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Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Until a decade ago it was believed that the wall of adult blood vessels exclusively contains terminally differentiated cell types. A paradigm shift was unavoidable since studies from different groups convincingly showed the presence of vascular wall-resident stem and progenitor cells (VW-SCs) which were identified to particularly reside in the sub-endothelial space and the so-called adventitial "vasculogenic zone". Data published during the last decade uncloaked the fact that VW-SCs have the capacity to differentiate into both vascular and non-vascular cell types. Up to date, little is known about the full capacity of VW-SCs, the exact composition of their endogenous niche and the mechanisms that govern their self-renewal, activation and differentiation. The aim of this review is to provide an overview about the current knowledge on VW-SCs and to highlight the impact of this endogenous niche on health and disease. In addition, we will discuss strategies how these adult stem cells could be manipulated in order to activate and expand them, ideally within their niche at sites of tissue damage and subsequently differentiate them into a desired cell type, e.g. an endothelial cell, a macrophage or a muscle cell. This would pave the way towards new pharmacological strategies for endogenous tissue repair and regeneration., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Direct cell conversion developed into an important paradigm for generating cells with enhanced differentiation capability. We combined a transcription-factor-based cell fate conversion strategy with the use of pharmacological compounds to derive early neuroepithelial progenitor cells from developmentally more restricted radial glia-type neural stem cells. By combining the small molecules CHIR99021, Tranylcypromine, SB431542 and valproic acid with viral transduction of the transcription factor c-Myc and the POU domain transcription factor Brn2, we dedifferentiated radial glia-type neural stem cells into an early neuroepithelial progenitor cell state within 6 days. Reverse transcription PCR analyses showed a rapid down-regulation of the radial glia markers Olig2 and Vimentin during conversion, whereas the neuroepithelial markers Dach1 and Sox1 were fastly up-regulated. Furthermore, a switch from N-Cadherin to E-Cadherin indicates a mesenchymal-to-epithelial transition. The differentiation of cells converted by Brn2/c-Myc yielded smooth muscle actin- and Peripherin-positive cells in addition to the neuronal marker TUJ1 and cells that are positive for the glial marker GFAP. This differentiation potential suggests that the applied reprogramming strategy induced an early neuroepithelial cell population, which might resemble cells of the neural border or even more primitive neuroepithelial cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated β-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27(KIP1) (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27(KIP1) gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene., (© 2016. Published by The Company of Biologists Ltd.)

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The biological phenomenon of cell fusion plays an important role in several physiological processes, like fertilization, placentation, or wound healing/tissue regeneration, as well as pathophysiological processes, such as cancer. Despite this fact, considerably less is still known about the factors and conditions that will induce the merging of two plasma membranes. Inflammation and proliferation has been suggested as a positive trigger for cell fusion, but it remains unclear, which of the factor(s) of the inflamed microenvironment are being involved. To clarify this we developed a reliable assay to quantify the in vitro fusion frequency of cells using a fluorescence double reporter vector (pFDR) containing a LoxP-flanked HcRed/DsRed expression cassette followed by an EGFP expression cassette. Because cell fusion has been implicated in cancer progression four human breast cancer cell lines were stably transfected with a pFDR vector and were co-cultured with the stably Cre-expressing human breast epithelial cell line. Cell fusion is associated with a Cre-mediated recombination resulting in induction of EGFP expression in hybrid cells, which can be quantified by flow cytometry. By testing a panel of different cytokines, chemokines, growth factors and other compounds, including exosomes, under normoxic and hypoxic conditions our data indicate that the proinflammatory cytokine TNF-α together with hypoxia is a strong inducer of cell fusion in human MDA-MB-435 and MDA-MB-231 breast cancer cells.

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

A comprehensive understanding of the functional network of transcription factors establishing and maintaining pluripotency is key for the development of biomedical applications of stem cells. Nanog plays an important role in early development and is essential to induce natural pluripotency in embryonic stem cells (ESCs). Inducible gain-of-function systems allowing a precise control over time and dosage of Nanog activity would be highly desirable to study its vital role in the establishment and maintenance of pluripotency at molecular level. Here we engineered a recombinant cell permeable version of Nanog by fusing it with the cell penetrating peptide TAT. Nanog-TAT can be readily expressed in and purified from E. coli and binds to a consensus Nanog DNA sequence. At cellular level it enhances proliferation and self-renewal of ESCs in the absence of leukemia inhibitory factor (LIF). Nanog-TAT together with LIF acts synergistically as judged by enhanced clonogenicity and activation of an Oct4-promoter-driven GFP reporter gene. Furthermore Nanog-TAT, in the absence of LIF, promotes pluripotency by inhibiting endodermal specification in a Stat3-independent manner. Our results demonstrate that Nanog protein transduction is an attractive tool allowing control over dose and time of addition to the cells for studying the molecular control of pluripotency without genetic manipulation., (Copyright © 2014. Published by Elsevier B.V.)

Background: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells., Results: A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide., Conclusions: Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs. Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-beta as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-beta gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-beta following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-beta is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-beta under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

Dendritic cells are able to take up exogenous Ags and present Ag-derived peptides on MHC class I molecules, a process termed cross-presentation. The mannose receptor (MR), an endocytic receptor expressed on a variety of APCs, has been demonstrated to target soluble Ags exclusively toward cross-presentation. In this study, we investigated the role of the murine nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor with immunomodulatory properties, in MR-mediated endocytosis and cross-presentation of the model Ag OVA. We could demonstrate both in vitro and in vivo that activation of PPARgamma resulted in increased MR expression, which in consequence led to enhanced MR-mediated endocytosis and elevated cross-presentation of soluble OVA. Concomitantly, activation of PPARgamma in dendritic cells induced up-regulation of the coinhibitory molecule B7H1, which, despite enhanced cross-presentation, caused an impaired activation of naive OVA-specific CD8(+) T cells and the induction of T cell tolerance. These data provide a mechanistic basis for the immunomodulatory action of PPARgamma which might open new possibilities in the development of therapeutic approaches aimed at the control of excessive immune responses, e.g., in T cell-mediated autoimmunity.

There is increasing evidence that dendritic cell (DC) immunogenicity is not only positively regulated by ligands of pattern recognition receptors, but also negatively by signals that prevent DC activation and full functional maturation. Depending on their activation status, DCs can induce either immunity or tolerance. In this study, we provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of DC maturation and function. Sustained PPARgamma activation in murine DCs reduced maturation-induced expression of costimulatory molecules and IL-12, and profoundly inhibited their capacity to prime naive CD4(+) T cells in vitro. Using PPARgamma-deficient DCs, generated by Cre-mediated ablation of the PPARgamma gene, agonist-mediated suppression of maturation-induced functional changes were abrogated. Moreover, absence of PPARgamma increased DC immunogenicity, suggesting a constitutive regulatory function of PPARgamma in DCs. Adoptive transfer of PPARgamma-activated Ag-presenting DCs induced CD4(+) T cell anergy, characterized by impaired differentiation resulting in absent Th1 and Th2 cytokine production and failure of secondary clonal expansion upon restimulation. Collectively, our data support the notion that PPARgamma is an efficient regulator of DC immunogenicity that may be exploited to deliberately target CD4(+) T cell-mediated immune responses.

Embryonic stem (ES) cells have become a major focus of scientific interest both as a potential donor source for regenerative medicine and as a model system for tissue development and pathobiology. Tight and efficient methods for genetic engineering are required to exploit ES cells as disease models and to generate specific somatic phenotypes by lineage selection or instruction. In 1990s, the application of site-specific recombinases (SSRs) such as Cre has revolutionized mammalian genetics by providing a reliable and efficient means to delete, insert, invert, or exchange chromosomal DNA in a conditional manner. Despite these significant advances, the available technology still suffers from limitations, including unwanted side effects elicited by the random integration of Cre expression vectors and leak activity of inducible or presumptive cell type-specific Cre expression systems. These challenges can be met by combining the Cre/loxP recombination system with direct intracellular delivery of Cre by protein transduction, thus enabling rapid and highly efficient conditional mutagenesis in ES cells and ES cell-derived somatic progeny. Modified recombinant variants of Cre protein induce recombination in virtually 100% of human ES (hES) and mouse ES (mES) cells. Here, we present methods for generating purified transducible Cre protein from Escherichia coli and its transduction into ES cells and their neural progeny.

Conditional mutagenesis is a powerful tool to analyze gene functions in mammalian cells. The site-specific recombinase Cre can be used to recombine loxP-modified alleles under temporal and spatial control. However, the efficient delivery of biologically active Cre recombinase to living cells represents a limiting factor. In this study we compared the potential of a hydrophobic peptide modified from Kaposi fibroblast growth factor with a basic peptide derived from HIV-TAT to promote cellular uptake of recombinant Cre. We present the production and characterization of a Cre protein that enters mammalian cells and subsequently performs recombination with high efficiency in a time- and concentration-dependent manner. Histidine-tagged Cre recombinase transduced inefficiently unless fused to a nuclear localization signal (NLS). Fusion of NLS-Cre to the fibroblast growth factor transduction peptide did not improve the transducibility, whereas fusion with the TAT peptide significantly enhanced cellular uptake and subsequent recombination. More than 95% recombination efficiency in fibroblast cells, as well as murine embryonic stem cells, was achieved after His-TAT-NLS-Cre transduction. Efficient recombination could also be obtained in primary splenocytes ex vivo. We expect that application of His-TAT-NLS-Cre, which can be produced readily in large quantities from a bacterial source, will expand our abilities to manipulate mammalian genomes.