Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Accelerating Medicines Partnership RA/SLE Network

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish (US)
Article number139
JournalArthritis Research and Therapy
Volume20
Issue number1
DOIs
StatePublished - Jul 11 2018

Fingerprint

RNA Sequence Analysis
Transcriptome
Phenotype
Rheumatoid Arthritis
Synovial Membrane
Fibroblasts
Pathology
Arthroplasty
B-Lymphocytes
Macrophages
Population
T-Lymphocytes
Biopsy
Antibody-Producing Cells
Cell Lineage
Stromal Cells
Dimethyl Sulfoxide
Digestion
Suspensions
Membrane Proteins

Keywords

  • Accelerating Medicines Partnership
  • Arthroplasty
  • CyTOF
  • Mass cytometry
  • Rheumatoid arthritis
  • RNA sequencing
  • Synovial biopsy
  • Synovial tissue

ASJC Scopus subject areas

  • Rheumatology
  • Immunology and Allergy
  • Immunology

Cite this

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue. / Accelerating Medicines Partnership RA/SLE Network.

In: Arthritis Research and Therapy, Vol. 20, No. 1, 139, 11.07.2018.

Research output: Contribution to journalArticle

Accelerating Medicines Partnership RA/SLE Network. / Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue. In: Arthritis Research and Therapy. 2018 ; Vol. 20, No. 1.
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title = "Methods for high-dimensonal analysis of cells dissociated from cyropreserved 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 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.",
keywords = "Accelerating Medicines Partnership, Arthroplasty, CyTOF, Mass cytometry, Rheumatoid arthritis, RNA sequencing, Synovial biopsy, Synovial tissue",
author = "{Accelerating Medicines Partnership RA/SLE Network} and Donlin, {Laura T.} and Rao, {Deepak A.} and Kevin Wei and Kamil Slowikowski and McGeachy, {Mandy J.} and Turner, {Jason D.} and Nida Meednu and Fumitaka Mizoguchi and Maria Gutierrez-Arcelus and Lieb, {David J.} and Joshua Keegan and Kaylin Muskat and Joshua Hillman and Cristina Rozo and Edd Ricker and Eisenhaure, {Thomas M.} and Shuqiang Li and Browne, {Edward P.} and Adam Chicoine and Danielle Sutherby and Akiko Noma and Chad Nusbaum and Stephen Kelly and Pernis, {Alessandra B.} and Ivashkiv, {Lionel B.} and Goodman, {Susan M.} and Robinson, {William H.} and Utz, {Paul J.} and Lederer, {James A.} and Gravallese, {Ellen M.} and Boyce, {Brendan F.} and Nir Hacohen and Costantino Pitzalis and Gregersen, {Peter K.} and Firestein, {Gary S.} and Soumya Raychaudhuri and Moreland, {Larry W.} and Holers, {V. Michael} and Bykerk, {Vivian P.} and Andrew Filer and Boyle, {David L.} and Brenner, {Michael B.} and Anolik, {Jennifer H.} and Jane Buckner and Derrick Todd and Michael Weisman and Artzi, {Ami Ben} and Lindsy Forbess and Joan Bathon and Chaim Putterman",
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T1 - Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

AU - Accelerating Medicines Partnership RA/SLE Network

AU - Donlin, Laura T.

AU - Rao, Deepak A.

AU - Wei, Kevin

AU - Slowikowski, Kamil

AU - McGeachy, Mandy J.

AU - Turner, Jason D.

AU - Meednu, Nida

AU - Mizoguchi, Fumitaka

AU - Gutierrez-Arcelus, Maria

AU - Lieb, David J.

AU - Keegan, Joshua

AU - Muskat, Kaylin

AU - Hillman, Joshua

AU - Rozo, Cristina

AU - Ricker, Edd

AU - Eisenhaure, Thomas M.

AU - Li, Shuqiang

AU - Browne, Edward P.

AU - Chicoine, Adam

AU - Sutherby, Danielle

AU - Noma, Akiko

AU - Nusbaum, Chad

AU - Kelly, Stephen

AU - Pernis, Alessandra B.

AU - Ivashkiv, Lionel B.

AU - Goodman, Susan M.

AU - Robinson, William H.

AU - Utz, Paul J.

AU - Lederer, James A.

AU - Gravallese, Ellen M.

AU - Boyce, Brendan F.

AU - Hacohen, Nir

AU - Pitzalis, Costantino

AU - Gregersen, Peter K.

AU - Firestein, Gary S.

AU - Raychaudhuri, Soumya

AU - Moreland, Larry W.

AU - Holers, V. Michael

AU - Bykerk, Vivian P.

AU - Filer, Andrew

AU - Boyle, David L.

AU - Brenner, Michael B.

AU - Anolik, Jennifer H.

AU - Buckner, Jane

AU - Todd, Derrick

AU - Weisman, Michael

AU - Artzi, Ami Ben

AU - Forbess, Lindsy

AU - Bathon, Joan

AU - Putterman, Chaim

PY - 2018/7/11

Y1 - 2018/7/11

N2 - 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.

AB - 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.

KW - Accelerating Medicines Partnership

KW - Arthroplasty

KW - CyTOF

KW - Mass cytometry

KW - Rheumatoid arthritis

KW - RNA sequencing

KW - Synovial biopsy

KW - Synovial tissue

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