Determination of Hydrophilic and Amphiphilic Organic Pollutants in the Aquatic Environment

Environmental chemists performing monitoring or process-oriented fate and behavior studies on organic micropollutants face the challenge of having to determine low concentrations of problem compounds in complex mixtures and difficult matrices, such as sewage sludge, surface and groundwater. Selective extraction and enrichment help to overcome sensitivity limitations and also to reduce the number of different species in the sample. A subsequent chromatographic separation step, together with analyte-specific detection, finally allows to identify and quantify single analytes in the presence of other organic material. This article describes a selection of analytical development work carried out at EAWAG for the determination of hydrophilic and amphiphilic organic pollutants in the aquatic environment

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays

Specification of haematopoietic stem cell fate via modulation of mitochondrial activity

Haematopoietic stem cells (HSCs) differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. However, whether this change in the metabolic program is the cause or the consequence of the unique function of HSCs remains unknown. Here we show that enforced modulation of energy metabolism impacts HSC self-renewal. Lowering the mitochondrial activity of HSCs by chemically uncoupling the electron transport chain drives self-renewal under culture conditions that normally induce rapid differentiation. We demonstrate that this metabolic specification of HSC fate occurs through the reversible decrease of mitochondrial mass by autophagy. Our data thus reveal a causal relationship between mitochondrial metabolism and fate choice of HSCs and also provide a valuable tool to expand HSCs outside of their native bone marrow niches

The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance

It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD(+)-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD(+)-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.Peer reviewe

Modeling hematopoietic stem cell dynamics in bioengineered niches

Bone marrow transplantation is a well-established medical procedure for the treatment of various hematologic diseases. However, the relatively low number of hematopoietic stem cells (HSCs) that can be harvested, especially from umbilical cord blood, limits even broader applicability of the procedure. A better understanding of the biology of HSCs, and in particular how these rare cells are regulated by microenvironmental niches in the bone marrow, could ultimately enable robust in vitro expansion of HSCs. In this thesis, several novel strategies were developed to study and manipulate HSC biology in vitro, through the regulation of key niche factors, cellular metabolism, and the complex multicellular crosstalk in a niche-mimicking context. First, we made use of gastruloids, an embryonic stem cell-based model of early mouse development, to mimic key aspects of embryonic hematopoiesis in vitro. Simultaneously with the establishment of a vascular network, we detected by immunophenotyping the emergence of primitive blood progenitor cells during the late developmental stages of gastruloids. These embryonic blood progenitors were spatially localized close to a vascular-like plexus in the anterior portion of the gastruloid. Colony-forming assays demonstrated an interesting differentiation potential of these cells. These data demonstrate the potential of gastruloids as an easily accessible in vitro model to study blood development in an embryo-like context. Second, a bioengineering approach was used to study HSC fate choices upon exposure to niche-specific ligands in a well-defined artificial environment. A combination of time-lapse microscopy and single-cell multigene expression analysis was used to define differentiation and cell-cycle states of mouse and human HSCs. Strikingly, selected artificial niches reduced proliferation and maintained the long-term multilineage potential of HSCs in vitro. These artificial niches hold significant potential for the study of mechanisms involved in HSC fate regulation. Third, the influence of the metabolism on fate choices of adult HSCs was studied, specifically focusing on fatty acid β-oxidation. Malonyl-CoA was used to reversibly block fatty acid β-oxidation in mouse HSCs. In vitro treatment of HSCs with malonyl-CoA promoted their expansion and increased lymphoid reconstitution upon in vivo transplantation. This study sheds new light on the role of the metabolic environment in HSC regulation. Surprisingly, exposure to only a single, readily available metabolite was found to be sufficient to strongly influence HSC behavior. Fourth, a miniaturized multicellular culture system was developed to mimic the hallmarks of the dynamics of HSCs in their native bone marrow. Primary human mesenchymal stem/progenitor cells and endothelial cells were aggregated in a high-throughput manner in biomimetic hydrogel microwells to form self-organizing bone marrow organoids. Immunostaining demonstrated the formation of branched vascular networks, rendering the organoids permissive for the homing of human hematopoietic stem and progenitor cells. These results suggest that bone marrow organoids may be a suitable platform for the modeling of physiological and clinically-relevant stem cell dynamics in vitro. Altogether, this thesis presents several innovative approaches for modeling key features of native stem cell microenvironments that will contribute to a better understanding of the biology of HSCs and their regulatory niche

p-Toluenesulfonate in Landfill Leachates : Leachability from Foundry Sands and Aerobic Biodegradation

p-Toluenesulfonate (pTS) was found at concentrations between 9.6 and 48.8 mg/L in leachates from two Swiss landfill sections where foundry wastes are stored. pTS represented ≤33% of the nonpurgable organic carbon (NPOC) in the leachates. A standard test showed that pTS is easily leached from foundry sands that contained 1% (w/w) of a technical hardener consisting of tolu enesulfonates. Hence, the hardener is a likely source of pTS in the landfill leachates. Furthermore, analyses using liquid chromatography coupled to mass spectrometry and UV absorption detection indicated that the hardener also was the source for toluene-2,4-disulfonate, benzenesulfonate and o- and m-toluenesulfonate, all of which were detected in the landfill leachates. Modified OECD screening tests with pure pTS or hardener as carbon source and landfill leachate as inoculum indicated that aerobic bacteria using pTS for growth are present in the landfill leachates. The intermediates p-sulfobenzyl alcohol and p-sulfobenzoate gives strong evidence for a degradation pathway common to Comamonas testosteroni

A single metabolite which modulates lipid metabolism alters hematopoietic stem/progenitor cell behavior and promotes lymphoid reconstitution

Fatty acid β-oxidation (FAO), the breakdown of lipids, is a metabolic pathway used by various stem cells. FAO levels are generally high during quiescence and downregulated with proliferation. The endogenous metabolite malonyl-CoA modulates lipid metabolism as a reversible FAO inhibitor and as a substrate for de novo lipogenesis. Here we assessed whether malonyl-CoA can be exploited to steer the behavior of hematopoietic stem/progenitor cells (HSPCs), quiescent stem cells of clinical relevance. Treatment of mouse HSPCs in vitro with malonyl-CoA increases HSPC numbers compared with nontreated controls and ameliorates blood reconstitution capacity when transplanted in vivo, mainly through enhanced lymphoid reconstitution. Similarly, human HSPC numbers also increase upon malonyl-CoA treatment in vitro. These data corroborate that lipid metabolism can be targeted to direct cell fate and stem cell proliferation. Physiological modulation of metabolic pathways, rather than genetic or pharmacological inhibition, provides unique perspectives for stem cell manipulations in health and disease

Yin Yang 1 sustains biosynthetic demands during brain development in a stage-specific manner

The transcription factor Yin Yang 1 (YY1) plays an important role in human disease. It is often overexpressed in cancers and mutations can lead to a congenital haploinsufficiency syndrome characterized by craniofacial dysmorphisms and neurological dysfunctions, consistent with a role in brain development. Here, we show that Yy1 controls murine cerebral cortex development in a stage-dependent manner. By regulating a wide range of metabolic pathways and protein translation, Yy1 maintains proliferation and survival of neural progenitor cells (NPCs) at early stages of brain development. Despite its constitutive expression, however, the dependence on Yy1 declines over the course of corticogenesis. This is associated with decreasing importance of processes controlled by Yy1 during development, as reflected by diminished protein synthesis rates at later developmental stages. Thus, our study unravels a novel role for Yy1 as a stage-dependent regulator of brain development and shows that biosynthetic demands of NPCs dynamically change throughout development

Yin Yang 1 sustains biosynthetic demands during brain development in a stage-specific manner

The transcription factor Yin Yang 1 (YY1) plays an important role in human disease. It is often overexpressed in cancers and mutations can lead to a congenital haploinsufficiency syndrome characterized by craniofacial dysmorphisms and neurological dysfunctions, consistent with a role in brain development. Here, we show that Yy1 controls murine cerebral cortex development in a stage-dependent manner. By regulating a wide range of metabolic pathways and protein translation, Yy1 maintains proliferation and survival of neural progenitor cells (NPCs) at early stages of brain development. Despite its constitutive expression, however, the dependence on Yy1 declines over the course of corticogenesis. This is associated with decreasing importance of processes controlled by Yy1 during development, as reflected by diminished protein synthesis rates at later developmental stages. Thus, our study unravels a novel role for Yy1 as a stage-dependent regulator of brain development and shows that biosynthetic demands of NPCs dynamically change throughout development.status: publishe