Marrow-derived Multipotent Mesenchymal Stem Cells Research Articles

Development of anticancer drug delivery system based on bone marrow-derived multipotent mesenchymal stem cells

Nowadays, a number of anticancer drugs are inadmissible due to their high toxicity. However, this matter can be addressed with improvements in pharmacokinetics, which would allow to generate localized high concentrations of antitumor drugs with minimal systemic toxicity. Such an effect can be achieved by utilizing high encapsulating properties of polyelectrolyte microcapsules with further modification of therapeutically relevant cells capable of pathotropism with these microcarriers. In the present work we studied the effect of drug carrier size on the migration properties of the human multipotent mesenchymal stromal cells (MMSCs). The conducted study included experiments on internalization efficiency, overall toxicity, migration index and speed in order to optimize the properties of the resulting cell-based drug delivery system. In addition to that the ability to directed migration of modified MMSCs along the gradient of tumor-associated chemokines (SDF-1) was explored using chemotaxis slides and 3D tumor tissue model. The results demonstrated that the combination of MMSCs and drug-loaded microcapsules resulted in a delivery system which possesses the ability to actively migrate into tumors, was not toxic towards cell carriers and overall had a therapeutic potential.

A Simple Method for Isolation, Propagation, Characterization, and Differentiation of Adult Mouse Bone Marrow-Derived Multipotent Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are spindle-shaped, adherent, clonogenic, non-phagocytic, fibroblastic and multipotent in nature with the intrinsic ability of self-renewal and proliferation. Bone marrow is the richest source of MSCs for studying the underlying processes of proliferation, self-renewal, and multiple-lineage differentiation. There have been several studies to improve the method of isolation, propagation, characterization and differentiation of mesenchymal stem cells from the mouse bone marrow, but none are widely acceptable. Owing to unavailability of universally acceptable method of MSCs culture continuous efforts are being made in this direction. Here, we report a simple method with some subtle modifications aiming to improve the overall method of isolation, culture, propagation and differentiation of MSCs in vitro. Following this protocol, we have isolated MSCs with typical spindle-shaped morphology as shown by scanning electron microscopy. These cells also showed expression of MSC-specific markers, CD29 (98.94% ± 0.67%), CD44 (84.27% ± 7.77%), Sca-1 (92.70% ± 3.81%) with negligible expression of HSC-specific markers such as CD45 (0.40% ± 0.10%), CD34 (0.15% ± 0.05%) and CD11b (0.45% ± 0.15%). MSCs were also found to differentiate into mesodermal lineages such as adipocytes, osteocytes and chondrocytes as well as ectodermal neuron-like cells. Moreover, MSCs showed differential basal expression of pluripotency-associated transcription factors such as Oct4, Nanog, Sox2 and Myc. Based on the above findings, we propose a simple protocol that can be used to isolate, culture, propagate and characterize multipotent MSCs from mouse bone marrow for experimental and application purposes.

AB037. Inhibition of extracellular signal-regulated kinase pathways by U0126 enhances osteogenic differentiation of bone marrow-derived multipotent mesenchymal stem cells via cross-talk with p38 pathway

Background and objective Bone marrow-derived multipotent mesenchymal stem cells (BM-MSCs) can differentiate into osteoblasts via signal transduction pathways that cause nuclear responses. The extracellular signal-regulated kinase (ERK) and p38 signaling pathways control a variety of important cellular events, especially, cell proliferation, differentiation and apoptosis. The aim of this study was to elucidate the role of ERK and p38 signaling pathways involved in the osteogenic differentiation of BM-MSCs.

Human Bone Marrow Mesenchymal Stem Cells Induce Collagen Production and Tongue Cancer Invasion

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.

Abstract 9673: Mesenchymal Stem Cells Modulate Inflammatory Responses in Murine Heart by Inhibition of the TNF System

Mesenchymal stem cells have been used to treat numerous different clinical conditions, although the mechanisms by which pathogenetic processes are affected are still poorly understood. We have recently demonstrated the homing of murine long-term renewing adult bone marrow-derived multipotent mesenchymal stem cells (BM-MASCs) to diseased tissues and postulated that BM-MASCs might modulate inflammatory reactions. We here show that administration of BM-MASCs modulates adverse cardiac remodeling inhibiting the TNF system in a transgenic mouse model of cardiac-specific expression of MCP-1-induced inflammatory dilative cardiomyopathy. Four-months old MCP-1 mice were analyzed before and after 4-week administration of MASCs, Etanercept (Amgen), Pentoxifylline (Sanofi-Aventis), or NaCl (sham). MRI, Histological (Masson's thricromic), immunofluorescent (CD45 and Collagen VI antibodies, and Lectin from Bandeiraera Simplicifolia), Western blot (sTNFR-I, TNF-β, Phosphorylated-nuclear factor k (NFkBp65), and Collagen VI), SILAC-base mass spectrometry, cytokine arrays, Mission shRNA system (NM_011609.2-1538s1c1, NM_011609.2-996s1c1, and NM_011609.2-1652s1c1), and survival analysis were performed. Our results show that administration of BM-MASCs to MCP-1 mice significantly reduced cardiac accumulation of infiltrating cells and fibrosis, improved capillary density and function, and extended lifespan. Comprehensive analysis of the secretome of MASCs unveiled the release of soluble tumor necrosis factor receptor I (sTNFR-I), which significantly suppressed activation of the NFκBp65 signaling pathway in MCP-1 hearts in vivo. Administration of TNFRI-knock-down BM-MASCs yielded no positive effects demonstrating the requirement of sTNFR-I for BM-MASCs-mediated improvement of inflammatory dilative cardiomyopathy. Substitution of BM-MASCs by recombinant soluble TNFR recapitulated several effects of BM-MASCs including lifespan expansion. We conclude that BM-MASCs suppress local inflammatory processes by release of numerous signaling molecules including sTNFR-I, which makes them useful tools for various clinical applications.

Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK

BackgroundIslet transplantation provides a promising cure for Type 1 diabetes; however it is limited by a shortage of pancreas donors. Bone marrow-derived multipotent mesenchymal stem cells (MSCs) offer renewable cells for generating insulin-producing cells (IPCs).MethodsWe used a four-stage differentiation protocol, containing neuronal differentiation and IPC-conversion stages, and combined with pellet suspension culture to induce IPC differentiation.ResultsHere, we report adding extracellular matrix proteins (ECM) such as fibronectin (FN) or laminin (LAM) enhances pancreatic differentiation with increases in insulin and Glut2 gene expressions, proinsulin and insulin protein levels, and insulin release in response to elevated glucose concentration. Adding FN or LAM induced activation of Akt and ERK. Blocking Akt or ERK by adding LY294002 (PI3K specific inhibitor), PD98059 (MEK specific inhibitor) or knocking down Akt or ERK failed to abrogate FN or LAM-induced enhancement of IPC differentiation. Only blocking both of Akt and ERK or knocking down Akt and ERK inhibited the enhancement of IPC differentiation by adding ECM.ConclusionsThese data prove IPC differentiation by MSCs can be modulated by adding ECM, and these stimulatory effects were mediated through activation of Akt and ERK pathways.

Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung

Recent studies have suggested that bone marrow-derived multipotent mesenchymal stem cells (MSCs) may have therapeutic applications in multiple clinical disorders including myocardial infarction, diabetes, sepsis, and hepatic and acute renal failure. Here, we tested the therapeutic capacity of human MSCs to restore alveolar epithelial fluid transport and lung fluid balance from acute lung injury (ALI) in an ex vivo perfused human lung preparation injured by E. coli endotoxin. Intra-bronchial instillation of endotoxin into the distal airspaces resulted in pulmonary edema with the loss of alveolar epithelial fluid transport measured as alveolar fluid clearance. Treatment with allogeneic human MSCs or its conditioned medium given 1 h following endotoxin-induced lung injury reduced extravascular lung water, improved lung endothelial barrier permeability and restored alveolar fluid clearance. Using siRNA knockdown of potential paracrine soluble factors, secretion of keratinocyte growth factor was essential for the beneficial effect of MSCs on alveolar epithelial fluid transport, in part by restoring amiloride-dependent sodium transport. In summary, treatment with allogeneic human MSCs or the conditioned medium restores normal fluid balance in an ex vivo perfused human lung injured by E. coli endotoxin.

Comparison of Human Placenta- and Bone Marrow–Derived Multipotent Mesenchymal Stem Cells

Bone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs), but placenta appears to be an alternative and more readily available source. This study comprehensively compared human placenta-derived MSC (hpMSC) and human bone marrow-derived MSC (hbmMSC) in terms of cell characteristics, optimal growth conditions and in vivo safety specifically to determine if hpMSC could represent a source of human MSC for clinical trial. MSC were isolated from human placenta (hpMSC) and human bone marrow (hbmMSC) and expanded ex vivo using good manufacturing practice-compliant reagents. hpMSC and hbmMSC showed similar proliferation characteristics in different basal culture media types, fetal calf serum (FCS) concentrations, FCS heat-inactivation experiments, flask types and media replacement responsiveness. However, hpMSC and hbmMSC differed with respect to their proliferation capabilities at different seeding densities, with hbmMSC proliferating more slowly than hpMSC in every experiment. hpMSC had greater long-term growth ability than hbmMSC. MSC from both sources exhibited similar light microscopy morphology, size, cell surface phenotype, and mesodermal differentiation ability with the exception that hpMSC consistently appeared less able to differentiate to the adipogenic lineage. A comparison of both hbmMSC and hpMSC from early and medium passage cultures using single-nucleotide polymorphism (SNP) GeneChip analysis confirmed GTG-banding data that no copy number changes had been acquired during sequential passaging. In three of three informative cases (in which the gender of the delivered baby was male), hpMSC were of maternal origin. Neither hpMSC nor hbmMSC caused any acute toxicity in normal mice when injected intravenously at the same, or higher, doses than those currently used in clinical trials of hbmMSC. This study suggests that human placenta is an acceptable alternative source for human MSC and their use is currently being evaluated in clinical trials.

Efficient Homing of Multipotent Adult Mesenchymal Stem Cells Depends on FROUNT-Mediated Clustering of CCR2

Circulating stem cells of different origin have been demonstrated to improve repair of various organs both after systemic and local application, although the mechanisms that cause these effects are still not fully understood. We have used a combination of DNA microarray analysis and in vitro migration assays to screen for molecules that mediate homing of long-term renewing adult bone marrow-derived multipotent mesenchymal stem cells (BM-MASCs). We show that the cytokine receptor CCR2 is necessary for organ-specific homing of bone marrow-derived MASCs to the heart in a transgenic mouse model and into hearts damaged by ischemia/reperfusion. Homing and migration of stem cells was dependent on the intracellular adaptor molecule FROUNT, which interacts with CCR2. FROUNT was required for polarization of MASCs, resulting in clustering of CCR2 and reorganization of the cytoskeleton. Recruited MASCs summoned by the CCR2 ligand MCP-1/CCL2 expressed SDF1, which might trap additional bone marrow-derived circulating cells to contribute to the complex process of homing and retention of circulating stem and progenitor cells to remodel diseased organs.

Stem Cell Therapy for Liver Disease: Parameters Governing the Success of Using Bone Marrow Mesenchymal Stem Cells

Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs and by complications associated with rejection and immunosuppression. There is increasing evidence to suggest the bone marrow is a transplantable source of hepatic progenitors. We previously reported that multipotent bone marrow-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells with almost 100% induction frequency under defined conditions, suggesting the potential for clinical applications. The aim of this study was to critically analyze the various parameters governing the success of bone marrow-derived mesenchymal stem cell-based therapy for treatment of liver diseases. Lethal fulminant hepatic failure in nonobese diabetic severe combined immunodeficient mice was induced by carbon tetrachloride gavage. Mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells were then intrasplenically or intravenously transplanted at different doses. Both mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells, transplanted by either intrasplenic or intravenous route, engrafted recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Intravenous transplantation was more effective in rescuing liver failure than intrasplenic transplantation. Moreover, mesenchymal stem cells were more resistant to reactive oxygen species in vitro, reduced oxidative stress in recipient mice, and accelerated repopulation of hepatocytes after liver damage, suggesting a possible role for paracrine effects. Bone marrow-derived mesenchymal stem cells can effectively rescue experimental liver failure and contribute to liver regeneration and offer a potentially alternative therapy to organ transplantation for treatment of liver diseases.