| 1. |
- Gyllborg, Daniel, et al.
(author)
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Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue
- 2020
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:19
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Journal article (peer-reviewed)abstract
- Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.
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| 2. |
- Hilscher, Markus M., et al.
(author)
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Spatial and temporal heterogeneity in the lineage progression of fine oligodendrocyte subtypes
- 2022
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In: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 20:1
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Journal article (peer-reviewed)abstract
- Background: Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.Results: We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.Conclusions: Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.
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| 3. |
- Horta, Sara, et al.
(author)
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Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes
- 2021
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:15, s. 6169-6177
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Journal article (peer-reviewed)abstract
- Antibody characterization is essential for understanding the immune system and development of diagnostics and therapeutics. Current technologies are mainly focusing on the detection of antigen-specific immunoglobulin G (IgG) using bulk singleplex measurements, which lack information on other isotypes and specificity of individual antibodies. Digital immunoassays based on nucleic acid amplification have demonstrated superior performance by allowing the detection of single molecules in a multiplex and sensitive manner. In this study, we demonstrate for the first time an immuno-rolling circle amplification (immunoRCA) assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM) and its integration with microengraving. To validate this approach, we used the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) as the model disease with anti-ADAMTS13 autoantibodies as the diagnostic target molecules. To identify the anti-ADAMTS13 autoantibody isotypes, we designed a pool of three unique antibody-oligonucleotide conjugates for identification and subsequent amplification and visualization via RCA. To validate this approach, we first confirmed an assay specificity of >88% and a low limit of detection of 0.3 ng/mL in the spiked buffer. Subsequently, we performed a dilution series of an iTTP plasma sample for the multiplex detection of the three isotypes with higher sensitivity compared to an enzyme-linked immunosorbent assay. Finally, we demonstrated single-cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving and achieved a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.
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| 4. |
- hower, lee, et al.
(author)
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investigating cell type changes in schizophrenia with spatially-resolved transcriptomics
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Other publication (pop. science, debate, etc.)abstract
- Schizophrenia is a heritable and genetically complex disorder with poorly understood aetiology. Previous study from our lab has shown that this disorder could affect a limited number of cell types in the brain1. Here, we build on these findings and present the first spatially-resolved transcriptomics study of schizophrenia with a large number of samples (17 in total). By comparing spatial cell type distributions in diseased and healthy prefrontal cortices, we discover that certain non-neuronal cell types change their co-localization patterns
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| 5. |
- La Manno, Gioele, et al.
(author)
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Molecular architecture of the developing mouse brain
- 2021
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 596:7870, s. 92-96
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Journal article (peer-reviewed)abstract
- The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions(1-8), the whole adult brain(9) and even entire embryos(10). Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system. A comprehensive single-cell transcriptomic atlas of the mouse brain between gastrulation and birth identifies hundreds of cellular states and reveals the spatiotemporal organization of brain development.
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| 6. |
- Langseth, Christoffer Mattsson, et al.
(author)
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Comprehensive in situ mapping of human cortical transcriptomic cell types
- 2021
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 4:1
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Journal article (peer-reviewed)abstract
- The ability to spatially resolve the cellular architecture of human cortical cell types over informative areas is essential to understanding brain function. We combined in situ sequencing gene expression data and single-nucleus RNA-sequencing cell type definitions to spatially map cells in sections of the human cortex via probabilistic cell typing. We mapped and classified a total of 59,816 cells into all 75 previously defined subtypes to create a first spatial atlas of human cortical cells in their native position, their abundances and genetic signatures. We also examined the precise within- and across-layer distributions of all the cell types and provide a resource for the cell atlas community. The abundances and locations presented here could serve as a reference for further studies, that include human brain tissues and disease applications at the cell type level.
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| 7. |
- lee, hower, et al.
(author)
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Open-source, high-throughput targeted in-situ transcriptomics for developmental and tissue biology
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Journal article (peer-reviewed)abstract
- Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.
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| 8. |
- Lee, Hower, et al.
(author)
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Open-source, high-throughput targeted in-situ transcriptomics for developmental biologists
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Other publication (other academic/artistic)abstract
- Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput, and large multiplexing. We validate the method across a number of species and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering, and annotation of cell types. By enabling easier access to high throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.
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| 9. |
- Li, Xiaofei, et al.
(author)
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Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin
- 2023
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In: Nature Neuroscience. - : Springer Nature. - 1097-6256 .- 1546-1726. ; 26:5, s. 891-901
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Journal article (peer-reviewed)abstract
- The authors created a comprehensive developmental cell atlas for spatiotemporal gene expression of the human spinal cord, revealed species-specific regulation during development and used the atlas to infer novel markers for pediatric ependymomas. The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.
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| 10. |
- Marco Salas, Sergio, et al.
(author)
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Matisse : a MATLAB-based analysis toolbox for in situ sequencing expression maps
- 2021
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In: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 22:1
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Journal article (peer-reviewed)abstract
- Background: A range of spatially resolved transcriptomic methods has recently emerged as a way to spatially characterize the molecular and cellular diversity of a tissue. As a consequence, an increasing number of computational techniques are developed to facilitate data analysis. There is also a need for versatile user friendly tools that can be used for a de novo exploration of datasets.Results: Here we present MATLAB-based Analysis toolbox for in situ sequencing (ISS) expression maps (Matisse). We demonstrate Matisse by characterizing the 2-dimensional spatial expression of 119 genes profiled in a mouse coronal section, exploring different levels of complexity. Additionally, in a comprehensive analysis, we further analyzed expression maps from a second technology, osmFISH, targeting a similar mouse brain region.Conclusion: Matisse proves to be a valuable tool for initial exploration of in situ sequencing datasets. The wide set of tools integrated allows for simple analysis, using the position of individual reads, up to more complex clustering and dimensional reduction approaches, taking cellular content into account. The toolbox can be used to analyze one or several samples at a time, even from different spatial technologies, and it includes different segmentation approaches that can be useful in the analysis of spatially resolved transcriptomic datasets.
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