Tumor-associated macrophages (TAM) form a major component of the tumor stroma. However, important concepts such as TAM heterogeneity and the nature of the monocytic TAM precursors remain speculative. Here, we show for the first time that mouse mammary tumors contained functionally distinct subsets of TAMs and provide markers for their identification. Furthermore, in search of the TAM progenitors, we show that the tumor-monocyte pool almost exclusively consisted of Ly6C hi CX 3 CR1 low monocytes, which continuously seeded tumors and renewed all nonproliferating TAM subsets. Interestingly, gene and protein profiling indicated that distinct TAM populations differed at the molecular level and could be classified based on the classic (M1) versus alternative (M2) macrophage activation paradigm. Importantly, the more M2-like TAMs were enriched in hypoxic tumor areas, had a superior proangiogenic activity in vivo, and increased in numbers as tumors progressed. Finally, it was shown that the TAM subsets were poor antigen presenters, but could suppress T-cell activation, albeit by using different suppressive mechanisms. Together, our data help to unravel the complexities of the tumor-infiltrating myeloid cell compartment and provide a rationale for targeting specialized TAM subsets, thereby optimally "re-educating" the TAM compartment. Cancer Res; 70(14); 5728-39. ©2010 AACR.

Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose-responsive, and reinstate normal glycemic control for up to 248 days. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3) expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by

High rates of glucose metabolism and mitochondrial electron transport have been associated with increased mitochondrial production of reactive oxygen species (ROS). This mechanism was also proposed as a possible cause for dysfunction and death of pancreatic ␤ cells exposed to high glucose levels. We examined whether high rates of glucose metabolism increase ROS production in purified rat ␤ cells. Glucose up to 20 mM did not stimulate H 2 O 2 or superoxide production, whereas it dose-dependently increased cellular NAD(P)H and FADH 2 levels with an EC 50 around 8 mM. On the contrary, glucose concentration-dependently suppressed H 2 O 2 and superoxide formation, with a major effect between 0 and 5 mM, parallel to an increase in cellular NAD(P)H levels. This suppressive effect was more marked in ␤ cells with higher NAD(P)H responsiveness to glucose; it was not observed in glucagon-containing ␣ cells, which lacked a glucose-induced increase in NAD(P)H. Suppression was also induced by the mitochondrial substrates leucine and succinate. Experiments with electron transport chain inhibitors indicate a role of respiratory complex I in ROS production at low mitochondrial activity and low NADH levels. Superoxide production at low glucose is potentially cytotoxic, because scavenging by the superoxide dismutase mimetic agent manganese(III)tetrakis(4-benzoic acid)porphyrin was found to reduce the rate of ␤ cell apoptosis. Analysis of islets cultured at 20 mM glucose confirmed that this condition does not induce ROS production in ␤ cells as a result of their increased rates of glucose metabolism. Our study indicates the need of ␤ cells for basal nutrients maintaining mitochondrial NADH production at levels that suppress ROS accumulation from an inadequate respiratory complex I activity and thus inhibit a potential apoptotic pathway.Chronically elevated glucose levels cause major lesions in retina, kidney, neurons, and vascular tissue (1). The toxic effects of hyperglycemia can be explained by mechanisms that involve increased glucose metabolism (2, 3). A primary role is often attributed to an excessive formation of reactive oxygen species (ROS) 1 at the mitochondrial electron transport chain (ETC) of cells in an early or late stage of glucose-induced dysfunction (4 -6). In endothelial cell lines, glucose-induced ROS production appeared a critical upstream event in the process of hyperglycemic damage (4). Re-oxidation of glucosegenerated NADH and FADH 2 by the ETC results in a progressively increasing proton gradient across the mitochondrial membrane. In isolated mitochondria such a condition increases superoxide production once the gradient reaches a critical threshold (3, 7). Elevated glucose levels are also suspected of causing dysfunction and/or death of insulin-producing ␤ cells through ROS formation (8, 9). It is unknown whether their toxicity in ␤ cells also involves glucose-induced elevations in cellular NADH and FADH 2 levels up to the point that mitochondrial energetic coupling is disrupted and followed by ROS formatio...

Therapy Consortium EU-FP7. Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

In this study, we present a new method to obtain pure, viable, freshly isolated hepatic stellate cells. Stellate cells were purified by cell sorting using their high side scatter (SSC) of incident light. Purity of the cells was established by light and transmission electron microscopy (TEM). Starting from stellate cells that were 50% to 70% enriched by centrifugation in 11% Nycodenz, the cell purity after sorting was found to be 96.6% ؎ 2.

Interleukin (IL)-1␤ is known to cause ␤-cell death in isolated rat islets. This effect has been attributed to induction of nitric oxide (NO) synthase in ␤-cells and subsequent generation of toxic NO levels; it was not observed, however, in dispersed rat ␤-cells. The present study demonstrates that IL-1␤ induces NO-dependent necrosis in rat ␤-cells cultured for 3 days at high cell density or in cell aggregates but not as single cells. Its cytotoxic condition is not explained by higher NO production rates but might result from higher intercellular NO concentrations in statically cultured cell preparations with cell-to-cell contacts; nitrite levels in collected culture medium are not a reliable index for these intercellular concentrations. Absence of IL-1-induced necrosis in rat ␣-cells or in human ␤-cells is attributed to the cytokine's failure to generate NO in these preparations, not to their reduced sensitivity to NO: the NO donor GEA 3162 (15 min, 50 -100 mol/l) exerts a comparable necrotic effect in rat and human ␣-or ␤-cells. In preparations in which IL-1␤ does not cause ␤-cell necrosis, its combination with ␥-interferon (IFN-␥) results in NO-independent apoptosis, starting after 3 days and increasing with the duration of exposure. Because IFN-␥ alone was apoptotic for rat ␣-cells, it is proposed that IL-1␤ can make ␤-cells susceptible to this effect, conceivably through altering their phenotype. It is concluded that IL-1␤ can cause NO-dependent necrosis or NO-independent apoptosis of islet cells, depending on the species and on the environmental conditions. The experiments in isolated human ␤-cell preparations suggest that these cells may preferentially undergo apoptosis when exposed to IL-1␤ plus IFN-␥ unless neighboring non-␤-cells produce toxic NO levels. Diabetes 50:551-557, 2001 I nterleukin (IL)-1␤ has been proposed as an effector in the immune-mediated destruction of pancreatic ␤-cells (1,2). Its putative role in the development of type 1 diabetes is based on in vitro observations indicating an IL-1-induced toxicity in isolated rodent islets (3,4). This cytotoxic effect involves induction of nitric oxide (NO) synthase in the islet ␤-cells and subsequent production of toxic NO concentrations (5-10). It is amplified by a simultaneous presence of ␥-interferon (IFN-␥) and tumor necrosis factor (TNF)-␣ (11,12). This combination of cytokines is also cytotoxic in human islet preparations (13,14). There is, however, a marked variation in the degree of direct IL-1 toxicity on islet ␤-cells (15). Failure to detect a destructive effect of IL-1␤ has been attributed to differences in species or in the functional state of the ␤-cells (16 -18). We previously noticed that IL-1 failed to kill single purified rat ␤-cells while causing the earlierdescribed cell death in intact rat islets (19). The IL-1-induced cytotoxicity was not observed in monolayer rat islet cells (11,20). These observations support the view that the IL-1-induced toxicity in intact islets is mediated, at least in part, through its effect on adjacen...

SummaryHuman stem cells represent a potential source for implants that replace the depleted functional beta cell mass (FBM) in diabetes patients. Human embryonic stem cell-derived pancreatic endoderm (hES-PE) can generate implants with glucose-responsive beta cells capable of reducing hyperglycemia in mice. This study with device-encapsulated hES-PE (4 × 106 cells/mouse) determines the biologic characteristics at which implants establish metabolic control during a 50-week follow-up. A metabolically adequate FBM was achieved by (1) formation of a sufficient beta cell number (>0.3 × 106/mouse) at >50% endocrine purity and (2) their maturation to a functional state comparable with human pancreatic beta cells, as judged by their secretory responses during perifusion, their content in typical secretory vesicles, and their nuclear NKX6.1-PDX1-MAFA co-expression. Assessment of FBM in implants and its correlation with in vivo metabolic markers will guide clinical translation of stem cell-derived grafts in diabetes.