic SCs, but has been poorly studied for other SC populations. Because the differentiation potential of human bone marrow mesenchymal stem cells (BM MSCs) remains controversial, we have explored the theory of lineage-priming applied to these cells. We show that proliferating primary layers and clones of BM MSCs have precise priming to the osteoblastic (O), chondrocytic (C), adipocytic (A), and the vascular smooth muscle (V) lineages, but not to skeletal muscle, cardiac muscle, hematopoietic, hepatocytic, or neural lineages. Priming was shown both at the mRNA (300 transcripts were evaluated) and the protein level. In particular, the master transactivator proteins PPARG, RUNX2, and SOX9 were coexpressed before differentiation induction in all cells from incipient clones. We further show that MSCs cultured in the presence of inducers differentiate into the lineages for which they are primed. Our data point out to a number of signaling pathways that might be activated in proliferating MSCs and would be responsible for the differentiation and proliferation potential of these cells. Our results extend the notion of lineage-priming and provide the molecular framework for inter-A, -O, -C, -V plasticity of BM MSCs. Our data highlight the use of BM MSCs for the cell therapy of skeletal or vascular disorders, but provide a word of caution about their use in other clinical indications.

3D environment on human mesenchymal stem cells differentiation for bone tissue engineering.

In this work a novel method was developed to create a three dimensional environment at a cellular level for bone tissue engineering. Biphasic calcium phosphate (BCP) particles of 140-200 microm were used in association with human mesenchymal stem cells (hMSCs). The cells seeded on these particles adhered and proliferated more rapidly in the first day of culture compared to culture on plastic. Analyses of hMSCs cultured without osteogenic factors on BCP particles revealed an abundant extracellular matrix production forming 3-dimensional (3D) hMSCs/BCP particles constructs after few days. Bone morphogenetic 2 (BMP-2), bone sialoprotein (BSP) and ALP gene expression using real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that expression profiles were modified by the culture substrate while the addition of osteogenic medium enhanced bone markers expression. These results indicate that BCP particles alone are able to induce an osteoblastic differentiation of hMSCs that might be of interest for bone tissue engineering.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients.

BACKGROUND AIMS: Advances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources. METHODS: We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit-fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels. RESULTS: MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential. CONCLUSIONS: Trabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.

Molecular profile of mouse stromal mesenchymal stem cells.

We determined a transcriptional profile specific for clonal stromal mesenchymal stem cells from adult and fetal hematopoietic sites. To identify mesenchymal stem cell-like stromal cell lines, we evaluated the adipocytic, osteoblastic, chondrocytic, and vascular smooth muscle differentiation potential and also the hematopoietic supportive (stromal) capacity of six mouse stromal cell lines from adult bone marrow and day 14.5 fetal liver. We found that two lines were quadripotent and also supported hematopoiesis, BMC9 from bone marrow and AFT024 from fetal liver. We then ascertained the set of genes differentially expressed in the intersection set of AFT024 and BMC9 compared with those expressed in the union set of two negative control lines, 2018 and BFC012 (both from fetal liver); 346 genes were upregulated and 299 downregulated. Using Ingenuity software, we found two major gene networks with highly significant scores. One network contained downregulated genes that are known to be implicated in osteoblastic differentiation, proliferation, or transformation. The other network contained upregulated genes that belonged to two categories, cytoskeletal genes and genes implicated in the transcriptional machinery. The data extend the concept of stromal mesenchymal stem cells to clonal cell populations derived not only from bone marrow but also from fetal liver. The gene networks described should discriminate this cell type from other types of stem cells and help define the stem cell state.

Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model.

Tissue-engineered constructs combining bone marrow mesenchymal stem cells with biodegradable osteoconductive scaffolds are very promising for repairing large segmental bone defects. Synchronizing and controlling the balance between scaffold-material resorption and new bone tissue formation are crucial aspects for the success of bone tissue engineering. The purpose of the present study was to determine, and compare, the osteogenic potential of ceramic scaffolds with different resorbability. Four clinically relevant granular biomaterial scaffolds (specifically, Porites coral, Acropora coral, beta-tricalcium phosphate and banked bone) with or without autologous bone marrow stromal cells were implanted in the ectopic, subcutaneous-pouch sheep model. Scaffold material resorption and new bone formation were assessed eight weeks after implantation. New bone formation was only detected when the biomaterial constructs tested contained MSCs. New bone formation was higher in the Porites coral and Acropora coral than in either the beta-tricalcium phosphate or the banked bone constructs; furthermore, there was a direct correlation between scaffold resorption and bone formation. The results of the present study provide evidence that, among the biomaterials tested, coral scaffolds containing MSCs promoted the best new bone formation in the present study.

Urokinase-type plasminogen activator receptor interaction with ß1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

INTRODUCTION: Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration. METHODS: Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect. RESULTS: We demonstrated that uPAR activation and its association with ß1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue. CONCLUSIONS: We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and ß1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.

Effect of high-dose irradiation on human bone-marrow-derived mesenchymal stromal cells.

Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of γ-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unit-fibroblasts. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a 16-week follow-up period. We found increased vascular endothelial growth factor (VEGF) levels and a decrease of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward γ-irradiation showed positive ß-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-16 weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p16) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose γ-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose γ-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.

Osteoblastic differentiation and potent osteogenicity of three-dimensional hBMSC-BCP particle constructs.

Bone tissue engineering usually consists of associating osteoprogenitor cells and macroporous scaffolds. This study investigated the in vitro osteoblastic differentiation and resulting in vivo bone formation induced by a different approach that uses particles as substrate for human bone marrow stromal cells (hBMSCs), in order to provide cells with a higher degree of freedom and allow them to synthesize a three-dimensional (3D) environment. Biphasic calcium phosphate (BCP) particles (35 mg, ~175 µm in diameter) were therefore associated with 4 × 10(5) hBMSCs. To discriminate the roles of BCP properties and cell-synthesized 3D environments, inert glass beads (GBs) of similar size were used under the same conditions. In both cases, high cell proliferation and extensive extracellular matrix (ECM) production resulted in the rapid formation of thick cell-synthesized 3D constructs. In vitro, spontaneous osteoblastic differentiation was observed in the 3D constructs at the mRNA and protein levels by monitoring the expression of Runx2, BMP2, ColI, BSP and OCN. The hBMSC-BCP particle constructs implanted in the subcutis of nude mice induced abundant ectopic bone formation after 8 weeks (~35%, n = 5/5). In comparison, only fibrous tissue without bone was observed in the implanted hBMSC-GB constructs (n = 0/5). Furthermore, little bone formation (~3%, n = 5/5) was found in hBMSC-macroporous BCP discs (diameter 8 × 3 mm). This study underlines the lack of correspondence between bone formation and in vitro differentiation assays. Furthermore, these results highlight the importance of using BCP as well as a 3D environment for achieving high bone yield of interest for bone engineering.

Granulocyte-colony-stimulating factor stimulation of bone marrow mesenchymal stromal cells promotes CD34+ cell migration via a matrix metalloproteinase-2-dependent mechanism.

Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models.

Consistent osteoblastic differentiation of human mesenchymal stem cells with bone morphogenetic protein 4 and low serum.

Providing fully mature and functional osteoblasts is challenging for bone tissue engineering and regenerative medicine. Such cells could be obtained from multipotent bone marrow mesenchymal stem cells (MSCs) after induction by different osteogenic factors. However, there are some discrepancies in results, notably due to the use of sera and to the type of osteogenic factor. In this study, we compared the osteogenic differentiation of bone marrow MSCs induced by dexamethasone (Dex) or bone morphogenetic proteins (BMPs) by assessing phenotypes in vitro and functional osteoblasts in vivo. Reducing the content of fetal calf serum from 10% to 2% significantly increased the mineral deposition and expression of osteoblastic markers during osteogenesis. In comparison to Dex condition, the addition of BMP4 greatly improved the differentiation of MSCs into fully mature osteoblasts as seen by high expression of Osterix. These results were confirmed in different supportive matrixes, plastic flasks, or biphasic calcium phosphate biomaterials. In contrast to Dex-derived osteoblasts, BMP4-derived osteoblasts from MSCs were significantly able to produce new bone in subcutis of nude mice in accordance with in vitro results. In conclusion, we describe a convenient ex vivo method to produce consistently mature functional osteoblasts from human MSCs with use of BMP4 and low serum.

The in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities.

Adult bone marrow (BM)-derived stem cells, including hematopoietic stem cells (HSCs) and MSCs, represent an important source of cells for the repair of a number of damaged tissues. In contrast to HSCs, the soluble factors able to induce MSC migration have not been extensively studied. In the present work, we compared the in vitro migration capacity of human BM-derived MSCs, preincubated or not with the inflammatory cytokines interleukin 1beta (IL1beta) and tumor necrosis factor alpha (TNFalpha), in response to 16 growth factors (GFs) and chemokines. We show that BM MSCs migrate in response to many chemotactic factors. The GFs platelet-derived growth factor-AB (PDGF-AB) and insulin-like growth factor 1 (IGF-1) are the most potent, whereas the chemokines RANTES, macrophage-derived chemokine (MDC), and stromal-derived factor-1 (SDF-1) have limited effect. Remarkably, preincubation with TNFalpha leads to increased MSC migration toward chemokines, whereas migration toward most GFs is unchanged. Consistent with these results, BM MSCs express the tyrosine kinase receptors PDGF-receptor (R) alpha, PDGF-Rbeta, and IGF-R, as well as the RANTES and MDC receptors CCR2, CCR3, and CCR4 and the SDF-1 receptor CXCR4. TNFalpha increases CCR2, CCR3, and CCR4 expression (as opposed to that of CXCR4), together with RANTES membrane binding. These data indicate that the migration capacity of BM MSCs is under the control of a large range of receptor tyrosine kinase GFs and CC and CXC chemokines. Most chemokines are more effective on TNFalpha-primed cells. Our results suggest that the mobilization of MSCs and their subsequent homing to injured tissues may depend on the systemic and local inflammatory state. Disclosure of potential conflicts of interest is found at the end of this article.

Real-time flow cytometry analysis of permeability transition in isolated mitochondria.

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.