Previous reports suggested that culture as 3D aggregates or as spheroids can increase the therapeutic potential of the adult stem/progenitor cells referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). Here we used a hanging drop protocol to prepare human MSCs (hMSCs) as spheroids that maximally expressed TNFα stimulated gene/protein 6 (TSG-6), the antiinflammatory protein that was expressed at high levels by hMSCs trapped in the lung after i.v. infusion and that largely explained the beneficial effects of hMSCs in mice with myocardial infarcts. The properties of spheroid hMSCs were found to depend critically on the culture conditions. Under optimal conditions for expression of TSG-6, the hMSCs also expressed high levels of stanniocalcin-1, a protein with both antiinflammatory and antiapoptotic properties. In addition, they expressed high levels of three anticancer proteins: IL-24, TNFα-related apoptosis inducing ligand, and CD82. The spheroid hMSCs were more effective than hMSCs from adherent monolayer cultures in suppressing inflammatory responses in a coculture system with LPS-activated macrophages and in a mouse model for peritonitis. In addition, the spheroid hMSCs were about one-fourth the volume of hMSCs from adherent cultures. Apparently as a result, larger numbers of the cells trafficked through the lung after i.v. infusion and were recovered in spleen, liver, kidney, and heart. The data suggest that spheroid hMSCs may be more effective than hMSCs from adherent cultures in therapies for diseases characterized by sterile tissue injury and unresolved inflammation and for some cancers that are sensitive to antiinflammatory agents.

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing β-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of β-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and β-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote β-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

IntroductionConsiderable efforts are currently being made to explore the therapeutic potentials of the stem/progenitor cells from bone marrow referred to initially as colony forming units-fibroblastic, then as marrow stromal cells, subsequently as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells (MSCs). [1][2][3][4][5][6] The cells are readily isolated from small aspirates of bone marrow from normal human donors or patients, they expand rapidly for 30 or more population doublings in culture, and they can differentiate into several cellular phenotypes in culture and in vivo. The therapeutic potentials of the cells have been tested in animal models and in clinical trials for a large number of diseases (see www.clinicaltrials.gov). Initially, it was assumed that the cells repaired tissues by engrafting and differentiating to replace injured cells. Engraftment with differentiation was observed in some animal models such as those with severe injuries to tissues, in embryos, or with local infusions of high concentrations of the cells. In most experimental situations, however, repair with functional improvements was observed without evidence of long-term engraftment. Therefore, most of the beneficial effects were explained by paracrine secretions or cell-to-cell contacts that had multiple effects including modulation of inflammatory or immune reactions. [6][7][8][9][10] Of special importance were the observations that although MSCs in culture secreted a large number of cytokines, 11,12 they were activated by cross-talk with injured cells to express high levels of additional therapeutic factors. 10,13 Previously we observed 14 that intravenously infused human MSCs (hMSCs) improved a mouse model for myocardial infarction in part because the hMSCs were trapped in the lung as microemboli and the injury produced by the microemboli activated the cells to secrete the anti-inflammatory protein TNF-␣-stimulated gene 6 protein (TSG-6). TSG-6 suppressed inflammatory reactions triggered by ischemia in the heart and thereby limited destruction of cardiomyocytes by invading neutrophils and monocytes/macrophages. TSG-6 is a 30 kD glycoprotein that was shown to produce anti-inflammatory effects in several animal models. 15,16 In transgenic mice, inactivation of the gene increased inflammatory responses, 17 and over-expression of the gene decreased inflammatory responses. 18 In addition, administration of the recombinant protein improved arthritis and decreased inflammation in several murine models. 19,20 To better understand the anti-inflammatory effects of hMSCs, we used the model of zymosan-induced peritonitis in mice. 21,22 The results indicated that hMSCs decreased inflammation in the model in part because the hMSCs were activated by the initial inflammatory microenvironment of the peritoneal cavity to secrete TSG-6. TSG-6 then produced a CD44-dependent decrease in the zymosan/ TLR2-mediated stimulation of NF-B signaling in resident macrophages. In effect, the hMSCs and TSG-6 generated a negativef...