Department of Medicine, Division of Rheumatology
Cell Biology | Cells | Immune System Diseases | Immunopathology | Investigative Techniques | Musculoskeletal Diseases | Pathology | Rheumatology | Skin and Connective Tissue Diseases | Tissues
BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples.
METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq.
RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 mug/ml of Liberase TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4(+) and CD8(+) T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified.
CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.
Accelerating Medicines Partnership, Arthroplasty, CyTOF, Mass cytometry, RNA sequencing, Rheumatoid arthritis, Synovial biopsy, Synovial tissue
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© The Author(s). 2018 Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
DOI of Published Version
Arthritis Res Ther. 2018 Jul 11;20(1):139. doi: 10.1186/s13075-018-1631-y. Link to article on publisher's site
Arthritis research and therapy
Donlin LT, Gravallese EM, Anolik JH. (2018). Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue. Open Access Articles. https://doi.org/10.1186/s13075-018-1631-y. Retrieved from https://escholarship.umassmed.edu/oapubs/3548
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Cell Biology Commons, Cells Commons, Immune System Diseases Commons, Immunopathology Commons, Investigative Techniques Commons, Musculoskeletal Diseases Commons, Pathology Commons, Rheumatology Commons, Skin and Connective Tissue Diseases Commons, Tissues Commons