High dimensional analyses 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 (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 TM 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 4 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.

Defining Inflammatory Cell States in Rheumatoid Arthritis Joint Synovial Tissues by Integrating Single-cell Transcriptomics and Mass Cytometry

“…Synovial tissue disaggregation yielded many viable cells (362,190 cells per tissue, S.E.M 7,687 cells) for downstream analyses. Applying a previously validated strategy for synovial cell sorting 24 ( Fig. 1a ), we separated cells into B cells (CD45 + CD3 − CD19 + ), T cells (CD45 + CD3 + ), monocytes (CD45 + CD14 + ), and stromal fibroblasts (CD45 − PDPN + ) ( Supplemental Fig.…”

“…We observed upregulation of chemokines ( CXCL8 , CXCL9 , and CXCL13 ), cytokines ( IFNG and IL15 ), and surface receptors ( PDGFRB and SLAMF7 ) in distinct immune and stromal cell populations, suggesting potential novel targets. This study was enabled by important advances in the statistical analysis of single-cell data 21,58–61 alongside rapid improvements in scaling single cell technologies 17,62 and our recent work optimizing robust methodologies for disaggregation of synovial tissue 24 .…”

“…To generate high-dimensional multi-modal single-cell data from synovial tissue samples, we developed a robust tissue analytical pipeline 24 in the Accelerating Medicines Partnership (AMP) RA/SLE consortium. We collected and disaggregated tissue samples from patients with RA and OA, and then subjected constituent cells to scRNA-seq, sorted-population bulk RNA-seq, mass cytometry, and flow cytometry.…”

Synovial Tissue Heterogeneity in Rheumatoid Arthritis and Changes With Biologic and Targeted Synthetic Therapies to Inform Stratified Therapy

“…We will soon see the outcomes of such initiatives [including STRAP—Stratification of Biologic Therapies for RA by Pathobiology (ISRCTN10618686) and R4-RA—A Randomized, open labeled study in anti-TNFa inadequate responders to investigate the mechanisms for Response—Resistance to Rituximab vs. Tocilizumab in RA (ISRCTN97443826)] that will inform future tissue driven trial design. These trials and other tissue-based programmes such as the recently established NIH Accelerating Medicines Partnership (AMP) RA/SLE network will also exploit high-dimensional analyses including mass cytometry, RNA-seq of selected cell populations, and single cell RNA-seq (83). Whilst the sheer volume of data in itself presents massive challenges in the clinically meaningful interpretation, the richness of data matched with improved sophisticated analytical techniques holds the promise of being able to join the field of personalized RA targeted therapy use.…”

Systems Biology and Kidney Disease

“…One of the key challenges in defining molecular mechanisms of kidney disease is the cellular heterogeneity of the kidney, second probably only to the human brain (56). Sustained efforts to overcome this challenge include microdissection of kidney tissue and biopsies for molecular analysis (57,58), development of profiling technologies for individual microdissected cells (59), and computational efforts to define cell type-specific signals from complex kidney biopsies signatures (60).…”