Functional analysis of Numb and redefinition of the human hematopoietic hierarchy

Hämatopoetische Stammzellen (HSZ) müssen in der Lage sein, die tägliche Produktion von Milliarden von spezialisierten Blutzellen aufrecht zu erhalten. Sie stellen die bislang am besten untersuchten somatischen Stammzellen dar, aber die Mechanismen, die ihre Entwicklung und ihre Differenzierung regulieren, sind weitgehend unklar. Vordaten aus unser Arbeitsgruppe deuten darauf hin, dass Numb, eine aus Drosophila als Antagonist des Notch-Signalweges bekannte Zellschicksalsdeterminante, auch an der Zellschicksalsspezifikation humaner hämatopoetischer Stamm- und Vorläuferzellen (HSVZ) beteiligt ist. Da der Notch-Signalweg ein wichtiger Faktor beim Selbsterhalt von HSZ in Säugern ist, war das primäre Ziel dieser Arbeit, die Funktion von Numb in humanen HSVZ (CD34+ Zellen) näher zu untersuchen und die Frage zu klären, ob Numb in der humanen Hämatopoese, ebenso wie in Drosophila, als Notch-Antagonist fungiert. In Übereinstimmung mit Vordaten aus unserer Gruppe konnte gezeigt werden, dass die Überexpression von Numb die Aufrechterhaltung primitiver Zellschicksale unterdrückt und die erythrozytäre Differenzierung induziert. Hingegen führte der funktionelle Knockdown von Numb und dem homologen Numblike zu höheren Anteilen primitiver hämatopoetischer Zellen. Weiterhin deutet sich an, dass Numb in humanen HSVZ nicht als Notch-Antagonist fungiert. Zudem wurde beobachtet, dass die Überexpression von Numb zu einer Herunterregulierung von CD133 führt, einem Oberflächenmarker, der auf dem Großteil humaner CD34+ Zellen ko-exprimiert wird. Um ein besseres Verständnis der erhaltenen Daten zu ermöglichen, entschlossen wir uns zur Analyse der Entwicklungspotenziale der Fraktionen CD34+CD133+ (CD133+) und CD34+CD133low (CD133low) in verschiedenen in vitro und in vivo Assays. Unerwarteterweise waren alle getesteten Linienpotenziale immer einer Fraktion zuzuordnen: HSVZ mit myeloischem und lymphatischem Langzeitpotenzial, CFU-GM Potenzial und SCID-repopulierende Zellen wurden nur in der CD133+ Fraktion detektiert. Hingegen fanden sich erythrozytäre, megakaryozytäre und CFU-MIX Potenziale in der CD133low Fraktion. Da Kolonien beider Fraktionen Granulozyten enthielten, wurde im Weiteren die Komposition Granulozyten-enthaltender Kolonien u. a. durchflusszytometrisch und mittels RT-PCR umfassend analysiert. Es zeigte sich, dass Neutrophile exklusiv von CD133+ Zellen gebildet wurden, während CD133low Zellen Basophile und Eosinophile hervorbringen. Zusammenfassend deutet das im Gegensatz zum klassischen Modell, das die strikte Trennung erythro-myeloischer von lymphatischen Zellen voraussagt, auf ein neues humanes hämatopoetisches Modell hin. Demnach erfolgt die erste Linienauftrennung zwischen erythro-myeloischen (CD133low) und lymphatisch-myeloischen (CD133+) Linienpotenzialen. Somit enstehen die verschiedenen Granulozyten-Subtypen in getrennten Linien. Eosinophile und Basophile werden von erythro-myeloischen Progenitoren (EMP), Neutrophile von Zellen mit LMPP-Potenzial (lymphoid-primed multipotent progenitors) gebildet. In einem Teilprojekt wurden zur funktionellen Analyse von Numb in der Maus-Hämatopoese Numb/Numblike Knockout Mäuse generiert. Die Mäuse waren lebensfähig, und ihre Hämatopoese war nur marginal beinflusst, was nahelegt, dass Numb im hämatopoetischen System der Maus keine wichtige Rolle spielt.Hematopoietic stem cells (HSCs) are required to sustain the daily production of billions of specialized blood cells fulfilling different functions. Even though they represent the best-studied somatic stem cell entity so far, there are many unresolved questions regarding the mechanisms that control their development including their differentiation. Preliminary studies in our group suggested that Numb, a cell fate determinant known from Drosophila as antagonist of the Notch signaling pathway, is involved in cell fate specification processes in human hematopoietic stem and progenitor cells (HSPCs), too. Since the Notch signaling pathway was shown to be essentially involved in mammalian HSC maintenance, the initial aim of this thesis was to further investigate the role of Numb and to clarify its potential involvement in regulation of the Notch signaling pathway in human HSPCs, i.e. CD34+ cells. In accordance with previous results from our group, it could be demonstrated that over-expression of Numb suppresses primitive hematopoietic cell fates and induces erythroid differentiation, while the functional knockdown of Numb and its homolog Numblike increased the content of primitive hematopoietic cells. Additional experimental data indicated that Numb function in human HSPCs is independent of Notch signaling. We further observed that over-expression of Numb resulted in downregulation of CD133, a surface marker expressed on the majority of human CD34+ cells. To facilitate profound interpretation of results in this context, we decided to compare the developmental capacities of the subpopulations CD34+CD133+ (CD133+) and CD34+CD133low (CD133low) in different functional assays in vitro and in vivo. Unexpectedly, almost all lineage potentials tested turned out to be limited to one of the two subpopulations: HSPCs with long-term myeloid, long-term lymphoid and granulocyte-macrophage colony-forming (CFU-GM) potentials as well as NOD/SCID-repopulating cells exclusively were detected in the CD133+ fraction. In contrast, most cells with erythroid, megakaryocytic and CFU-MIX potentials were found in the CD133low fraction. Since granulocytes were formed within colonies from both populations, we comprehensively compared the composition of granulocyte-containing colonies by different methods including flow-cytometry and RT-PCR. We identified neutrophils to derive exclusively from CD133+ cells, while CD133low cells mainly gave rise to basophils and eosinophils. Taken together, in contrast to the classical model that postulates the first lineage separation to occur between erythro-myeloid and lymphoid cell fates, our data suggest a revised model of human hematopoiesis. Accordingly, the first lineage branch point is marked by segregation of eythro-myeloid (CD133low) from lympho-myeloid (CD133+) potentials. Furthermore, the different granulocyte subtypes arise in distinct lineages, with eosinophils and basophils developing from erythro-myeloid progenitors (EMPs), while neutrophils develop from lymphoid-primed multipotent progenitors (LMPPs). In another sub-project of the thesis, we generated Numb/Numblike knock-out mice to study the role of Numb in mouse hematopoiesis. The mice were viable and we detected only mild effects, indicating that Numb doesn’t seem to be essentially involved in cell fate specifictation processes in mouse HSPCs

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

A compendium of single extracellular vesicle flow cytometry

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost