Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

High dimensional analyses of cells dissociated from cryopreserved synovial tissue

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
doi: https://doi.org/10.1101/284844
Laura T. Donlin
1Hospital for Special Surgery, New York, NY, 10021 USA
2Weill Cornell Medical College, New York, NY, 10065 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Deepak A. Rao
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kevin Wei
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kamil Slowikowski
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mandy J. McGeachy
5University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jason D. Turner
6University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nida Meednu
7University of Rochester Medical Center, Rochester, NY, 14642, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fumitaka Mizoguchi
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maria Gutierrez-Arcelus
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David J. Lieb
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joshua Keegan
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kaylin Muskat
8University of California San Diego School of Medicine, La Jolla, CA, 92093 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joshua Hillman
8University of California San Diego School of Medicine, La Jolla, CA, 92093 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Cristina Rozo
1Hospital for Special Surgery, New York, NY, 10021 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Edd Ricker
1Hospital for Special Surgery, New York, NY, 10021 USA
2Weill Cornell Medical College, New York, NY, 10065 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thomas M. Eisenhaure
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shuqiang Li
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Edward P. Browne
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Adam Chicoine
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Danielle Sutherby
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Akiko Noma
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Chad Nusbaum
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stephen Kelly
10Mile End Hospital, Barts Health NHS Trust, London, E1 1BB, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alessandra B. Pernis
1Hospital for Special Surgery, New York, NY, 10021 USA
2Weill Cornell Medical College, New York, NY, 10065 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lionel B. Ivashkiv
1Hospital for Special Surgery, New York, NY, 10021 USA
2Weill Cornell Medical College, New York, NY, 10065 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susan M. Goodman
1Hospital for Special Surgery, New York, NY, 10021 USA
2Weill Cornell Medical College, New York, NY, 10065 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
William H. Robinson
11Stanford University School of Medicine, Stanford, CA, 94305 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul J. Utz
11Stanford University School of Medicine, Stanford, CA, 94305 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
James A. Lederer
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ellen M. Gravallese
12University of Massachusetts Memorial Medical Center, Worcester, MA, 01605 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Brendan F. Boyce
7University of Rochester Medical Center, Rochester, NY, 14642, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nir Hacohen
4Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, MA, 02142 USA
13Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Costantino Pitzalis
14Queen Mary University of London, London, E1 4NS, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Peter K. Gregersen
15The Feinstein Institute for Medical Research, Manhasset, New York, 11030, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gary S. Firestein
8University of California San Diego School of Medicine, La Jolla, CA, 92093 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Soumya Raychaudhuri
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Larry W. Moreland
5University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
V. Michael Holers
16University of Colorado of Denver School of Medicine, Aurora, CO, 80045, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Vivian P. Bykerk
1Hospital for Special Surgery, New York, NY, 10021 USA
2Weill Cornell Medical College, New York, NY, 10065 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrew Filer
6University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David L. Boyle
8University of California San Diego School of Medicine, La Jolla, CA, 92093 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael B. Brenner
3Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115 USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer H. Anolik
7University of Rochester Medical Center, Rochester, NY, 14642, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: jennifer_anolik@urmc.rochester.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

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 (AMP) 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 100µg/mL of Liberase TL™ enzyme. 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: 1) diverse fibroblast phenotypes, 2) distinct populations of memory B cells and antibody-secreting cells, and 3) multiple CD4+ and CD8+ T cell activation states. Bulk RNA sequencing 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.

Conclusion 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted March 19, 2018.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
High dimensional analyses of cells dissociated from cryopreserved synovial tissue
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
High dimensional analyses of cells dissociated from cryopreserved synovial tissue
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
bioRxiv 284844; doi: https://doi.org/10.1101/284844
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
High dimensional analyses of cells dissociated from cryopreserved synovial tissue
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
bioRxiv 284844; doi: https://doi.org/10.1101/284844

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Immunology
Subject Areas
All Articles
  • Animal Behavior and Cognition (4076)
  • Biochemistry (8749)
  • Bioengineering (6467)
  • Bioinformatics (23310)
  • Biophysics (11710)
  • Cancer Biology (9131)
  • Cell Biology (13224)
  • Clinical Trials (138)
  • Developmental Biology (7402)
  • Ecology (11357)
  • Epidemiology (2066)
  • Evolutionary Biology (15074)
  • Genetics (10390)
  • Genomics (13997)
  • Immunology (9106)
  • Microbiology (22018)
  • Molecular Biology (8767)
  • Neuroscience (47303)
  • Paleontology (350)
  • Pathology (1418)
  • Pharmacology and Toxicology (2480)
  • Physiology (3701)
  • Plant Biology (8041)
  • Scientific Communication and Education (1427)
  • Synthetic Biology (2206)
  • Systems Biology (6009)
  • Zoology (1246)