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Single-Cell RNA-Seq Analysis Reveals the Progression of Human Osteoarthritis Ann Rheum Dis: first published as 10.1136/annrheumdis-2017-212863 on 19 July 2018. Downloaded from TRANSLATIONAL SCIENCE Single-cell RNA-seq analysis reveals the progression of human osteoarthritis Quanbo Ji,1,2 Yuxuan Zheng,2,3,4 Guoqiang Zhang,1 Yuqiong Hu,2,3,4 Xiaoying Fan,2,3 Yu Hou,2,3 Lu Wen,2,3 Li Li,2,3 Yameng Xu,5 Yan Wang,1 Fuchou Tang2,3,4 Handling editor Josef S ABSTRact of critical importance. Nevertheless, intense efforts Smolen Objectives Understanding the molecular mechanisms have focused on the transplantation of tissue-en- underlying human cartilage degeneration and gineered cartilage derived from stem cells and ► Additional material is published online only. To view regeneration is helpful for improving therapeutic chondrocytes for cartilage regeneration in treating please visit the journal online strategies for treating osteoarthritis (OA). Here, we report OA.6–8 However, OA cartilage cell-type compo- (http:// dx. doi. org/ 10. 1136/ the molecular programmes and lineage progression sition, biochemical markers that can effectively annrheumdis- 2017- 212863). patterns controlling human OA pathogenesis using predict OA and the cellular heterogeneity leading For numbered affiliations see single-cell RNA sequencing (scRNA-seq). to OA progression remain largely unknown. end of article. Methods We performed unbiased transcriptome- Articular cartilage is derived from condensed wide scRNA-seq analysis, computational analysis and mesenchymal stem cells (MSCs), which subsequently Correspondence to histological assays on 1464 chondrocytes from 10 differentiate into chondrocytes.9 10 Following this Prof. Yan Wang, Department of patients with OA undergoing knee arthroplasty surgery. chondrogenesis, cells produce a collagenous extra- Orthopaedics, General Hospital We investigated the relationship between transcriptional of Chinese People’s Liberation cellular matrix, a ground substance containing Army, Beijing 100853, China; programmes of the OA landscape and clinical outcome abundant collagen and proteoglycans. Studies yanwang301@ 126. com and using severity index and correspondence analysis. have shown that the chondrocyte subtypes in artic- Prof. Fuchou Tang, Beijing Results We identified seven molecularly defined ular cartilage include proliferative chondrocytes Advanced Innovation Center populations of chondrocytes in the human OA cartilage, (ProCs), prehypertrophic chondrocytes (preHTCs) for Genomics (ICG), College of 11–13 Life Science, Peking University, including three novel phenotypes with distinct functions. and hypertrophic chondrocytes (HTCs). Beijing 100871, China; We presented gene expression profiles at different ProCs are mainly found in the proliferative zone tangfuchou@ pku. edu. cn OA stages at single-cell resolution. We found a of growth plates, preHTCs have the capacity to potential transition among proliferative chondrocytes, modulate the onset of hypertrophic differentia- QJ and YZ contributed equally. prehypertrophic chondrocytes and hypertrophic tion, while HTCs could regulate the mineralisation Received 16 December 2017 chondrocytes (HTCs) and defined a new subdivision of the surrounding matrix in cartilage. Recently, Revised 21 May 2018 within HTCs. We revealed novel markers for cartilage senescent cells (SNCs) and cartilage progenitor cells Accepted 28 May 2018 progenitor cells (CPCs) and demonstrated a relationship (CPCs) were identified.2 5 14–17 SNCs assembled in Published Online First between CPCs and fibrocartilage chondrocytes using 19 July 2018 OA cartilage were found to be cell-cycle-arrested computational analysis. Notably, we derived predictive and to exhibit features of a senescence-associated targets with respect to clinical outcomes and clarified secretory phenotype.14 Selective elimination of http://ard.bmj.com/ the role of different cell types for the early diagnosis and SNCs attenuates OA development,14 15 whereas treatment of OA. CPCs, which are specialised in their capacity for Conclusions Our results provide new insights into self-renewal, have the ability to differentiate along chondrocyte taxonomy and present potential clues for multiple lineages, express stem-cell-related surface effective and functional manipulation of human OA markers and contribute to the maintenance of OA cartilage regeneration that could lead to improved cartilage repair and homeostasis.5 17 These different health. chondrocyte subtypes were historically defined on October 1, 2021 by guest. Protected copyright. by a combination of their developmental origin, localisation, physical properties, morphology and INTRODUCTION molecular functions. However, because of their Osteoarthritis (OA) is the most common chronic phenotypic heterogeneity and the limited number condition associated with ageing and progressive of available markers to identify, isolate and manipu- joint dysfunction and the one with the greatest late these cells, how to define the types of articular socioeconomic cost. OA is primarily character- cartilage chondrocytes in human OA has still not ised by disordered articular cartilage homeostasis been fully determined. These unknown parameters with subsequent inflammation and degradation.1–4 affect the ontogeny and function of each chondro- Cartilage is a physiologically non-self-renewing cyte population in OA pathogenesis. http:// dx. doi. org/ 10.1136/ 5 ► avascular tissue and consists of chondrocytes. To study the molecular programmes involved annrheumdis- 2018- 213864 Despite accumulating reports on chondrocytes that in the relationships among cell populations, have identified strategies to predict and modify OA we performed single-cell RNA sequencing progression, effective measures of disease-modi- (scRNA-seq). This allowed us to better elucidate To cite: Ji Q, Zheng Y, fying OA diagnoses and outcomes are still lacking. the endogenous heterogeneity of chondrocytes Zhang G, et al. A better understanding of the pathophysiology of in human OA cartilage, uncover novel pheno- Ann Rheum Dis inflammation and the mechanisms underlying the types of OA chondrocytes with defined markers 2019;78:100–110. role of chondrocytes in the process leading to OA is and refine existing classifications. Notably, using 100 Ji Q, et al. Ann Rheum Dis 2019;78:100–110. doi:10.1136/annrheumdis-2017-212863 Osteoarthritis Ann Rheum Dis: first published as 10.1136/annrheumdis-2017-212863 on 19 July 2018. Downloaded from computational analysis, we identified the relationship between and supplementary table S1). In total, we sequenced 1600 indi- the transcriptional programmes of OA landscapes and clinical vidual chondrocytes and retained 1464 chondrocytes for subse- outcomes. Therefore, our results offer an unbiased atlas of carti- quent analysis after rigorous filtration (online supplementary lage chondrocytes and shed light on diagnostic and therapeutic figure S2A–C and supplementary table S2). options for human OA. To reveal transcriptional states during direct conversion between stages of OA progression, we analysed OA chondro- MatERIALS AND METHODS cytes using principal component analysis (PCA) and identified Single-cell RNA-seq library construction and sequencing a strong disease-stage progression along PC2 (figure 1C,D and Sequencing libraries were generated following a modified single- online supplementary figure S2D). OA chondrocytes at all stages cell tagged reverse transcription (STRT) protocol as previously were spread out along PC2 and followed an expected timing, reported.18–20 Briefly, after chondrocyte isolation, a single chon- with chondrocytes at the OA early stages (0 and 1) occupying drocyte was put into the lysis buffer using a mouth pipette. the negative region of the axis and chondrocytes at the OA late Reverse transcription reactions were performed using a 25 nt stages (3 and 4) occupying the positive region (figure 1D). Since oligo(dT) primer anchored with an 8 nt cell-specific barcode then, genes in PC2 reflected the enrichment of OA chondrocyte and 8 nt unique molecular identifiers.21–23 First-strand synthesis disease associated genes. We then analysed the top 50 positively was performed and second-strand cDNAs were then synthesised, correlated genes and top 50 negatively correlated genes along followed by 16 cycles of amplification. The amplified cDNAs PC2 and PC1 in these 1464 chondrocytes using hierarchical of single cells were then pooled together. Biotinylated prein- clustering. We identified four clusters (A, B, C and D) along dexed primers were applied for further amplification of the PC2 and three clusters (1, 2 and 3) along PC1 (figure 1E and products by 4-cycle PCR to introduce biotin tags to the 3ʹ ends online supplementary figure S2E). Cluster 1 highly expressed of the amplified cDNAs. Approximately 300 ng cDNA was then genes showing a negative correlation along PC1 (ID3, HES1, sheared to 300 bp using Covaris S2 (Covaris). The 3ʹ terminals JUN and others) which are mainly involved in protein binding of amplified cDNAs were purified using Dynabeads MyOne and RNA metabolic process (online supplementary figure S2E Streptavidin C1 beads (Thermo Fisher Scientific). Libraries were and supplementary table S3), while cluster 2 highly expressed constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and genes revealing a positive correlation along PC1 (KLHL21, were then submitted to 150 bp paired-end sequencing on an Illu- SGMS2, ITGA5ID3 and others) which are associated with angio- mina HiSeq 4000 platform (Novogene). genesis and cell motility. Moreover, clusters
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