Nanostructured Bifunctional Hydrogels as Potential Instructing Platform for Hematopoietic Stem Cell Differentiation

Hematopoietic stem cells (HSCs) are blood forming cells which possess the ability to differentiate into all types of blood cells. T cells are one important cell type HSCs can differentiate into, via corresponding progenitor cells. T cells are part of the adaptive immune system as they mediate cellular immune responses. Due to this crucial function, in vitro differentiated T cells are the subject of current studies in the biomedical field in terms of cell transplantation. Studies show that the density of the immobilized Notch ligand Delta-like 1 (DLL1) presented in HSCs' environment can stimulate their differentiation toward T cells. The development of reliable synthetic cell culture systems presenting variable densities of DLL1 is promising for the future expansion of T cells' clinical applications. Here we introduce bifunctional polyethylene glycol-based (PEG-based) hydrogels as a potential instructing platform for the differentiation of human hematopoietic stem and progenitor cells (HSPCs) to T cells. PEG hydrogels bearing the cell adhesion supporting motif RGD (arginyl-glycyl-aspartic acid) were synthesized by UV-light induced radical copolymerization of PEG diacrylate and RGD modified PEG acrylate. The hydrogels were furthermore nanostructured by incorporation of gold nanoparticle arrays that were produced by block copolymer micelle nanolithography (BCML). BCML allows for the decoration of surfaces with gold nanoparticles in a hexagonal manner with well-defined interparticle distances. To determine the impact of DLL1 density on the cell differentiation, hydrogels with particle distances of ~40 and 90 nm were synthesized and the gold nanoparticles were functionalized with DLL1. After 27 days in culture, HSPCs showed an unphysiological differentiation status and, therefore, the differentiation was evaluated as atypical T lymphoid differentiation. Cluster of differentiation (CD) 4 was the only tested T cell marker which was expressed clearly in all samples. Thus, although the applied nanopatterned hydrogels affected two important signaling pathways (integrins and Notch) for T cell differentiation, it appears that more functionalities that control T cell differentiation in nature need to be considered for achieving fully synthetic in vitro T cell differentiation strategies

Rebuilding the hematopoietic stem cell niche: Recent developments and future prospects

Hematopoietic stem cells (HSCs) have proven their clinical relevance in stem cell transplantation to cure patients with hematological disorders. Key to their regenerative potential is their natural microenvironment – their niche – in the bone marrow (BM). Developments in the field of biomaterials enable the recreation of such environments with increasing preciseness in the laboratory. Such artificial niches help to gain a fundamental understanding of the biophysical and biochemical processes underlying the interaction of HSCs with the materials in their environment and the disturbance of this interplay during diseases affecting the BM. Artificial niches also have the potential to multiply HSCs in vitro, to enable the targeted differentiation of HSCs into mature blood cells or to serve as drug-testing platforms. In this review, we will introduce the importance of artificial niches followed by the biology and biophysics of the natural archetype. We will outline how 2D biomaterials can be used to dissect the complexity of the natural niche into individual parameters for fundamental research and how 3D systems evolved from them. We will present commonly used biomaterials for HSC research and their applications. Finally, we will highlight two areas in the field of HSC research, which just started to unlock the possibilities provided by novel biomaterials, in vitro blood production and studying the pathophysiology of the niche in vitro. With these contents, the review aims to give a broad overview of the different biomaterials applied for HSC research and to discuss their potentials, challenges and future directions in the field. Statement of significance Hematopoietic stem cells (HSCs) are multipotent cells responsible for maintaining the turnover of all blood cells. They are routinely applied to treat patients with hematological diseases. This high clinical relevance explains the necessity of multiplication or differentiation of HSCs in the laboratory, which is hampered by the missing natural microenvironment – the so called niche. Biomaterials offer the possibility to mimic the niche and thus overcome this hurdle. The review introduces the HSC niche in the bone marrow and discusses the utility of biomaterials in creating artificial niches. It outlines how 2D systems evolved into sophisticated 3D platforms, which opened the gateway to applications such as, expansion of clinically relevant HSCs, in vitro blood production, studying niche pathologies and drug testing

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Search for stop and higgsino production using diphoton Higgs boson decays

Results are presented of a search for a "natural" supersymmetry scenario with gauge mediated symmetry breaking. It is assumed that only the supersymmetric partners of the top-quark (stop) and the Higgs boson (higgsino) are accessible. Events are examined in which there are two photons forming a Higgs boson candidate, and at least two b-quark jets. In 19.7 inverse femtobarns of proton-proton collision data at sqrt(s) = 8 TeV, recorded in the CMS experiment, no evidence of a signal is found and lower limits at the 95% confidence level are set, excluding the stop mass below 360 to 410 GeV, depending on the higgsino mass

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia

Measurement of associated W plus charm production in pp collisions at √s=7 TeV

Peer reviewe

Multifunktionale nanostrukturierte Hydrogele zur Kultur hämatopoetischer Stammzellen

Um die funktionellen Interaktionen hämatopoetischer Stamm- und Progenitorzellen (HSPZ) mit ihrer physiologischen Nische im Knochenmark zu verstehen, sind artifizielle Nischenmodelle zur Untersuchung einzelner biochemischer und biophysikalischer Eigenschaften der Nische hervorragend geeignet. In dieser Arbeit wurde ein zweidimensionales, nanostrukturiertes, heparinbasiertes Biohybridhydrogel entwickelt, um multiple Funktionen für die in vitro HSPZ-Kultur nachzustellen. Dabei standen drei elementare Funktionalitäten der Nische im Vordergrund: (i) die Matrixelastizität, (ii) die Bindung und Freisetzung von Zytokinen und (iii) die nanostrukturelle Präsentation von extrazellulären bzw. zellulären α4β1 Integrin-Peptidliganden. Bei unveränderter Matrixelastizität innerhalb der physiologischen Knochenmarkssteifigkeit wurden durch Variation der Nanostrukturierung unterschiedliche Dichten von zellulärem IDSP oder extrazellulärem LDV als Integrin α4β1-spezifische Bindemotive realisiert. Zusätzlich war die Gelmatrix in der Lage, das Chemokin SDF-1α spezifisch zu binden und wieder freizusetzen. Der Einfluss der multifunktionalen Geleigenschaften wurde an humanen CD34+ HSPZ in Form von Zellpolarisierung, Zellmotilität, Proliferation und Ausprägung der Differenzierung untersucht. Dabei konnte in dieser Arbeit Folgendes beobachtet werden. Der Polarisierungsgrad und die Motilität der HSPZ-Kulturen zeigten eine deutliche Abhängigkeit vom Ligandentyp, waren jedoch unabhängig von der Ligandendichte. SDF-1α beeinflusste fördernd die Zellpolarisation, aber nicht die Motilität der Zellen. Die Proliferation von HSPZ insgesamt und die Expansion von CD34+ Zellen zeigten keine Abhängigkeit von der Nanostrukturierung. Auf die Differenzierung hatte SDF-1α einen geringen reduzierenden Effekt. Für die nanostrukturierte Präsentation von IDSP ergab sich eine ausgeprägte negative Korrelation zwischen dem Differenzierungsgrad und dem Nanopartikelabstand, der die IDSP-Ligandendichte bestimmt. Die Ergebnisse zeigen eine deutliche Wechselwirkung zwischen HSPZ und dem hier untersuchten Biohybridhydrogelsystem. Dabei können die unterschiedlichen Effekte von zellulärem und extrazellulärem Integrinbindemotiv vorrangig mit der unterschiedlichen Bindungsaffinität des aktivierten α4β1 Integrins für die verschiedenen Liganden erklärt werden. Bei der durch die IDSP-Ligandendichte beeinflussten Differenzierung bleibt es unklar, ob bei gegebener Matrixelastizität eine effizientere α4β1 Integrin-Aktivierung bei hohen oder niedrigen Ligandendichten stattfindet. Schließlich konnte ein kooperativer Effekt zwischen gleichzeitig aktiviertem α4β1 Integrin- und SDF-1α-Rezeptor auf HSPZ im Sinne einer Synergie für die Zellpolarisation bei beiden Integrinliganden festgestellt werden. Dagegen scheint der resultierende Effekt von SDF-1α in Kombination mit dem IDSP-Liganden auf die Differenzierung antagonistischer Natur zu sein. Damit tragen die Ergebnisse auf Basis nanostrukturierter Integrinliganden zu einem besseren Verständnis über das komplexe Zusammenspiel der HSPZ mit ihrer Knochenmarksnische bei. Durch die ermöglichte simultane Anpassung mehrerer Nischefaktoren in solchen Biohybridhydrogelsystemen könnte zukünftig das Verhalten von HSPZ ex vivo gezielter untersucht und beeinflusst werden.To understand the functional interactions of hematopoietic stem and progenitor cells (HSPCs) with their physiological niche in bone marrow, artificial niche models are excellent for studying individual biochemical and biophysical niche properties. In this work, a two-dimensional, nanostructured, heparin-based biohybrid hydrogel was developed to mimic multiple functions for in vitro HSPC culture. Three essential functionalities of the niche were in the focus of this study: (i) matrix elasticity, (ii) cytokine binding and release, and (iii) nanostructured presentation of extracellular or cellular α4β1 integrin peptide ligands. With unchanged matrix elasticity within physiological bone marrow stiffness, different densities of cellular IDSP or extracellular LDV as integrin α4β1-specific binding motifs were realized by varying the nanostructure. In addition, the gel matrix was able to specifically bind and release the chemokine SDF-1α. The influence of the multifunctional gel properties on human CD34+ HSPCs was investigated in terms of cell polarization, cell motility, proliferation and degree of differentiation. The following cell behavior was observed in this work. The degree of polarization and motility of HSPC cultures showed a clear dependence on ligand type, but were independent of ligand density. SDF-1α promotively affected cell polarization but not cell motility. Proliferation of total HSPCs and expansion of CD34+ cells both showed no dependence on nanostructuring. SDF-1α had only a small reducing effect on differentiation. The nanostructured presentation of IDSP revealed a pronounced negative correlation between the degree of differentiation and nanoparticle spacing, which determines IDSP ligand density. The results show a clear interplay between HSPCs and the biohybrid hydrogel system studied here. The different effects of the cellular and extracellular integrin binding motif can be primarily explained by the different binding affinity of the activated α4β1 integrin for the different ligands. For differentiation affected by IDSP ligand density, it remains unclear whether, for a given matrix elasticity, more efficient α4β1 integrin activation occurs at high or low ligand densities. Finally, a synergetic cooperative effect between simultaneously activated α4β1 integrin and SDF-1α receptor on HSPCs on cell polarization was detected for both integrin ligands. In contrast, the resulting effect of SDF-1α in combination with the IDSP ligand on differentiation appears to be antagonistic in nature. Thus, the results based on nanostructured integrin ligands contribute to a better understanding of the complex interplay of HSPCs with their bone marrow niche. By enabling simultaneous tailoring of multiple niche factors in such biohybrid hydrogel systems, the behavior of HSPCs ex vivo could be more specifically studied and influenced in the future.BMBF/Werkstoffinnovationen für Industrie und Gesellschaft/13N12968/E

Nanostructured Bifunctional Hydrogels as Potential Instructing Platform for Hematopoietic Stem Cell Differentiation

Hematopoietic stem cells (HSCs) are blood forming cells which possess the ability to differentiate into all types of blood cells. T cells are one important cell type HSCs can differentiate into, via corresponding progenitor cells. T cells are part of the adaptive immune system as they mediate cellular immune responses. Due to this crucial function, in vitro differentiated T cells are the subject of current studies in the biomedical field in terms of cell transplantation. Studies show that the density of the immobilized Notch ligand Delta-like 1 (DLL1) presented in HSCs' environment can stimulate their differentiation toward T cells. The development of reliable synthetic cell culture systems presenting variable densities of DLL1 is promising for the future expansion of T cells' clinical applications. Here we introduce bifunctional polyethylene glycol-based (PEG-based) hydrogels as a potential instructing platform for the differentiation of human hematopoietic stem and progenitor cells (HSPCs) to T cells. PEG hydrogels bearing the cell adhesion supporting motif RGD (arginyl-glycyl-aspartic acid) were synthesized by UV-light induced radical copolymerization of PEG diacrylate and RGD modified PEG acrylate. The hydrogels were furthermore nanostructured by incorporation of gold nanoparticle arrays that were produced by block copolymer micelle nanolithography (BCML). BCML allows for the decoration of surfaces with gold nanoparticles in a hexagonal manner with well-defined interparticle distances. To determine the impact of DLL1 density on the cell differentiation, hydrogels with particle distances of ~40 and 90 nm were synthesized and the gold nanoparticles were functionalized with DLL1. After 27 days in culture, HSPCs showed an unphysiological differentiation status and, therefore, the differentiation was evaluated as atypical T lymphoid differentiation. Cluster of differentiation (CD) 4 was the only tested T cell marker which was expressed clearly in all samples. Thus, although the applied nanopatterned hydrogels affected two important signaling pathways (integrins and Notch) for T cell differentiation, it appears that more functionalities that control T cell differentiation in nature need to be considered for achieving fully synthetic in vitro T cell differentiation strategies