Intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs.

BACKGROUND: Mesenchymal stem/progenitor cells (MSC) have shown beneficial effects in many models of disease in part by modulating excessive inflammatory and immune responses. Frequently the beneficial effects of MSC persist long after their disappearance from host tissues, suggesting that MSC interact with intermediate cells in the host that relay or amplify their effects. The cells have usually been injected intravenously, but beneficial effects have also been reported with intraperitoneal (IP) injection of MSC. However the fate of IP injection of MSC has not been examined. METHODS: The fate of the human MSC injected IP into immune-competent mice was studied. In vivo imaging was used to track green fluorescent protein-labeled MSC in the peritoneal cavity. In addition, their retention in peritoneal tissues was measured by real-time polymerase chain reaction for human GAPDH mRNA. To describe the effects of human MSC on the immune system of the peritoneum, the peritoneal lavage, omentum, lymph nodes and mesenteric tissues were collected. Flow cytometry was used to evaluate the immune cell populations, while cytokine/chemokine production was measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Challenge with lipopolysaccharide at 3 days after the administration of MSC was used to evaluate the preconditioning of the immune system. RESULTS: Within 20 min, single MSC were no longer detected in peritoneal lavage fluid. Instead they were recovered as aggregates of varying size that contained mouse macrophages and a few B220+ lymphocytes. After 1 day, most of the aggregates containing live MSC were attached to sites throughout the peritoneal cavity including the omentum and mesentery. Less than 0.05 % of the live injected cells were detected in the spleen and jejunal lymph nodes. In all locations, MSC colocalized with mouse macrophages and B220+ lymphocytes. Attachment to the omentum and mesentery was accompanied by the recruitment of immune cells and changes in the production of a series of mouse cytokines. A similar increase in mouse cytokines in the peritoneum was seen after IP injections of human fibroblasts. CONCLUSIONS: IP injected human MSC rapidly formed aggregates with mouse macrophages and B220+ lymphocytes and attached to the walls of the peritoneal cavity. The formation of the aggregates probably limits access of the cells to the systemic circulation.

TSG-6 as a biomarker to predict efficacy of human mesenchymal stem/progenitor cells (hMSCs) in modulating sterile inflammation in vivo.

Human mesenchymal stem/progenitor cells (hMSCs) from bone marrow and other tissues are currently being administered to large numbers of patients even though there are no biomarkers that accurately predict their efficacy in vivo. Using a mouse model of chemical injury of the cornea, we found that bone-marrow-derived hMSCs isolated from different donors varied widely in their efficacy in modulating sterile inflammation. Importantly, RT-PCR assays of hMSCs for the inflammation-modulating protein TSG-6 expressed by the TNFα-stimulated gene 6 (TSG-6 or TNFAIP6) predicted their efficacy in sterile inflammation models for corneal injury, sterile peritonitis, and bleomycin-induced lung injury. In contrast, the levels of TSG-6 mRNA were negatively correlated with their potential for osteogenic differentiation in vitro and poorly correlated with other criteria for evaluating hMSCs. Also, a survey of a small cohort suggested that hMSCs from female donors compared with male donors more effectively suppressed sterile inflammation, expressed higher levels of TSG-6, and had slightly less osteogenic potential.

Phase-directed therapy: TSG-6 targeted to early inflammation improves bleomycin-injured lungs.

Previous reports demonstrated that bleomycin-induced injury of lungs in mice can be improved by the administration of murine multipotent adult stem/progenitor cells (MSCs) from the bone marrow. Recently some of the beneficial effects of MSCs have been explained by the cells being activated by signals from injured tissues to express the inflammation modulating protein TNF-α-stimulated gene/protein 6 (TSG-6). In this study, we elected to test the hypothesis that targeting the early phase of bleomycin-induced lung injury with systemic TSG-6 administration may produce therapeutic effects such as preventing the deterioration of lung function and increasing survival by modulation of the inflammatory cascade. Lung injury in C57Bl/6J mice was induced by intratracheal administration of bleomycin. Mice then received intravenous injections of TSG-6 or sham controls. Pulse oximetry was used to monitor changes in lung function. Cell infiltration was evaluated by flow cytometry, cytokine expression was measured by ELISA assays, and lungs were assessed for histological attributes. The results demonstrated that intravenous infusion of TSG-6 during the early inflammatory phase decreased cellular infiltration into alveolar spaces. Most importantly, it improved both the subsequent decrease in arterial oxygen saturation levels and the survival of the mice. These findings demonstrated that the beneficial effects of TSG-6 in a model of bleomycin-induced lung injury are largely explained by the protein modulating the early inflammatory phase. Similar phase-directed strategy with TSG-6 or other therapeutic factors that MSCs produce may be useful for other lung diseases and diseases of other organs.

Hypoxia and extracellular matrix proteins influence angiogenesis and lymphangiogenesis in mouse embryoid bodies.

Regulatory mechanisms for angiogenesis are relatively well established compared to lymphangiogenesis. Few studies have shown that a combination of vascular endothelial growth factor VEGF-A/C with hypoxia or collagen matrix promotes lymphatic structures along with blood vessel development in mouse embryoid bodies (EB). In this study we tested the hypothesis that while hypoxia combined with prolonged VEGF-A/C treatment would induce early lymphangiogenesis in addition to angiogenesis in mouse EBs, under similar conditions specific extracellular matrix (ECM) proteins would promote lymphatic vessel-like structures over angiogenesis. EBs were subjected to four conditions and were maintained under normoxia and hypoxia (21% and 2.6% O(2), respectively) with or without VEGF-A/C. Microarray analyses of normoxic and hypoxic EBs, and immunofluorescence data showed very low expression of early lymphatic endothelial cell (LEC) markers, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and prospero-related homeobox 1 (Prox1) at early time points. Double immunofluorescence using MECA-32 and Prox1/LYVE1 demonstrated that combined hypoxia and VEGF-A/C treatment promoted formation of blood vessel-like structures, whereas only Prox1(+)/LYVE1(+) LECs were detected in EBs at E22.5. Furthermore, EBs were grown on laminin or collagen-I coated plates and were subjected to the four treatments as described above. Results revealed that LECs in EBs at E36.5 attached better to collagen-I, resulting in an organized network of lymphatic vessel-like structures as compared to EBs grown on laminin. However, blood vessel-like structures were less favored under these same conditions. Collectively, our data demonstrate that hypoxia combined with growth factors promotes angiogenesis, whereas combination of these conditions with specific ECM proteins favors lymphangiogenesis processes in mouse EBs.

Cardiomyocyte contractile status is associated with differences in fibronectin and integrin interactions.

Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K(+) (tonically contracted), or low-Ca(2+) (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte alpha(3)beta(1)- and/or alpha(5)beta(1)-integrins was 39.6 +/- 1.3 pN. The binding specificity between FN and alpha(3)beta(1)- and alpha(5)beta(1)-integrins was verified by using monoclonal antibodies against alpha(3)-, alpha(5)-, alpha(3) + alpha(5)-, or beta(1)-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K(+) solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca(2+) solution. However, it could be partially prevented by high-K(+) bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to FN is modulated by the contractile state of cardiac myocytes.