| Title: |
Beyond the nuclear border:single-cell analysis of in situ sequenced human brain tissue using cellular features |
| Authors: |
Kotah, Janssen M; Rust, Thomas; id_orcid 0000-0003-2000-8141; van Weering, Hilmar R J; Bosma, Janneke; id_orcid 0009-0002-3820-6293; Woudstra, Amber L; Kooistra, Susanne M; Eggen, Bart J L; id_orcid 0000-0001-8941-0353 |
| Source: |
Kotah, J M, Rust, T, van Weering, H R J, Bosma, J, Woudstra, A L, Kooistra, S M & Eggen, B J L 2025, 'Beyond the nuclear border : single-cell analysis of in situ sequenced human brain tissue using cellular features', Communications biology, vol. 8, no. 1, 1089. |
| Publisher Information: |
2025-07-22 |
| Document Type: |
Electronic Resource |
| 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. |
| Index Terms: |
Humans; Single-Cell Analysis/methods; Brain/metabolism; Microglia/metabolism; Transcriptome; Cell Nucleus; Gene Expression Profiling/methods; Neurons/metabolism; info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion |
| URL: |
https://research.rug.nl/en/publications/58a646d3-4f83-42e5-98b6-e21423f76cb8; https://doi.org/10.1038/s42003-025-08518-6; https://hdl.handle.net/11370/58a646d3-4f83-42e5-98b6-e21423f76cb8; https://pure.rug.nl/ws/files/1413561786/s42003-025-08518-6.pdf; info:eu-repo/semantics/altIdentifier/doi/10.1038/s42003-025-08518-6; info:eu-repo/semantics/altIdentifier/pmid/40696148; info:eu-repo/semantics/altIdentifier/hdl/https://hdl.handle.net/11370/58a646d3-4f83-42e5-98b6-e21423f76cb8; info:eu-repo/semantics/altIdentifier/pissn/2399-3642; info:eu-repo/semantics/altIdentifier/eissn/2399-3642 |
| Availability: |
Open access content. Open access content; info:eu-repo/semantics/openAccess; http://creativecommons.org/licenses/by-nc-nd/4.0 |
| Note: |
application/pdf; Kotah, J M, Rust, T, van Weering, H R J, Bosma, J, Woudstra, A L, Kooistra, S M & Eggen, B J L 2025, 'Beyond the nuclear border : single-cell analysis of in situ sequenced human brain tissue using cellular features', Communications biology, vol. 8, no. 1, 1089. https://doi.org/10.1038/s42003-025-08518-6; English |
| Other Numbers: |
GRU oai:pure.rug.nl:publications/58a646d3-4f83-42e5-98b6-e21423f76cb8; 1553661341 |
| Contributing Source: |
UNIV OF GRONINGEN; From OAIster®, provided by the OCLC Cooperative. |
| Accession Number: |
edsoai.on1553661341 |
| Database: |
OAIster |