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Thrombopoietin (Thpo)/myeloproliferative leukemia virus oncogene (Mpl) signaling controls hematopoietic stem cell (HSC) self-renewal and quiescence; however, how these 2 seemingly opposing functions are controlled is not well understood. By transplantation of lentiviral-transduced hematopoietic cells in the Mpl-deficient mouse model, we addressed whether known or predicted Thpo target genes were able to rescue the Mpl-deficient phenotype of the mice. Among the tested genes, we identified endothelial protein C receptor (Epcr) to expand HSCs with the long-term (LT)-HSC surface phenotype in Mpl−/− mice and to enable secondary transplantation of Mpl-deficient bone marrow (BM). Epcr-transduced Mpl−/− HSCs enter quiescence earlier after transplantation than control-transduced Mpl−/− cells, and upregulated expression of the anti-apoptotic gene Bcl-xL. Also, in the wild-type background, Epcr expression marked the engrafting population in the BM. Furthermore, Epcr expression in Mpl−/− hematopoiesis increased the number of megakaryocytes in the BM. In vitro Thpo supported the surface expression of Epcr on primary murine hematopoietic stem and progenitor cells. With these data, we add new insights into Thpo-dependent influence on HSC engraftment after transplantation. This may be of use for the in vitro manipulation of HSCs, also in the context of gene therapy.

Until now, hues as dynamic as those adorning the Apatura emperor butterflies have never been encountered in the painting world. Unlike and unmatched by the chemical pigments traditionally found on the painter's palette, the emperor's wings are studded with strongly reflecting iridescent scales that are structured like those of the iconic Morpho butterflies. The scale ridges act as diffractive multilayers, giving rise to narrow-band reflectance spectra. All scales together create a vividly purple iridescent wing colouration that is observed within a narrow angular range only. Recently, synthetic structures analogous to the multilayer reflectors found on butterfly wings have been developed, referred to as effect pigments. Artists can obtain vital clues for how to adapt and adopt these challenging new materials for painting, by tracing the origin of biomimetics back to the ancient concept of mimesis and building on the knowledge accumulated by optical studies. By selecting various effect pigments, and using the lesser purple emperor butterfly, Apatura ilia, as exemplar, we have accurately mimicked the butterfly's iridescence in art. The resulting artwork, like the butterfly, fluctuates in perceived colour depending on the direction of illumination and viewing. These nature-inspired-colouration and biomimetic-application methods extend the canon of art.

Background Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. Objectives To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). Methods To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. Results Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. Conclusion We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

Until now, hues as dynamic as those adorning the Apatura emperor butterflies have never been encountered in the painting world. Unlike and unmatched by the chemical pigments traditionally found on the painter's palette, the emperor's wings are studded with strongly reflecting iridescent scales that are structured like those of the iconic Morpho butterflies. The scale ridges act as diffractive multilayers, giving rise to narrow-band reflectance spectra. All scales together create a vividly purple iridescent wing colouration that is observed within a narrow angular range only. Recently, synthetic structures analogous to the multilayer reflectors found on butterfly wings have been developed, referred to as effect pigments. Artists can obtain vital clues for how to adapt and adopt these challenging new materials for painting, by tracing the origin of biomimetics back to the ancient concept of mimesis and building on the knowledge accumulated by optical studies. By selecting various effect pigments, and using the lesser purple emperor butterfly, Apatura ilia, as exemplar, we have accurately mimicked the butterfly's iridescence in art. The resulting artwork, like the butterfly, fluctuates in perceived colour depending on the direction of illumination and viewing. These nature-inspired-colouration and biomimetic-application methods extend the canon of art.

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Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro.

Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site. In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.

Long-term hematopoietic stem cells (LT-HSC) persist in quiescence to maintain their hematopoietic potential throughout life. In the case of need LT-HSC can be activated to replenish the pool of blood cells. We investigated the impact of acute influenza A virus (IAV) infection on hematopoiesis in C57Bl/6N mice, focusing on the most immature HSC and progenitors. Mice were infected with a lethal dose of IAV PR/8/1934 H1N1 (humane endpoints reached within 6 days post infection (dpi)). In two further groups, mice were treated daily with oseltamivir (antiviral neuraminidase inhibitor, dpi 0-4) or were vaccinated with single-cycle vesicular stomatitis virus replicon particles expressing a miss-matched neuraminidase from influenza A virus Yamaguchi/7/2004 H5N1 four weeks prior to infection. Both treatments rescued mice from infection-induced mortality. Every day 6-9 mice were analyzed for differences in the bone marrow (BM) and blood by flow cytometry and multiplex cytokine assays as well as in the lung to determine viral tissue titers and histopathology. HSC functionality was analyzed in a competitive BM transplantation of infected and non-infected mice. Irrespective of the treatment, high IAV lung tissue titers (≥5x106 tissue culture infectious dose 50) in the first days post infection (dpi 1-5) were associated with activation of HSC into the cell cycle. LT-HSCs (LSK, CD150+, CD34- and CD48-) were 50% less quiescent and shifted into the G1/S-G2-M phase (dpi 2-6) and returned to quiescence state after virus clearance (dpi 10). Furthermore, we detected 1.5-fold increase in proliferation of phenotypic LT-HSC. Differentiation was increased towards lymphoid progenitors (≥3-fold more compared to non-infected mice) during the acute phase of infection in untreated and oseltamivir treated mice and myeloid progenitors were reduced ~50% in all groups (dpi 4-8). We found the inflammatory cytokines IFNγ, IL-1α, IL-6, and TNFα to be significantly upregulated in the BM of untreated and oseltamivir treated mice but less in vaccinated animals (dpi 2-4). IL-1α or IL-6 stimulation of LT-HSCs was sufficient to initiate proliferation in cell culture. In all groups the initial drop of the peripheral platelet count (~30% lower compared to non-infected mice, dpi 2) was replenished with an excessive production of platelets (~45% increased, dpi 8-15). Histopathology and electron microscopy revealed the sequestration and accumulation of platelets in pulmonary capillaries and vessels. Subsequently, we detected twice as many mature megakaryocytes in the BM (dpi 2-4) and elevated CD41 expression on HSCs (LSK, CD150+ and CD34-; 3-fold more compared to non-infected mice dpi 2-6) indicating a myeloid/platelet-biased HSC compartment in response to infection. Competitive whole BM transplantation with activated LT-HSCs from the acute phase of infection vs non-infected mice showed delayed reconstitution of T-cells but a preferential differentiation towards platelets in recipient mice. Taken together, local IAV infection in the lung substantially affected LT-HSC quiescence and differentiation by inflammatory cytokines with systemic consequences and a myeloid/platelet-biased lineage output. Disclosures No relevant conflicts of interest to declare.

Retroviral vectors have shown their curative potential in clinical trials correcting monogenetic disorders. However, therapeutic benefits were compromised due to vector-induced dysregulation of cellular genes and leukemia development in a subset of patients. Bromodomain and extraterminal domain (BET) proteins act as cellular cofactors that tether the murine leukemia virus (MLV) pre-integration complex to host chromatin via interaction with the MLV integrase (IN) and thereby define the typical gammaretroviral integration distribution. We engineered next-generation BET-independent (Bin) MLV vectors to retarget their integration to regions where they are less likely to dysregulate nearby genes. We mutated MLV IN to uncouple BET protein interaction and fused it with chromatin-binding peptides. The addition of the CBX1 chromodomain to MLV INW390A efficiently targeted integration away from gene regulatory elements. The retargeted vector produced at high titers and efficiently transduced CD34+ hematopoietic stem cells, while fewer colonies were detected in a serial colony-forming assay, a surrogate test for genotoxicity. Our findings underscore the potential of the engineered vectors to reduce the risk of insertional mutagenesis without compromising transduction efficiency. Ultimately, combined with other safety features in vector design, next-generation BinMLV vectors can improve the safety of gammaretroviral vectors for gene therapy. publisher: Elsevier articletitle: Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy journaltitle: Molecular Therapy - Nucleic Acids articlelink: http://dx.doi.org/10.1016/j.omtn.2017.04.002 content_type: article copyright: © 2017 The Authors. ispartof: Molecular Therapy-Nucleic Acids vol:7 pages:231-245 ispartof: location:United States status: published

The shifting rainbow hues of iridescence have, until recently, remained exclusive to nature. Now, the latest advances in nanotechnology enable the introduction of novel, bio-inspired color-shifting flakes into painting—thereby affording artists potential access to the full spectacle of iridescence. Unfortunately, existing rules of easel painting do not apply to the new medium; but, as nature inspired the technology, an exploration of natural phenomena can best inform how to overcome this hurdle. Thus, by adopting a biomimetic approach, this paper outlines the optical principles underlying iridescence and provides technical ground rules for its incorporation into painting.

For centuries artists and natural scientists have been captivated by the colour changing effects of iridescence. Producing brilliant fl ashes of colour in the natural world, the phenomenon is best known in the displays of ‘living jewels’, e.g. tropical birds and butterfl ies, where the colour perceived changes with viewing angle. Such striking effects are not produced by chemical pigments but by complex physical structures interplaying with light. Until now, artists have tried to capture these luminous, oscillating colours with varying degrees of success. However, for the fi rst time, latest advances in ‘pigment’ technology offer artists the exciting, but challenging, potential to introduce the full spectacle of iridescence into painting. These ‘pigments’ (developed with lucrative industrial applications in mind) currently remain restricted to commercial usage. The major drawback seriously impeding their advancement in art is that they do not adhere to colour theory as applied in painting. Having worked on adapting iridescent technology from its inception, gradual emergence and now rapid expansion, the author traces the sustained effort necessary on her part to overcome the many inherent challenges. Interweaving the fi ndings of art theory, physics and personal studio practice, an attempt is made to position the new technology within the wider discourse on colour. And readers are furnished with an increased understanding of the scientifi c and aesthetic principles governing iridescence.

Color as dramatic, dynamic and dazzling as the iridescent hues on the wings of certain butterflies has never been encountered in the art world. Unlike and unmatched by the chemical pigments of the artists’ palette, this changeable color is created by transparent, colorless nanostructures that, as with prisms, diffract and reflect light to render spectral color visible. Until now, iridescent colors, by their very nature, have defied artists’ best efforts to fully capture these rainbow hues. Now, for the first time, the artist and researcher Franziska Schenk employs latest nature-inspired color-shift technology to actually simulate the iridescence of butterflies and beetles on canvas. Crucially, studying the ingenious ways in which a range of such displays are created by insects has provided the artist with vital clues on how to adapt and adopt these challenging optical nano-materials for painting. And indeed, after years of meticulous and painstaking research both in the lab and studio, the desired effect is achieved. The resulting paintings, like an iridescent insect, do in fact fluctuate in perceived color - depending on the light and viewing angle. In tracing the artist’s respective biomimetic approach, the paper not only provides an insight into the new color technology’s evolution and innovative artistic possibilities, but also suggests what artists can learn from nature.

Colours as dynamic as the metallic-like hues adorning the Japanese jewel beetle have never been captured on canvas before. Unlike, and unmatched by, the chemical pigments of the artist's palette, the effect is generated by layered microstructures that refract and reflect light to make colour visible. Exclusive to nature for millions of years, such jewel-like colouration is only now being introduced to art. Sustained scientific research into nature's iridescent multilayer reflectors has recently led to the development and manufacture of analogous synthetic structures, notably innovative light interference flakes. For the first time this novel technology offers artists the exciting, yet challenging, potential to accurately depict nature's iridescence. Mimicking the Japanese jewel beetle by using paints with embedded flakes, we demonstrate that the resulting painting, just like the model, displays iridescent colours that shift with minute variation of the angle of light and viewing.