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.

Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations. METHODS: MSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells. RESULTS: MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in α-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death. CONCLUSIONS: We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.

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.