Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue
Abstract 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 c...
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2018-07-01
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Series: | Arthritis Research & Therapy |
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Online Access: | http://link.springer.com/article/10.1186/s13075-018-1631-y |
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language |
English |
format |
Article |
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DOAJ |
author |
Laura T. Donlin Deepak A. Rao Kevin Wei Kamil Slowikowski Mandy J. McGeachy Jason D. Turner Nida Meednu Fumitaka Mizoguchi Maria Gutierrez-Arcelus David J. Lieb Joshua Keegan Kaylin Muskat Joshua Hillman Cristina Rozo Edd Ricker Thomas M. Eisenhaure Shuqiang Li Edward P. Browne Adam Chicoine Danielle Sutherby Akiko Noma Accelerating Medicines Partnership RA/SLE Network Chad Nusbaum Stephen Kelly Alessandra B. Pernis Lionel B. Ivashkiv Susan M. Goodman William H. Robinson Paul J. Utz James A. Lederer Ellen M. Gravallese Brendan F. Boyce Nir Hacohen Costantino Pitzalis Peter K. Gregersen Gary S. Firestein Soumya Raychaudhuri Larry W. Moreland V. Michael Holers Vivian P. Bykerk Andrew Filer David L. Boyle Michael B. Brenner Jennifer H. Anolik |
spellingShingle |
Laura T. Donlin Deepak A. Rao Kevin Wei Kamil Slowikowski Mandy J. McGeachy Jason D. Turner Nida Meednu Fumitaka Mizoguchi Maria Gutierrez-Arcelus David J. Lieb Joshua Keegan Kaylin Muskat Joshua Hillman Cristina Rozo Edd Ricker Thomas M. Eisenhaure Shuqiang Li Edward P. Browne Adam Chicoine Danielle Sutherby Akiko Noma Accelerating Medicines Partnership RA/SLE Network Chad Nusbaum Stephen Kelly Alessandra B. Pernis Lionel B. Ivashkiv Susan M. Goodman William H. Robinson Paul J. Utz James A. Lederer Ellen M. Gravallese Brendan F. Boyce Nir Hacohen Costantino Pitzalis Peter K. Gregersen Gary S. Firestein Soumya Raychaudhuri Larry W. Moreland V. Michael Holers Vivian P. Bykerk Andrew Filer David L. Boyle Michael B. Brenner Jennifer H. Anolik Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue Arthritis Research & Therapy Rheumatoid arthritis Synovial tissue Accelerating Medicines Partnership RNA sequencing CyTOF Mass cytometry |
author_facet |
Laura T. Donlin Deepak A. Rao Kevin Wei Kamil Slowikowski Mandy J. McGeachy Jason D. Turner Nida Meednu Fumitaka Mizoguchi Maria Gutierrez-Arcelus David J. Lieb Joshua Keegan Kaylin Muskat Joshua Hillman Cristina Rozo Edd Ricker Thomas M. Eisenhaure Shuqiang Li Edward P. Browne Adam Chicoine Danielle Sutherby Akiko Noma Accelerating Medicines Partnership RA/SLE Network Chad Nusbaum Stephen Kelly Alessandra B. Pernis Lionel B. Ivashkiv Susan M. Goodman William H. Robinson Paul J. Utz James A. Lederer Ellen M. Gravallese Brendan F. Boyce Nir Hacohen Costantino Pitzalis Peter K. Gregersen Gary S. Firestein Soumya Raychaudhuri Larry W. Moreland V. Michael Holers Vivian P. Bykerk Andrew Filer David L. Boyle Michael B. Brenner Jennifer H. Anolik |
author_sort |
Laura T. Donlin |
title |
Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue |
title_short |
Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue |
title_full |
Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue |
title_fullStr |
Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue |
title_full_unstemmed |
Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue |
title_sort |
methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue |
publisher |
BMC |
series |
Arthritis Research & Therapy |
issn |
1478-6362 |
publishDate |
2018-07-01 |
description |
Abstract 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 μg/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. |
topic |
Rheumatoid arthritis Synovial tissue Accelerating Medicines Partnership RNA sequencing CyTOF Mass cytometry |
url |
http://link.springer.com/article/10.1186/s13075-018-1631-y |
work_keys_str_mv |
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doaj-f1834e16a4ee4cddb44b1feb039eb1182020-11-25T00:37:36ZengBMCArthritis Research & Therapy1478-63622018-07-0120111510.1186/s13075-018-1631-yMethods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissueLaura T. Donlin0Deepak A. Rao1Kevin Wei2Kamil Slowikowski3Mandy J. McGeachy4Jason D. Turner5Nida Meednu6Fumitaka Mizoguchi7Maria Gutierrez-Arcelus8David J. Lieb9Joshua Keegan10Kaylin Muskat11Joshua Hillman12Cristina Rozo13Edd Ricker14Thomas M. Eisenhaure15Shuqiang Li16Edward P. Browne17Adam Chicoine18Danielle Sutherby19Akiko Noma20Accelerating Medicines Partnership RA/SLE NetworkChad Nusbaum21Stephen Kelly22Alessandra B. Pernis23Lionel B. Ivashkiv24Susan M. Goodman25William H. Robinson26Paul J. Utz27James A. Lederer28Ellen M. Gravallese29Brendan F. Boyce30Nir Hacohen31Costantino Pitzalis32Peter K. Gregersen33Gary S. Firestein34Soumya Raychaudhuri35Larry W. Moreland36V. Michael Holers37Vivian P. Bykerk38Andrew Filer39David L. Boyle40Michael B. Brenner41Jennifer H. Anolik42Hospital for Special SurgeryBrigham and Women’s Hospital, Harvard Medical SchoolBrigham and Women’s Hospital, Harvard Medical SchoolBrigham and Women’s Hospital, Harvard Medical SchoolUniversity of Pittsburgh School of MedicineUniversity of Birmingham, Queen Elizabeth HospitalUniversity of Rochester Medical CenterBrigham and Women’s Hospital, Harvard Medical SchoolBrigham and Women’s Hospital, Harvard Medical SchoolBroad Institute of MIT and Harvard UniversityBrigham and Women’s Hospital, Harvard Medical SchoolUniversity of California San Diego School of MedicineUniversity of California San Diego School of MedicineHospital for Special SurgeryHospital for Special SurgeryBroad Institute of MIT and Harvard UniversityBroad Institute of MIT and Harvard UniversityBroad Institute of MIT and Harvard UniversityBrigham and Women’s Hospital, Harvard Medical SchoolBroad Institute of MIT and Harvard UniversityBroad Institute of MIT and Harvard UniversityBroad Institute of MIT and Harvard UniversityMile End Hospital, Barts Health NHS TrustHospital for Special SurgeryHospital for Special SurgeryHospital for Special SurgeryStanford University School of MedicineStanford University School of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolUniversity of Massachusetts Medical SchoolUniversity of Rochester Medical CenterBroad Institute of MIT and Harvard UniversityQueen Mary University of LondonThe Feinstein Institute for Medical ResearchUniversity of California San Diego School of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolUniversity of Pittsburgh School of MedicineUniversity of Colorado of Denver School of MedicineHospital for Special SurgeryUniversity of Birmingham, Queen Elizabeth HospitalUniversity of California San Diego School of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolUniversity of Rochester Medical CenterAbstract 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 μg/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.http://link.springer.com/article/10.1186/s13075-018-1631-yRheumatoid arthritisSynovial tissueAccelerating Medicines PartnershipRNA sequencingCyTOFMass cytometry |