Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features
Abstract Spatial transcriptomics has advanced our understanding of cellular heterogeneity at single-cell resolution. Here, we assess the suitability of in situ sequencing (ISS) for analyzing formalin-fixed, paraffin-embedded (FFPE) postmortem human brain tissue. A key challenge in ISS data analysis...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-08518-6 |
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| author | Janssen M. Kotah Thomas Rust Hilmar R. J. van Weering Janneke Bosma Amber L. Woudstra Susanne M. Kooistra Bart J. L. Eggen |
| author_facet | Janssen M. Kotah Thomas Rust Hilmar R. J. van Weering Janneke Bosma Amber L. Woudstra Susanne M. Kooistra Bart J. L. Eggen |
| author_sort | Janssen M. Kotah |
| collection | DOAJ |
| description | Abstract Spatial transcriptomics has advanced our understanding of cellular heterogeneity at single-cell resolution. Here, we assess the suitability of in situ sequencing (ISS) for analyzing formalin-fixed, paraffin-embedded (FFPE) postmortem human brain tissue. A key challenge in ISS data analysis is optimizing transcript allocation while minimizing misallocation, particularly in the morphologically complex central nervous system (CNS). We compared geospatial methods using nuclear and expanded nuclear boundaries for segmentation and transcript allocation. While overall cell-type proportions remained comparable, transcript allocation methods affected specific cell types, including microglia, neurons, and neurovascular cells. To enhance specificity, we integrated fluorescent imaging data targeting 18S RNA and IBA1 protein to direct transcript allocation toward RNA-rich cells (e.g., neurons) and microglia, respectively. We demonstrate how this approach, paired with secondary allocation of transcripts outside imaging masks, improved both the number of microglia detected and the specificity of microglial transcripts assigned. Our method offers a flexible and efficient strategy for targeted transcript allocation based on cellular morphology, optimizing CNS cell segmentation in FFPE-preserved human brain tissue. |
| format | Article |
| id | doaj-art-45abdf0f8d994cecb965d7d883f4b4e2 |
| institution | DOAJ |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-45abdf0f8d994cecb965d7d883f4b4e22025-08-20T03:06:01ZengNature PortfolioCommunications Biology2399-36422025-07-018111210.1038/s42003-025-08518-6Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular featuresJanssen M. Kotah0Thomas Rust1Hilmar R. J. van Weering2Janneke Bosma3Amber L. Woudstra4Susanne M. Kooistra5Bart J. L. Eggen6Department of Biomedical Sciences, University of Groningen, University Medical Center GroningenDepartment of Biomedical Sciences, University of Groningen, University Medical Center GroningenDepartment of Biomedical Sciences, University of Groningen, University Medical Center GroningenDepartment of Biomedical Sciences, University of Groningen, University Medical Center GroningenDepartment of Biomedical Sciences, University of Groningen, University Medical Center GroningenDepartment of Biomedical Sciences, University of Groningen, University Medical Center GroningenDepartment of Biomedical Sciences, University of Groningen, University Medical Center GroningenAbstract Spatial transcriptomics has advanced our understanding of cellular heterogeneity at single-cell resolution. Here, we assess the suitability of in situ sequencing (ISS) for analyzing formalin-fixed, paraffin-embedded (FFPE) postmortem human brain tissue. A key challenge in ISS data analysis is optimizing transcript allocation while minimizing misallocation, particularly in the morphologically complex central nervous system (CNS). We compared geospatial methods using nuclear and expanded nuclear boundaries for segmentation and transcript allocation. While overall cell-type proportions remained comparable, transcript allocation methods affected specific cell types, including microglia, neurons, and neurovascular cells. To enhance specificity, we integrated fluorescent imaging data targeting 18S RNA and IBA1 protein to direct transcript allocation toward RNA-rich cells (e.g., neurons) and microglia, respectively. We demonstrate how this approach, paired with secondary allocation of transcripts outside imaging masks, improved both the number of microglia detected and the specificity of microglial transcripts assigned. Our method offers a flexible and efficient strategy for targeted transcript allocation based on cellular morphology, optimizing CNS cell segmentation in FFPE-preserved human brain tissue.https://doi.org/10.1038/s42003-025-08518-6 |
| spellingShingle | Janssen M. Kotah Thomas Rust Hilmar R. J. van Weering Janneke Bosma Amber L. Woudstra Susanne M. Kooistra Bart J. L. Eggen Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features Communications Biology |
| title | Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features |
| title_full | Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features |
| title_fullStr | Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features |
| title_full_unstemmed | Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features |
| title_short | Beyond the nuclear border: single-cell analysis of in situ sequenced human brain tissue using cellular features |
| title_sort | beyond the nuclear border single cell analysis of in situ sequenced human brain tissue using cellular features |
| url | https://doi.org/10.1038/s42003-025-08518-6 |
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