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"Cytology Experiments."
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Cell and microbe science fair projects : revised and expanded using the scientific method
A collection of science experiments about cells and microbes with emphasis on using the scientific method.
Book
Visualizing the invisible : imaging techniques for the structural biologist
Knowledge of the microscopic structure of biological systems is the key to understanding their physiological properties. Most of what we now know about this subject has been generated by techniques that produce images of the materials of interest, one way or another, and there is every reason to believe that the impact of these techniques on the biological sciences will be every bit as important in the future as they are today. Thus the 21st-century biologist needs to understand how microscopic imaging techniques work, as it is likely that sooner or later he or she will have to use one or another of them, or will otherwise become dependent on the information that they provide. This book introduces readers to the many techniques now available for imaging biological materials—crystallography, optical microscopy, and electron microscopy—at a level that will enable them to use them effectively to do research. Since all of these experimental methods are best understood in terms of Fourier transformations, this book explains the relevant concepts from this branch of mathematics, and then illustrates their elegance and power by applying them to each of the techniques presented.
eBook
Hierarchical organization of cortical and thalamic connectivity
The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource
1
, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.
Using mouse lines in which subsets of neurons are genetically labelled, the authors provide generalized anatomical rules for connections within and between the cortex and thalamus.
Journal Article
Comprehensive characterization of four different populations of human mesenchymal stem cells as regards their immune properties, proliferation and differentiation
In the present study, we compared mesenchymal stem cells (MSCs) derived from 4 different sources, human bone marrow (BM), adipose tissue (AT), umbilical cord Wharton's Jelly (WJ) and the placenta (PL), in order to determine which population of MSCs displayed the most prominent immunosuppressive effects on phytohemagglutinin-induced T cell proliferation, and which one had the highest proliferative and differentiation potential. MSC and T lymphocyte co-culture (mixed culture) was used to determine whether the MSCs inhibit T cell proliferation, as well as which population of MSCs has the strongest inhibitory ability. The expression of immune-related genes was analyzed by RT-PCR and RT-qPCR. The proliferation and differentiation potential of the MSCs were determined using standard methods. Following MSC and T cell co-culture, mitogen-induced T cell proliferation was effectively suppressed by all 4 populations of MSCs. This occurred through soluble factors rather than direct contact inhibition. Among the 4 populations of MSCs, the WJ-MSC has the strongest suppression effects. On immune related genes, WJ-MSC has the weakest expression of MHC II genes, TLR4, TLR3, JAG1, NOTCH2 and NOTCH3. To compare the proliferation potential, WJ-MSCs showed the most rapid growth rate followed by the AT-, PL- and BM-MSCs. As regards differentiation potential, the WJ-MSCs had the strongest osteogenetic ability followed by PL, AT and BM-MSC. AT-MSC has the strongest adipogenetic ability followed by the WJ-, BM- and PL-MSCs. These data indicated that the WJ-MSCs had the strongest immunomodulatory and immunosuppressive potential. In light of these observations, we suggest that WJ-MSCs are the most attractive cell population for use in immune cellular therapy when immunosuppressive action is required.
Journal Article
The single-cell transcriptional landscape of mammalian organogenesis
Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution. Using single-cell combinatorial indexing, we profiled the transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting ‘mouse organogenesis cell atlas’ (MOCA) provides a global view of developmental processes during this critical window. We use Monocle 3 to identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. We explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle.
Data from single-cell combinatorial-indexing RNA-sequencing analysis of 2 million cells from mouse embryos between embryonic days 9.5 and 13.5 are compiled in a cell atlas of mouse organogenesis, which provides a global view of developmental processes occurring during this critical period.
Journal Article
In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm Identity
Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated in vitro from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate in vivo development. Similar to NMPs in vivo, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial mesoderm in vitro and in vivo. The neural cells produced by NMPs have spinal cord but not anterior neural identity and can differentiate into spinal cord motor neurons. This is consistent with the shared origin of spinal cord and somites and the distinct ontogeny of the anterior and posterior nervous system. Systematic analysis of the transcriptome during differentiation identifies the molecular correlates of each of the cell identities and the routes by which they are obtained. Moreover, we take advantage of the system to provide evidence that Brachyury represses neural differentiation and that signals from mesoderm are not necessary to induce the posterior identity of spinal cord cells. This indicates that the mesoderm inducing and posteriorising functions of Wnt signalling represent two molecularly separate activities. Together the data illustrate how reverse engineering normal developmental mechanisms allows the differentiation of specific cell types in vitro and the analysis of previous difficult to access aspects of embryo development.
Journal Article
Probabilistic cell typing enables fine mapping of closely related cell types in situ
Understanding the function of a tissue requires knowing the spatial organization of its constituent cell types. In the cerebral cortex, single-cell RNA sequencing (scRNA-seq) has revealed the genome-wide expression patterns that define its many, closely related neuronal types, but cannot reveal their spatial arrangement. Here we introduce probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages previous scRNA-seq classification to identify cell types using multiplexed in situ RNA detection. We applied this method by mapping the inhibitory neurons of mouse hippocampal area CA1, for which ground truth is available from extensive previous work identifying their laminar organization. Our method identified these neuronal classes in a spatial arrangement matching ground truth, and further identified multiple classes of isocortical pyramidal cell in a pattern matching their known organization. This method will allow identifying the spatial organization of closely related cell types across the brain and other tissues.
Probabilistic cell typing by in situ sequencing (pciSeq), leverages previous single-cell RNA sequencing classification and multiplexed in situ RNA detection to spatially map cell types accurately in the mouse hippocampus and isocortex.
Journal Article
Systematic comparison of single-cell and single-nucleus RNA-sequencing methods
The scale and capabilities of single-cell RNA-sequencing methods have expanded rapidly in recent years, enabling major discoveries and large-scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single-cell and/or single-nucleus profiling—selecting representative methods based on their usage and our expertise and resources to prepare libraries—including two low-throughput and five high-throughput methods. We tested the methods on three types of samples: cell lines, peripheral blood mononuclear cells and brain tissue, generating 36 libraries in six separate experiments in a single center. To directly compare the methods and avoid processing differences introduced by the existing pipelines, we developed scumi, a flexible computational pipeline that can be used with any single-cell RNA-sequencing method. We evaluated the methods for both basic performance, such as the structure and alignment of reads, sensitivity and extent of multiplets, and for their ability to recover known biological information in the samples.
Seven methods for single-cell RNA sequencing are benchmarked on cell lines, primary cells and mouse cortex.
Journal Article
Systemic AAV vectors for widespread and targeted gene delivery in rodents
We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.
Having developed AAV capsids that target sites throughout the body, here the authors describe how to produce and systemically administer these AAVs to rodents to label and/or genetically manipulate cells in the nervous system and visceral organs.
Journal Article
Combinatory differentiation of human induced pluripotent stem cells generates functional thymic epithelium driving dendritic cell and CD4/CD8 T cell development
The thymus educates thymocytes through a selection process mediated by thymic epithelial cells (TECs). Recent advances have made the generation of T lymphocytes from induced pluripotent stem cells (iPSc) a promising therapeutic strategy. However, current approaches often fail to replicate the thymic niche, leading to impaired T cell generation. Here we address the production of functional mature iPSc-derived TECs supporting in vitro T cell generation. We optimize thymic lineage differentiation through an unbiased multifactorial experimental design. By modulating specific signaling pathways, we generate progenitors that mature into medullary and cortical TECs. Co-culture with primary hematopoietic progenitors in a 3D thymic organoid setup induces their differentiation into CD4
+
and CD8
+
T cells. Importantly, thymic organoids support multilineage differentiation, with dendritic cell populations also emerging. Thus, the presented thymic organoid model provides a practical platform for studying thymic cellular interactions and thymopoiesis in vitro, and opens further research perspectives towards cell-based therapies.
In vitro methods for thymic organoid cultures are useful to examine requirements for T cell development and for generating large numbers of cells for therapeutic purposes. Here the authors use human induced pluripotent stem cells, differentiate these into thymic epithelial organoid cultures and utilise haematopoietic progenitors to show development of T cells in vitro.
Journal Article