Abstract
Tendinopathy accounts for over 30% of primary care consultations and represents a growing healthcare challenge in an active and increasingly ageing population. Recognising critical cells involved in tendinopathy is essential in developing therapeutics to meet this challenge. Tendon cells are heterogenous and sparsely distributed in a dense collagen matrix; limiting previous methods to investigate cell characteristics ex vivo. We applied next generation CITE-sequencing; combining surface proteomics with in-depth, unbiased gene expression analysis of > 6400 single cells ex vivo from 11 chronically tendinopathic and 8 healthy human tendons. Immunohistochemistry validated the single cell findings. For the first time we show that human tendon harbours at least five distinct COL1A1/2 expressing tenocyte populations in addition to endothelial cells, T-cells, and monocytes. These consist of KRT7/SCX+ cells expressing microfibril associated genes, PTX3+ cells co-expressing high levels of pro-inflammatory markers, APOD+ fibro–adipogenic progenitors, TPPP3/PRG4+ chondrogenic cells, and ITGA7+ smooth muscle-mesenchymal cells. Surface proteomic analysis identified markers by which these sub-classes could be isolated and targeted in future. Chronic tendinopathy was associated with increased expression of pro-inflammatory markers PTX3, CXCL1, CXCL6, CXCL8, and PDPN by microfibril associated tenocytes. Diseased endothelium had increased expression of chemokine and alarmin genes including IL33.
Highlights
Tendinopathy accounts for over 30% of primary care consultations and represents a growing healthcare challenge in an active and increasingly ageing population
Each cluster was further annotated by combining the top differentially expressed genes with a set of literature-defined gene markers and revealed five COL1A1/2 expressing tenocyte clusters, monocytes, Tc lymphocytes and a group of combined endothelial cells (Fig. 1B)
It is possible that these clusters were generated as a result of variation in gene expression that is not directly related to tendon function and as such they represent an artefact of single cell RNA sequencing
Summary
Tendinopathy accounts for over 30% of primary care consultations and represents a growing healthcare challenge in an active and increasingly ageing population. Research into patients with Marfan syndrome demonstrated that tendon cells are surrounded by pericellular matrix microfibrils formed by fibrillin chains and bound ancillary proteins (including versican, fibulin, matrix associated glycoproteins) They represent a mechanism for altering growth factor signalling (e.g. sequestering TGFβ), controlling morphogenic gradients, and influencing cell interactions with the extracellular collagen matrix[8,9,10]. Single cell RNA sequencing offers an unbiased and sensitive inventory of the transcriptome of individual cells and allows characterisation of cell subtypes based on shared and differential gene expression d ata[13]. This approach has been successfully used to characterise cell types is mouse tissue[14,15,16,17]
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