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.

Learning-related congruent and incongruent changes of excitation and inhibition in distinct cortical areas.

Excitatory and inhibitory neurons in diverse cortical regions are likely to contribute differentially to the transformation of sensory information into goal-directed motor plans. Here, we investigate the relative changes across mouse sensorimotor cortex in the activity of putative excitatory and inhibitory neurons-categorized as regular spiking (RS) or fast spiking (FS) according to their action potential (AP) waveform-comparing before and after learning of a whisker detection task with delayed licking as perceptual report. Surprisingly, we found that the whisker-evoked activity of RS versus FS neurons changed in opposite directions after learning in primary and secondary whisker motor cortices, while it changed similarly in primary and secondary orofacial motor cortices. Our results suggest that changes in the balance of excitation and inhibition in local circuits concurrent with changes in the long-range synaptic inputs in distinct cortical regions might contribute to performance of delayed sensory-to-motor transformation.

Sphingolipids control dermal fibroblast heterogeneity.

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

Highlights from the 2016-2020 NEUBIAS training schools for Bioimage Analysts: a success story and key asset for analysts and life scientists.

NEUBIAS, the European Network of Bioimage Analysts, was created in 2016 with the goal of improving the communication and the knowledge transfer among the various stakeholders involved in the acquisition, processing and analysis of biological image data, and to promote the establishment and recognition of the profession of Bioimage Analyst. One of the most successful initiatives of the NEUBIAS programme was its series of 15 training schools, which trained over 400 new Bioimage Analysts, coming from over 40 countries. Here we outline the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. We discuss the future of the materials created during this programme and some of the new initiatives emanating from the community of NEUBIAS-trained analysts, such as the NEUBIAS Academy. Overall, we elaborate on how this training programme played a key role in collectively leveraging Bioimaging and Life Science research by bringing the latest innovations into structured, frequent and intensive training activities, and on why we believe this should become a model to further develop in Life Sciences.

Biotin-NeutrAvidin Mediated Immobilization of Polymer Micro- and Nanoparticles on T Lymphocytes.

Cells are powerful carriers that can help to improve the delivery of nanomedicines. One approach to use cells as carriers is to immobilize the nanoparticulate cargo on the cell surface. While a plethora of chemical conjugation strategies are available to bind nanoparticles to cell surfaces, only relatively little is known about the effects of particle size and cell type on the surface immobilization of nanoparticles. This study investigates the biotin-NeutrAvidin mediated immobilization of model polymer nanoparticles with sizes ranging from 40 nm to 1 µm on two different T cell lines, viz., human Jurkat cells as well as mouse SJL/PLP7 T cells, which are of potential interest for drug delivery across the blood-brain barrier. The nanoparticle cell surface immobilization and the particle surface concentration and distribution were analyzed by flow cytometry and confocal microscopy. The functional properties of nanoparticle-modified SJL/PLP7 T cells were assessed in an ICAM-1 binding assay as well as in a two-chamber setup in which the migration of the particle-modified T cells across an in vitro model of the blood-brain barrier was studied. The results of these experiments highlight the effects of particle size and cell line on the surface immobilization of nanoparticles on living cells.

T Cell-Mediated Transport of Polymer Nanoparticles across the Blood-Brain Barrier.

Delivery of therapeutics to the central nervous system (CNS) is challenging due to the presence of the blood-brain barrier (BBB). Amongst various approaches that have been explored to facilitate drug delivery to the CNS, the use of cells that have the intrinsic ability to cross the BBB is relatively unexplored, yet very attractive. This paper presents a first proof-of-concept that demonstrates the feasibility of activated effector/memory CD4+ helper T cells (CD4+ TEM cells) as carriers for the delivery of polymer nanoparticles across the BBB. This study shows that CD4+ TEM cells can be decorated with poly(ethylene glycol)-modified polystyrene nanoparticles using thiol-maleimide coupling chemistry, resulting in the immobilization of ≈105 nanoparticles per cell as determined by confocal microscopy. The ability of these cells to serve as carriers to transport nanoparticles across the BBB is established in vitro and in vivo. Using in vitro BBB models, CD4+ TEM cells are found to be able to transport nanoparticles across the BBB both under static conditions as well as under physiological flow. Finally, upon systemic administration, nanoparticle-modified T cells are shown to enter the brain parenchyma of mice, demonstrating the brain delivery potential of this T cell subset in allogeneic hosts.

Capturing Cardiogenesis in Gastruloids.

Organoids are powerful models for studying tissue development, physiology, and disease. However, current culture systems disrupt the inductive tissue-tissue interactions needed for the complex morphogenetic processes of native organogenesis. Here, we show that mouse embryonic stem cells (mESCs) can be coaxed to robustly undergo fundamental steps of early heart organogenesis with an in-vivo-like spatiotemporal fidelity. These axially patterned embryonic organoids (gastruloids) mimic embryonic development and support the generation of cardiovascular progenitors, including first and second heart fields. The cardiac progenitors self-organize into an anterior domain reminiscent of a cardiac crescent before forming a beating cardiac tissue near a putative primitive gut-like tube, from which it is separated by an endocardial-like layer. These findings unveil the surprising morphogenetic potential of mESCs to execute key aspects of organogenesis through the coordinated development of multiple tissues. This platform could be an excellent tool for studying heart development in unprecedented detail and throughput.

Hookworms Evade Host Immunity by Secreting a Deoxyribonuclease to Degrade Neutrophil Extracellular Traps.

Hookworms cause a major neglected tropical disease, occurring after larvae penetrate the host skin. Neutrophils are phagocytes that kill large pathogens by releasing neutrophil extracellular traps (NETs), but whether they target hookworms during skin infection is unknown. Using a murine hookworm, Nippostrongylus brasiliensis, we observed neutrophils being rapidly recruited and deploying NETs around skin-penetrating larvae. Neutrophils depletion or NET inhibition altered larvae behavior and enhanced the number of adult worms following murine infection. Nevertheless, larvae were able to mitigate the effect of NETs by secreting a deoxyribonuclease (Nb-DNase II) to degrade the DNA backbone. Critically, neutrophils were able to kill larvae in vitro, which was enhanced by neutralizing Nb-DNase II. Homologs of Nb-DNase II are present in other nematodes, including the human hookworm, Necator americanus, which also evaded NETs in vitro. These findings highlight the importance of neutrophils in hookworm infection and a potential conserved mechanism of immune evasion.

DEVILS: a tool for the visualization of large datasets with a high dynamic range.

The number of grey values that can be displayed on monitors and be processed by the human eye is smaller than the dynamic range of image-based sensors. This makes the visualization of such data a challenge, especially with specimens where small dim structures are equally important as large bright ones, or whenever variations in intensity, such as non-homogeneous staining efficiencies or light depth penetration, becomes an issue. While simple intensity display mappings are easily possible, these fail to provide a one-shot observation that can display objects of varying intensities. In order to facilitate the visualization-based analysis of large volumetric datasets, we developed an easy-to-use ImageJ plugin enabling the compressed display of features within several magnitudes of intensities. The Display Enhancement for Visual Inspection of Large Stacks plugin (DEVILS) homogenizes the intensities by using a combination of local and global pixel operations to allow for high and low intensities to be visible simultaneously to the human eye. The plugin is based on a single, intuitively understandable parameter, features a preview mode, and uses parallelization to process multiple image planes. As output, the plugin is capable of producing a BigDataViewer-compatible dataset for fast visualization. We demonstrate the utility of the plugin for large volumetric image data.

A Derived Positional Mapping of Inhibitory Subtypes in the Somatosensory Cortex.

Obtaining a catalog of cell types is a fundamental building block for understanding the brain. The ideal classification of cell-types is based on the profile of molecules expressed by a cell, in particular, the profile of genes expressed. One strategy is, therefore, to obtain as many single-cell transcriptomes as possible and isolate clusters of neurons with similar gene expression profiles. In this study, we explored an alternative strategy. We explored whether cell-types can be algorithmically derived by combining protein tissue stains with transcript expression profiles. We developed an algorithm that aims to distribute cell-types in the different layers of somatosensory cortex of the developing rat constrained by the tissue- and cellular level data. We found that the spatial distribution of major inhibitory cell types can be approximated using the available data. The result is a depth-wise atlas of inhibitory cell-types of the rat somatosensory cortex. In principle, any data that constrains what can occur in a particular part of the brain can also strongly constrain the derivation of cell-types. This draft inhibitory cell-type mapping is therefore dynamic and can iteratively converge towards the ground truth as further data is integrated.

Open Source Tools for Biological Image Analysis.

Visiting the Bio Imaging Search Engine (BISE) (Bio, BISE, Engine, http://biii.eu/, Imaging, Search) website at the time of writing this article, almost 1200 open source assets (components, workflows, collections) were found. This overwhelming range of offer difficults the fact of making a reasonable choice, especially to newcomers. In the following chapter, we briefly sketch the advantages of the open source software (OSS) particularly used for image analysis in the field of life sciences. We introduce both the general OSS idea as well as some programs used for image analysis. Even more, we outline the history of ImageJ as it has served as a role model for the development of more recent software packages. We focus on the programs that are, to our knowledge, the most relevant and widely used in the field of light microscopy, as well as the most commonly used within our facility. In addition, we briefly discuss recent efforts and approaches aimed to share and compare algorithms and introduce software and data sharing good practices as a promising strategy to facilitate reproducibility, software understanding, and optimal software choice for a given scientific problem in the future.

Cellular Uptake and Intracellular Trafficking of Poly( N-(2-Hydroxypropyl) Methacrylamide).

Cellular uptake and intracellular trafficking of polymer conjugates or polymer nanoparticles is typically monitored using fluorescence-based techniques such as confocal microscopy. While these methods have provided a wealth of insight into the internalization and trafficking of polymers and polymer nanoparticles, they require fluorescent labeling of the polymer or polymer nanoparticle. Because in biological media fluorescent dyes may degrade, be cleaved from the polymer or particle, or even change uptake and trafficking pathways, there is an interest in fluorescent label-free methods to study the interactions between cells and polymer nanomedicines. This article presents a first proof-of-concept that demonstrates the feasibility of NanoSIMS to monitor the intracellular localization of polymer conjugates. For the experiments reported here, poly( N-(2-hydroxypropyl) methacrylamide)) (PHPMA) was selected as a prototypical polymer-drug conjugate. This PHPMA polymer contained a 19F-label at the α-terminus, which was introduced in order to allow NanoSIMS analysis. Prior to the NanoSIMS experiments, the uptake and intracellular trafficking of the polymer was established using confocal microscopy and flow cytometry. These experiments not only provided detailed insight into the kinetics of these processes but were also important to select time points for the NanoSIMS analysis. For the NanoSIMS experiments, HeLa cells were investigated that had been exposed to the PHPMA polymer for a period of 4 or 15 h, which was known to lead to predominant lysosomal accumulation of the polymer. NanoSIMS analysis of resin-embedded and microtomed samples of the cells revealed a punctuated fluorine signal, which was found to colocalize with the sulfur signal that was attributed to the lysosomal compartments. The localization of the polymer in the endolysosomal compartments was confirmed by TEM analysis on the same cell samples. The results of this study illustrate the potential of NanoSIMS to study the uptake and intracellular trafficking of polymer nanomedicines.

Podosomes, But Not the Maturation Status, Determine the Protease-Dependent 3D Migration in Human Dendritic Cells.

Dendritic cells (DC) are professional Antigen-Presenting Cells scattered throughout antigen-exposed tissues and draining lymph nodes, and survey the body for pathogens. Their ability to migrate through tissues, a 3D environment, is essential for an effective immune response. Upon infection, recognition of Pathogen-Associated Molecular Patterns (PAMP) by Toll-like receptors (TLR) triggers DC maturation. Mature DC (mDC) essentially use the protease-independent, ROCK-dependent amoeboid mode in vivo, or in collagen matrices in vitro. However, the mechanisms of 3D migration used by human immature DC (iDC) are still poorly characterized. Here, we reveal that human monocyte-derived DC are able to use two migration modes in 3D. In porous matrices of fibrillar collagen I, iDC adopted the amoeboid migration mode. In dense matrices of gelled collagen I or Matrigel, iDC used the protease-dependent, ROCK-independent mesenchymal migration mode. Upon TLR4 activation by LPS, mDC-LPS lose the capacity to form podosomes and degrade the matrix along with impaired mesenchymal migration. TLR2 activation by Pam3CSK4 resulted in DC maturation, podosome maintenance, and efficient mesenchymal migration. Under all these conditions, when DC used the mesenchymal mode in dense matrices, they formed 3D podosomes at the tip of cell protrusions. Using PGE2, known to disrupt podosomes in DC, we observed that the cells remained in an immature status and the mesenchymal migration mode was abolished. We also observed that, while CCL5 (attractant of iDC) enhanced both amoeboid and mesenchymal migration of iDC, CCL19 and CCL21 (attractants of mDC) only enhanced mDC-LPS amoeboid migration without triggering mesenchymal migration. Finally, we examined the migration of iDC in tumor cell spheroids, a tissue-like 3D environment. We observed that iDC infiltrated spheroids of tumor cells using both migration modes. Altogether, these results demonstrate that human DC adopt the mesenchymal mode to migrate in 3D dense environments, which relies on their capacity to form podosomes independent of their maturation status, paving the way of further investigations on in vivo DC migration in dense tissues and its regulation during infections.

DeconvolutionLab2: An open-source software for deconvolution microscopy.

Images in fluorescence microscopy are inherently blurred due to the limit of diffraction of light. The purpose of deconvolution microscopy is to compensate numerically for this degradation. Deconvolution is widely used to restore fine details of 3D biological samples. Unfortunately, dealing with deconvolution tools is not straightforward. Among others, end users have to select the appropriate algorithm, calibration and parametrization, while potentially facing demanding computational tasks. To make deconvolution more accessible, we have developed a practical platform for deconvolution microscopy called DeconvolutionLab. Freely distributed, DeconvolutionLab hosts standard algorithms for 3D microscopy deconvolution and drives them through a user-oriented interface. In this paper, we take advantage of the release of DeconvolutionLab2 to provide a complete description of the software package and its built-in deconvolution algorithms. We examine several standard algorithms used in deconvolution microscopy, notably: Regularized inverse filter, Tikhonov regularization, Landweber, Tikhonov-Miller, Richardson-Lucy, and fast iterative shrinkage-thresholding. We evaluate these methods over large 3D microscopy images using simulated datasets and real experimental images. We distinguish the algorithms in terms of image quality, performance, usability and computational requirements. Our presentation is completed with a discussion of recent trends in deconvolution, inspired by the results of the Grand Challenge on deconvolution microscopy that was recently organized.

Antibody-mediated trapping of helminth larvae requires CD11b and Fcγ receptor I.

Infections with intestinal helminths severely impact on human and veterinary health, particularly through the damage that these large parasites inflict when migrating through host tissues. Host immunity often targets the motility of tissue-migrating helminth larvae, which ideally should be mimicked by anti-helminth vaccines. However, the mechanisms of larval trapping are still poorly defined. We have recently reported an important role for Abs in the rapid trapping of tissue-migrating larvae of the murine parasite Heligmosomoides polygyrus bakeri. Trapping was mediated by macrophages (MΦ) and involved complement, activating FcRs, and Arginase-1 (Arg1) activity. However, the receptors and Ab isotypes responsible for MΦ adherence and Arg1 induction remained unclear. Using an in vitro coculture assay of H. polygyrus bakeri larvae and bone marrow-derived MΦ, we now identify CD11b as the major complement receptor mediating MΦ adherence to the larval surface. However, larval immobilization was largely independent of CD11b and instead required the activating IgG receptor FcγRI (CD64) both in vitro and during challenge H. polygyrus bakeri infection in vivo. FcγRI signaling also contributed to the upregulation of MΦ Arg1 expression in vitro and in vivo. Finally, IgG2a/c was the major IgG subtype from early immune serum bound by FcγRI on the MΦ surface, and purified IgG2c could trigger larval immobilization and Arg1 expression in MΦ in vitro. Our findings reveal a novel role for IgG2a/c-FcγRI-driven MΦ activation in the efficient trapping of tissue-migrating helminth larvae and thus provide important mechanistic insights vital for anti-helminth vaccine development.

Correlative multicolor 3D SIM and STORM microscopy.

Within the last decade, super-resolution methods that surpass the diffraction limit of light microscopy have provided invaluable insight into a variety of biological questions. Each of these approaches has inherent advantages and limitations, such that their combination is a powerful means to transform them into versatile tools for the life sciences. Here, we report the development of a combined SIM and STORM setup that maintains the optimal resolution of both methods and which is coupled to image registration to localize biological structures in 3D using multicolor labeling. We utilized this workflow to determine the localization of Bld12p/CrSAS-6 in purified basal bodies of Chlamydomonas reinhardtii with utmost precision, demonstrating its usefulness for accurate molecular mapping in 3D.

Tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) by Hck regulates macrophage function.

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck(-/-) bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck(-/-) BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP(-/-) BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.

Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages.

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH (-/-)) or activating Fc receptors (FcRγ(-/-)) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Adipocyte-derived fibroblasts promote tumor progression and contribute to the desmoplastic reaction in breast cancer.

Cancer-associated fibroblasts (CAF) comprise the majority of stromal cells in breast cancers, yet their precise origins and relative functional contributions to malignant progression remain uncertain. Local invasion leads to the proximity of cancer cells and adipocytes, which respond by phenotypical changes to generate fibroblast-like cells termed as adipocyte-derived fibroblasts (ADF) here. These cells exhibit enhanced secretion of fibronectin and collagen I, increased migratory/invasive abilities, and increased expression of the CAF marker FSP-1 but not α-SMA. Generation of the ADF phenotype depends on reactivation of the Wnt/ß-catenin pathway in response to Wnt3a secreted by tumor cells. Tumor cells cocultivated with ADFs in two-dimensional or spheroid culture display increased invasive capabilities. In clinical specimens of breast cancer, we confirmed the presence of this new stromal subpopulation. By defining a new stromal cell population, our results offer new opportunities for stroma-targeted therapies in breast cancer.

Macrophage mesenchymal migration requires podosome stabilization by filamin A.

Filamin A (FLNa) is a cross-linker of actin filaments and serves as a scaffold protein mostly involved in the regulation of actin polymerization. It is distributed ubiquitously, and null mutations have strong consequences on embryonic development in humans, with organ defects which suggest deficiencies in cell migration. We have reported previously that macrophages, the archetypal migratory cells, use the protease- and podosome-dependent mesenchymal migration mode in dense three-dimensional environments, whereas they use the protease- and podosome-independent amoeboid mode in more porous matrices. Because FLNa has been shown to localize to podosomes, we hypothesized that the defects seen in patients carrying FLNa mutations could be related to the capacity of certain cell types to form podosomes. Using strategies based on FLNa knock-out, knockdown, and rescue, we show that FLNa (i) is involved in podosome stability and their organization as rosettes and three-dimensional podosomes, (ii) regulates the proteolysis of the matrix mediated by podosomes in macrophages, (iii) is required for podosome rosette formation triggered by Hck, and (iv) is necessary for mesenchymal migration but dispensable for amoeboid migration. These new functions assigned to FLNa, particularly its role in mesenchymal migration, could be directly related to the defects in cell migration described during the embryonic development in FLNa-defective patients.