Background
The destructive inflammatory environment in rheumatoid arthritis (RA) synovium results from the activity of various cell types, including synovial fibroblasts, macrophages, lymphocytes, osteoclasts, and vascular endothelial cells [
1‐
5]. Multiple pathways can be targeted to treat RA, including inhibition of tumor necrosis factor (TNF) or interleukin-6 (IL-6) signaling, blockade of T-cell costimulation, depletion of B cells, and inhibition of the JAK/STAT pathway [
6]. However, despite the advent of biologic therapies, up to ~ 2/3 of RA patients do not achieve sustained disease remission [
7], and there are no reliable biomarkers that serve to guide selection of specific therapeutic options for the individual patient. A more comprehensive interrogation of cells present in rheumatoid synovium may identify additional pathologic pathways targetable by therapeutics and aid in stratifying patients into disease subsets and treatment response categories based on informative biomarkers [
8,
9]. Such studies have previously been limited by the lack of methods to simultaneously recover and assay the diversity of cell types in the synovium and by the challenge of applying high-dimensional single cell analytics to sufficient numbers of patient samples. In addition, comparisons of identical cell lineages isolated from targeted tissues in patients with RA and other diseases such as systemic lupus erythematosus (SLE) have not been performed.
In recognition of these needs, the NIH Accelerating Medicines Partnership (AMP) RA/SLE Network was assembled with the goal of generating detailed analyses of synovial tissue samples from a large number of RA patients. The RA Working Group of the AMP RA/SLE Network (or AMP RA network) has developed a pipeline to study RA synovial tissue samples through parallel high-dimensional analyses including mass cytometry, bulk RNA-seq of selected cell populations, and single cell RNA-seq. Mass cytometry can define the cellular landscape of tissues, while RNA-seq delves deeper into the gene expression profile for each cell type. When performed at scale on a large patient cohort, these assays have the potential to aid in developing personalized therapeutic approaches for RA.
To amass an RA cohort of sufficient size, numerous clinical collection sites contributed synovial tissue samples. The network set out to develop a pipeline for uniform and reproducible sample handling involving cryopreservation of intact synovial tissue samples. The frozen tissue fragments were then shipped to a central processing site capable of performing multiple high-dimensional analyses in parallel for each sample. The protocol was optimized for two sources of tissue: synovial biopsy obtained for research and clinically indicated excision during arthroplasty.
Here, we report the AMP RA network protocol for dissociation and analysis of immune and stromal cells from cryopreserved synovial tissue by multiple high-dimensional technologies. We describe a consensus protocol for synovial tissue disaggregation that results in high yields of viable cells with preserved surface marker expression—for both larger arthroplasty and millimeter-sized biopsy samples. We also describe an experimental pipeline to analyze each sample in parallel using single-cell RNA-seq and low-input bulk RNA-seq on selected populations, as well as mass cytometry with defined markers for synovial cells. This pipeline allows for cytometric identification and quantification of numerous immune and stromal cell populations, as well as robust transcriptomic analyses of small populations of cells from pathologic synovial samples. These methods are adaptable for use in multiple sites and have been adopted and utilized by the AMP RA network.
Methods
Human patient samples
Patients with RA fulfilled the ACR 2010 Rheumatoid Arthritis classification criteria or were identified by the participating site rheumatologist as having clinical RA [
6]. A diagnosis of osteoarthritis (OA) was defined by the treating surgeon and confirmed by chart review. Synovial tissue samples were acquired by two different methods: arthroplasty and ultrasound-guided synovial biopsy. Arthroplasty samples were acquired after removal as part of standard of care at four US institutions (Hospital for Special Surgery, NY; University California San Diego, CA; University of Pittsburgh, PA; and University of Rochester, NY). All synovial biopsy samples were acquired from clinically inflamed joints under research protocols at the University of Birmingham, UK and Barts & the London School of Medicine and Dentistry, UK. The study received institutional review board approval at each site.
Arthroplasty synovial tissue collection
Synovial tissue excised as standard of care during arthroplasty was transported from the operating room to a laboratory in ice-cold PBS. The tissue was identified as synovium by characteristic features: a fibrous or elastic tissue, pink or reddish in appearance. Small fragments (~ 1–2 mm
3) were generated by dissecting with forceps or surgical scissors. Six fragments (~ 150 mg total) across the tissue were randomly combined to make individual aliquots [
10,
11].
Synovial biopsy procedures
Synovial biopsy samples were obtained under ultrasound guidance by an experienced interventionist using either a needle biopsy or portal and forceps. For needle biopsy procedures, a 14G or 16G spring-loaded Cook Quick-Core® Biopsy needle was used to obtain multiple fragments [
12,
13]. For the portal-based approach, a portal (Merit Medical Prelude 7F) was initially inserted into the synovial cavity with multiple samples and then retrieved using flexible 2.0–2.2 mm forceps [
14,
15]. Six biopsy fragments were randomly allocated per aliquot.
Synovial tissue cryopreservation and thawing
Synovial tissues were either (1) disaggregated immediately followed by cryopreservation of dissociated cells or (2) cut into fragments that were cryopreserved for subsequent disaggregation at a central processing site. Dissociated synovial cells were resuspended in CryoStor® CS10 (BioLife Solutions) at ~ 2 million cells/ml to viably freeze them. Intact synovial tissue samples were divided into fragments as already described and transferred to a cryovial (1.5 ml; Nalgene) containing 1 ml of CryoStor® CS10 for viable freezing. Cryovials were then placed in an insulated container with isopropanol in the bottom chamber for slow freezing (Mr. Frosty; Nalgene), which comprised incubation at 4 °C for 10 min followed by 1 day at − 80 °C. The samples were then either shipped on dry ice or transferred into liquid nitrogen for long-term storage.
Synovial fragments were thawed by rapidly warming the cryovial in a 37 °C water bath. The preservation media was filtered out through a 70-μm strainer. The tissue was then rinsed through a series of incubations in a six-well culture plate: 10 min in 10% FBS/RPMI at room temperature with intermittent swirling, a quick rinse in 10% FBS/RPMI, and a final rinse in serum-free RPMI. Frozen synovial cells were thawed rapidly in a 37 °C water bath and transferred into 20 ml of 10% FBS/RPMI, centrifuged to pellet cells, and then resuspended in media for downstream analyses.
Dissociation of synovial tissue
Both arthroplasty and synovial biopsy samples were dissociated by a combination of enzymatic digestion and mechanical disruption with various test conditions described in Results. The final consensus AMP RA network enzyme digestion uses RPMI media with Liberase™ TL enzyme preparations (100 μg/ml; Roche) and DNase I (100 μg/ml; Roche).
Arthroplasty tissue
To achieve consistent mechanical disruption and proteolytic enzyme exposure across a fragment of tissue, large synovial specimens (> 150 mg) were cut into small fragments (~ 2 mm3) using surgical scissors. Mechanical disruption of arthroplasty samples (six tissue fragments totaling ≥ 500 mg) was carried out by a gentleMACS dissociator system (Miltenyi) with the m_Spleen 04.01 setting, which involves churning and shredding in a sterile disposable tube. Enzymatic digestion was performed in RPMI medium at 37 °C for 30 min. A large volume of 5% FBS/RPMI was then added to terminate the enzymatic reaction. The tissue was ground through a 70-μm filter using the flat plastic end of a 3-ml syringe plunger to disperse the remaining intact tissue and dispense dissociated cells.
Synovial biopsy tissue
For the samples obtained by synovial biopsy, tissue fragments were minced into small fragments (~1 mm3) using a scalpel. Samples were then subjected to enzymatic digestion at 37 °C while being exposed to continuous stirring in a U-bottom polystyrene tube (12 × 75 mm2) with a magnetic stir bar for 30 min. Halfway through the enzymatic digestion, samples were passed gently through a 16G syringe needle 10 times for additional mechanical disruption.
For samples used in the aforementioned dissociation protocol, synovial tissue fragments were also preserved for whole tissue RNA extraction. Three whole tissue replicates were collected for each sample, with six fragments placed into a cryovial containing 1 ml of RNALater (Qiagen) and inverted three times. The cryovials were incubated overnight at 4 °C. The next day, the cryovials were spun at ~ 1000×g for 30 s and most of the RNALater was removed, leaving only enough RNALater to cover the tissue. The cryovials were then placed in storage at − 70 °C. For RNA extraction, samples were thawed and fragments transferred into RLT lysis buffer (Qiagen) + 1% β-mercaptoethanol (Sigma) and homogenized using a TissueLyser II (Qiagen) before RNA isolation using RNeasy columns.
Flow cytometry cell sorting
Synovial cell suspensions were stained with an 11-color flow cytometry panel designed to identify synovial stromal and leukocyte populations. Antibodies included anti-CD45-FITC (HI30), anti-CD90-PE(5E10), anti-podoplanin-PerCP/eFluor710 (NZ1.3), anti-CD3-PECy7 (UCHT1), anti-CD19-BV421 (HIB19), anti-CD14-BV510 (M5E2), anti-CD34-BV605 (4H11), anti-CD4-BV650 (RPA-T4), anti-CD8-BV711 (SK1), anti-CD31-AlexaFluor700 (WM59), anti-CD27-APC (M-T271), anti-CD235a-APC/AF750, TruStain FcX, and propidium iodide. Cells were stained in HEPES-buffered saline (20 mM HEPES, 137 mM NaCl, 3 mM KCl, 1 mM CaCl2) with 1% bovine serum albumin (BSA) for 30 min, then washed once, resuspended in the same buffer with propidium iodide added, vortexed briefly, and passed through a 100-μm filter.
Cells were sorted on a three-laser BD FACSAria Fusion cell sorter. Intact cells were gated according to FSC-A and SSC-A. Doublets were excluded by serial FSC-H/FSC-W and SSC-H/SSC-W gates. Nonviable cells were excluded based on propidium iodide uptake. Cells were sorted through a 100-μm nozzle at 20 psi.
A serial sorting strategy was used to sequentially capture cells for bulk RNA-seq and then single-cell RNA-seq if sufficient numbers of cells were present. First, 1000 cells of the targeted cell type were sorted for low-input RNA-seq into a 1.7-ml Eppendorf tube containing 350 μl of RLT lysis buffer (Qiagen) + 1% β-mercaptoethanol. Once 1000 cells of a particular cell type were collected, the sort was stopped and the tube was exchanged for a second tube containing FACS buffer. Sorting was then resumed and the rest of the cells of that type were collected into the second tube as viable cells. This process was carried out for four targeted populations. Live cells of each population that were sorted into FACS buffer were then resorted as single cells into wells of 384-well plates containing 1 μl of 1% NP-40, targeting up to 144 cells of each type per sample.
RNA from sorted bulk cell populations was isolated using RNeasy columns (Qiagen). RNA from up to 1000 cells was treated with DNase I (New England Biolabs), and then concentrated using Agencourt RNAClean XP beads (Beckman Coulter). Full-length cDNA and sequencing libraries were prepared using the Smart-Seq2 protocol as described previously [
16]. Libraries were sequenced on a MiSeq (Illumina) to generate 25-base-pair, paired-end reads totaling a fragment length of 50 base pairs.
Single-cell RNA sequencing
Single cell RNAseq (scRNA-Seq) was performed using the CEL-Seq2 method [
17] with the following modifications. Single cells were sorted into 384-well plates containing 0.6 μl of 1% NP-40 buffer in each well. Then, 0.6 μl dNTPs (10 mM each; NEB) and 5 nl of barcoded reverse transcription primer (1 μg/μl) were added to each well along with 20 nl of ERCC spike-in (diluted 1:800,000). Reactions were incubated at 65 °C for 5 min, and then moved immediately to ice. Reverse transcription reactions were carried out as described previously [
17] and cDNA was purified using 0.8× volumes of AMPure XP beads (Beckman Coulter). In-vitro transcription reactions (IVT) were performed as described followed by EXO-SAP treatment. Amplified RNA (aRNA) was fragmented at 80 °C for 3 min and purified using RNAClean XP beads (Beckman Coulter). The purified aRNA was converted to cDNA using an anchored random primer and Illumina adaptor sequences were added by PCR. The final cDNA library was purified using AMPure XP beads (Beckman Coulter). Libraries were sequenced on a Hiseq 2500 (Illumina) in Rapid Run Mode to generate paired-end reads. Forward reads (35 nt) were mapped to human reference genome hg19 using STAR v2.5 [
18], while reverse reads (15 nt) included cellular and molecular barcodes (6 nt each). Basic analyses of single-cell RNA-seq data, including quantification of genes detected per cell, filtering of cells with less than 1000 detected genes, and principal components analyses, were performed using RSEM [
19].
Differential expression
To examine differential gene expression profiles between RA and OA synovial tissues processed through the consensus disaggregation protocol, tissue samples were collected from 10 RA patients and 10 OA patients. From these 20 patients, 2–8 replicates were generated per donor for a total of 87 samples. We performed differential expression analysis by fitting a linear mixed model to each gene, in which we controlled for unwanted technical variation with donor and site as random effects.
Gene Ontology term enrichment analysis
We tested Gene Ontology (GO) enrichment with differentially expressed genes. We used Ensembl gene IDs downloaded in April 2016, including 9797 GO terms and 15,693 genes. The minimal hypergeometric test was used to test for significance [
20].
Mass cytometry
Synovial cells were resuspended in PBS/1%BSA with primary antibody cocktails at 1:100 dilution for 30 min (Additional file
1: Table S1). All antibodies were obtained from the Longwood Medical Area CyTOF Antibody Resource Core (Boston, MA, USA). Cisplatin was added at 1:400 dilution for the last 5 min of the stain to assess viability. Cells were then washed and fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were then washed and incubated with Ebioscience Transcription Factor Fix/Perm Buffer for 30 min, washed in Ebioscience perm buffer, and stained for intracellular markers at 1:100 for 30 min. Cells were refixed in 1.6% paraformaldehyde for 10 min and stored overnight in PBS/1%BSA. The following day, cells were incubated with MaxPar Intercalator-Ir 500 μM 1:4000 in PBS for 20 min, and then washed twice with MilliQ water, filtered, and analyzed on a Helios instrument (Fluidigm). Mass cytometry data were normalized using EQ™ Four Element Calibration Beads (Fluidigm) as described previously [
21]. viSNE analysis was performed using the Barnes-Hut SNE implementation on Cytobank (
www.cytobank.org). Gated live cells (DNA-positive, cisplatin-negative) were analyzed using all available protein markers. Biaxial gating was performed using FlowJo 10.0.7.
Discussion
Here we describe the development of an experimental pipeline to analyze synovial tissue with multiple high-dimensional technologies, aimed at deconstructing the cellular and molecular features of RA. This standardized and validated strategy enables single-cell analytics on large sample numbers collected from numerous clinical sites.
The integration of contemporary high-dimensional assays provides the opportunity to dissect the properties of tissues affected by rheumatic disease with a resolution not previously possible. Histologic and immunohistochemical assessments of synovium have revealed marked heterogeneity in RA synovial pathology [
13,
30]. Gene expression analyses of synovium by microarray have added gene expression signatures to these histologic patterns [
31‐
34]. However, broad analyses of gene expression in synovium have thus far been limited mainly to assessments of whole tissue, which cannot distinguish which cell types express the relevant genes [
31,
35‐
38]. The analysis pipeline described here enables simultaneous quantification of a wide range of stromal and leukocyte subpopulations by mass cytometry, paired with genome-wide transcriptional analyses, both of targeted cell populations and of single cells. Mass cytometry alone provides a powerful, broad assessment of the cell types and phenotypes within the tissue on a relatively large number of cells [
5]. Transcriptomic analyses of sorted subpopulations can identify the cell types contributing specific gene expression signatures derived in the synovium, including signatures specific to RA synovium. Single-cell transcriptomics allow a high-powered characterization of the heterogeneity of cells across a tissue [
39] and within cell subsets that were previously classified as the same cell type [
25,
26,
40]. We propose that integrating the multiple high-dimensional datasets acquired with this pipeline will reveal how distinct cell types relate to specific aspects of RA pathology. Applied to a large patient cohort, these analyses have the potential to reveal functional pathologic markers that distinguish patients with distinct clinical trajectories and therapeutic responses [
5].
Implementing these technologies requires robust tissue processing protocols. The validation studies described here, which were carried out by a collaboration among laboratories with expertise in synovial tissue and RA, provide a widely applicable, reproducible method for obtaining single cells from RA synovium that performs well in cytometric and transcriptomic analyses. Classically, synovial tissue has been disaggregated by crude collagenase preparations, which often cleave cell surface proteins and may contain bacterial products that stimulate immune cells. Here, varied concentrations of highly purified proteolytic enzyme preparations were assessed for preservation of vulnerable cell surface proteins required for downstream assays, as well as cell viability, RNA integrity, and applicability to next-generation sequencing. Combining limited enzyme incubation time with mechanical disaggregation yielded viable cell populations from disparate lineages that performed well in downstream assays. It is important to recognize that dissociating tissues into cell suspensions may introduce processing signatures [
24]. However, differences in transcriptome between intact and dissociated tissue may arise from a number of sources. These may include better representation of genes from cells difficult to extract in the intact tissue samples, in addition to potentially adverse consequences such as a cellular response to tissue manipulation. Importantly, with the synovial tissue disaggregation protocols developed here, mass cytometry of the single cells obtained clearly identified multiple expected cell populations in striking detail. RNA-seq transcriptomic analyses also robustly distinguished cells of different types at both the low-input and single-cell level. Further, transcriptomic analysis of synovial cells identified RA-specific gene signatures even in the relatively small sample set presented with the largest source of variation in the transcriptome due to donor biological differences. While dissociation-induced gene signatures are identifiable, they can be adjusted for computationally if necessary. Taken together, these observations strongly argue for the potential of this approach to discover new aspects of RA cellular pathology, which can be further validated in intact tissues.
The ability to effectively cryopreserve synovial tissue marks a major technical advance for synovial research. The viability and flow cytometric phenotypes of cells from cryopreserved synovial tissue samples were comparable to those from freshly processed tissues. Cells obtained from cryopreserved tissue samples were largely viable and readily used for all of the mass cytometric and RNA-seq analyses presented in this report. Cryopreservation of intact synovial tissue provides a simple, rapid technique to preserve and transport affected tissues for cellular studies in a multisite consortium. Tissue fragment cryopreservation enables sample collection from numerous clinical sites, including those lacking the resources for tissue dissociation. This approach also eliminates site-specific processing effects, allowing samples to be processed at a central site that possesses multiple high-dimensional technologies, and to utilize batch processing of samples to minimize technical variation and confounding effects. While it remains to be determined how long cryopreserved synovial tissue can be stored, samples have been routinely stored in liquid nitrogen for several months, and a small number of samples have been analyzed over 1 year after freezing without obvious decreases in cell yield.
In an ongoing phase I study, the AMP RA Network has implemented this synovial tissue pipeline to study cryopreserved synovial samples from a pilot cohort of over 40 RA and OA patients with the goal of defining RA disease-specific cellular populations and pathways. Greater than half of the synovial samples processed yielded sufficient cells for both mass cytometry and RNA-seq analyses, with cell viabilities comparable to those reported here. Analysis of cells from these phase I samples is in progress.
The phase I pilot study will be followed by a phase II study involving a much larger cohort of RA patients, including those with early disease, with a focus on identifying RA tissue biomarkers and novel tissue drug targets. A similar strategy of tissue cryopreservation has been adopted by the AMP for single-cell transcriptomic analyses of kidney biopsy samples from patients with lupus nephritis, including development of a dissociation protocol optimized for human kidney tissue [
41]. Although different tissues and cellular subsets may respond to cryopreservation differently, the strategy of analyzing cells from cryopreserved tissue appears readily adaptable to multicenter studies of tissues in other inflammatory diseases.
Acknowledgements
The authors acknowledge the many clinical coordinators and patients who helped to provide samples for analysis in this study. They appreciate the many efforts of Mina Pichavant, Clinical Research Project Manager for the AMP, and Bill Apruzzese, Associate Director of Operations and Management for the AMP. Dr. Anolik gratefully acknowledges the UR Flow Cytometry Core and the Genomics Research Center. The following additional AMP members have contributed to the consortium through a variety of efforts: Jane Buckner, Derrick Todd, Michael Weisman, Ami Ben Artzi, Lindsy Forbess, Joan Bathon, John Carrino, Oganna Nwawka, Eric Matteson, Robert Darnell, Dana Orange, Rahul Satija, Diane Horowitz, Harris Perlman, Art Mandelin, Louis Bridges, Laura B. Hughes, Arnold Ceponis, Peter Lowry, Paul Emery, Ahmed Zayat, Amir Aslaam, Karen Salomon-Escoto, David Fox, Robert Ike, Andy Cordle, Aaron Wise, John Ashton, Javier Rangel-Moreno, Christopher Ritchlin, Darren Tabechian, Ralf Thiele, Deborah Parks, John Akinson, Chiam Putterman, Evan Der, Elena Massarotti, Michael Weisman, David Hildeman, Richard Furie, Betty Diamond, Michelle Petri, Diane Kamen, Melissa Cunningham, Jill Buyon, Iris Lee, Hasan Salameh, Maureen McMahon, Ken Kalunian, Maria Dall’Era, David Wofsy, Mattias Kretzler, Celine Berthier, William McCune, Ruba Kado, Wiliam Pendergraft, Dia Waguespack, Yanyan Liu, Gerald Watts, Arnon Arazi, Rohit Gupta, Holden Maecker, Patrick Dunn, Rong Mao, Mina Pichavant, Quan Chen, John Peyman, Ellen Goldmuntz, Justine Buschman, Jennifer Chi, Su-Yau Mao, Susana Serrate-Sztein, Yan Wang, Thomas Tuschl, Yvonne Lee, Chamith Fonseka, Fan Zhang, Ilya Korsunskiy, Judith A. James, and Joel Guthridge.