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Microglia,the immune cells of the brain,are crucial to proper development and maintenance of the CNS,and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology,we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes,expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca(2+) transients,whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines. View Publication

Economic development has become a prominent issue for state governments. Nevertheless,states vary in the economic policies they choose. Two general approaches to the issue are discussed: the maintenance/attraction strategy and the creation strategy. Factor analysis allows us to gauge state effort on these two criteria. Regression analysis shows that political culture is an important factor in predicting which approach a state chooses,with traditionalistic states favoring the maintenance/attraction strategy,and moralistic states favoring the creation alternative. Other predictors of state policy choices include the condition of the economy and the diffusion of innovations. Also discussed is the interaction of political culture with other relevant variables in shaping state policies.

Tissue-repair regulatory T cells (trTregs) comprise a specialized cell subset essential for tissue homeostasis and repair. While well-studied in sterile injury models,their role in infection-induced tissue damage and antimicrobial immunity is less understood. We investigated trTreg dynamics during acute Trypanosoma cruzi infection,marked by extensive tissue damage and strong CD8+ immunity. Unlike sterile injury models,trTregs significantly declined in secondary lymphoid organs and non-lymphoid target tissues during infection,correlating with systemic and local tissue damage,and downregulation of function-associated genes in skeletal muscle. This decline was linked to decreased systemic IL-33 levels,a key trTreg growth factor,and promoted by the Th1 cytokine IFN-γ. Early recombinant IL-33 treatment increased trTregs,type 2 innate lymphoid cells,and parasite-specific CD8+ cells at specific time points after infection,leading to reduced tissue damage,lower parasite burden,and improved disease outcome. Our findings not only provide novel insights into trTregs during infection but also highlight the potential of optimizing immune balance by modulating trTreg responses to promote tissue repair while maintaining effective pathogen control during infection-induced injury. Author summaryDuring Chagas’ disease,caused by the protozoan Trypanosoma cruzi,severe organ damage is generated by the interplay between the parasite and the immune response. In our investigation,we examined the role of tissue-repair regulatory T cells (trTregs) during the acute phase of T. cruzi infection in mice. Surprisingly,we observed a reduction in trTregs at the peak of tissue damage,contrary to their usual accumulation after injury in other contexts. This decline aligned with decreased levels of interleukin-33,a critical factor for trTreg survival,and was promoted by the effector cytokine IFN-γ. Administering interleukin-33 at early infection times not only boosted trTregs but also expanded other reparative and antiparasitic immune cells. Consequently,these treated mice exhibited reduced damage and lower parasite levels in tissues. Our findings provide new insights into how trTreg function during infection-related injury,paving the way for strategies that balance the immune response to support tissue repair without weakening the body’s ability to fight the infection. This approach could have broader implications for treating infectious diseases and conditions involving tissue damage. View Publication

Bone marrow mesenchymal stem cells (MSC) have potent immunosuppressive properties and have been advocated for therapeutic use in humans. The nature of their suppressive capacity is poorly understood but is said to be a primitive stem cell function. Demonstration that adult stromal cells such as fibroblasts (Fb) can modulate T cells would have important implications for immunoregulation and cellular therapy. In this report,we show that dermal Fb inhibit allogeneic T cell activation by autologously derived cutaneous APCs and other stimulators. Fb mediate suppression through soluble factors,but this is critically dependent on IFN-gamma from activated T cells. IFN-gamma induces IDO in Fb,and accelerated tryptophan metabolism is at least partly responsible for suppression of T cell proliferation. T cell suppression is reversible,and transient exposure to Fb during activation reprograms T cells,increasing IL-4 and IL-10 secretion upon restimulation. Increased Th2 polarization by stromal cells is associated with amelioration of pathological changes in a human model of graft-vs-host disease. Dermal Fb are highly clonogenic in vitro,suggesting that Fb-mediated immunosuppression is not due to outgrowth of rare MSC,although dermal Fb remain difficult to distinguish from MSC by phenotype or transdifferentiation capacity. These results suggest that immunosuppression is a general property of stromal cells and that dermal Fb may provide an alternative and accessible source of cellular therapy. View Publication

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy,including generation of pancreatic β-cells for diabetes research and treatment. In this study,we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation,and then combined treatment with retinoic acid,a bone morphogenic protein inhibitor,and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover,several compounds,including forskolin,dexamethasone,and a TGF-β inhibitor,were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds,more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition,all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol. View Publication

The enteric nervous system (ENS) is recognized as a second brain because of its complexity and its largely autonomic control of bowel function. Recent progress in studying the interactions between the ENS and the central nervous system (CNS) has implicated alterations of the gut/brain axis as a possible mechanism in the pathophysiology of autism spectrum disorders (ASDs),Parkinson's disease (PD) and other human CNS disorders,whereas the underlying mechanisms are largely unknown because of the lack of good model systems. Human induced pluripotent stem cells (hiPSCs) have the ability to proliferate indefinitely and differentiate into cells of all three germ layers,thus making iPSCs an ideal source of cells for disease modelling and cell therapy. Here,hiPSCs were induced to differentiate into neural crest stem cells (NCSCs) efficiently. When co-cultured with smooth muscle layers of ganglionic gut tissue,the NCSCs differentiated into different subtypes of mature enteric-like neurons expressing nitric oxide synthase (nNOS),vasoactive intestinal polypeptide (VIP),choline acetyltransferase (ChAT) or calretinin with typical electrophysiological characteristics of functional neurons. Furthermore,when they were transplanted into aneural or aganglionic chick,mouse or human gut tissues in ovo,in vitro or in vivo,hiPSC-derived NCSCs showed extensive migration and neural differentiation capacity,generating neurons and glial cells that expressed phenotypic markers characteristic of the enteric nervous system. Our results indicate that enteric NCSCs derived from hiPSCs supply a powerful tool for studying the pathogenesis of gastrointestinal disorders and brain/gut dysfunction and represent a potentially ideal cell source for enteric neural transplantation treatments.Molecular Psychiatry advance online publication,25 October 2016; doi:10.1038/mp.2016.191. View Publication

The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases,their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients,enabling the study of disease-associated mutations and,when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately,the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study,we optimized our previously published protocol by fine-tuning the initial cell number,exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple,cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB,HNF4?,and CYP3A4) and protein (ALB) expression,as well as significantly elevated inducible CYP3A4 activity. Graphical Abstract View Publication

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However,attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions,in up to 63% of 59 low-volume UCB units,cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition,osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However,in contrast to MSCs,MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours,a net volume of more than 33 ml,and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations,we regard UCB as an additional stem cell source for experimental and potentially clinical purposes. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

NOTCH signaling is critical for specifying the intestinal epithelial cell lineage and for initiating colorectal adenomas and colorectal cancers (CRC). Based on evidence that NOTCH is important for the maintenance and self-renewal of cancer-initiating cells in other malignancies,we studied the role of NOTCH signaling in colon cancer-initiating cells (CCIC). Tumors formed by CCICs maintain many properties of the primary CRCs from which they were derived,such as glandular organization,cell polarity,gap junctions,and expression of characteristic CRC molecular markers. Furthermore,CCICs have the property of self-renewal. In this study,we show that NOTCH signaling is 10- to 30-fold higher in CCIC compared with widely used colon cancer cell lines. Using small-molecule inhibition and short hairpin RNA knockdown,we show that NOTCH prevents CCIC apoptosis through repression of cell cycle kinase inhibitor p27 and transcription factor ATOH1. NOTCH is also critical to intrinsic maintenance of CCIC self-renewal and the repression of secretory cell lineage differentiation genes such as MUC2. Our findings describe a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest that inhibition of NOTCH signaling may improve CRC chemoprevention and chemotherapy. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication