Transcriptome analysis of synovial osteoprogenitor populations in mice with antigen-induced arthritis and non-immunized mice

The aim of this experiment was to analyze differences in gene expression between potential osteoprogenitor populations in synovial compartment of non-immunized mice and mice with antigen-induced arthritis (AIA). We analyzed transcriptome of TER119–CD31–CD45–CD51+CD200+CD105– cells from nonimmunized mice, TER119–CD31–CD45–CD51+CD200+CD105– cells from mice with arthritis, and TER119–CD31–CD45–CD51+CD200–CD105+ from mice with arthritis. Comparison between nonimmunized and arthritic mice served to determine changes in TER119–CD31–CD45–CD51+CD200+CD105–population induced by arthritis. Comparison of TER119–CD31–CD45–CD51+CD200+CD105– and TER119–CD31–CD45–CD51+CD200–CD105+ from mice with arthritis aimed to define transcriptomic differences between those two populations. AIA was induced in C57BL6 mice by immunization with methylated bovine serum albumin (mBSA) and subsequent intra-articular injection of mBSA. Non-immunized mice (NI) were injected with phosphate buffered saline (PBS) at all timepoints. Ten days after intra-articular injection knee joints were harvested and synovial cells were released by injection of collagenase into joint cavities. Released cells were labeled with anti-mouse CD90.2-FITC, CD200-PE, CD105-PECy7, CD31-APC, CD45-APC, TER119-APC, CD51-biotin, streptavidin-APCeF780 and DAPI. 4 samples of TER119–CD31–CD45-CD51+CD200+CD105- cells from nonimmunized mice, 5 samples of TER119–CD31–CD45-CD51+CD200+CD105- cells from mice with arthritis, and 5 samples of TER119–CD31–CD45-CD51+CD200-CD105+ from mice with arthritis were sorted using BD FASCAria IIu. In each sample, 200-500 live (DAPI-) cells of described phenotypes were sorted directly into cell lysis buffer (Smartseq v4 Ultra® Low Input RNA Kit for Sequencing, TakaRa) according to the manufacturer’s instructions. ERCC RNA Spike-In Mix (Invitrogen) was added to lysed cell samples. cDNA amplicons were created using SmartSeq v4 Ultra® Low Input RNA Kit for Sequencing (TakaRa) and libraries for sequencing were prepared using Nextera XT DNA Library Preparation Kit (Illumina) and sequenced on NextSeq 500 (Illumina) instrument.