Single cell transcriptomics reveals chondrocyte differentiation dynamicsin vivoandin vitro

Abstract

SummaryThe consistent production ofin vitrochondrocytes that faithfully recapitulatein vivodevelopment would be of great benefit for musculoskeletal disease modelling and regenerative medicine. Current efforts are often limited by off-target differentiation, resulting in a heterogeneous product. Furthermore, the lack of comparison to human embryonic tissue, precludes detailed evaluation ofin vitrocells. Here, we perform single-cell RNA sequencing of embryonic long bones dissected from first trimester hind limbs from a range of gestational ages. We combine this with publicly available data to form a detailed atlas of endochondral ossification, which we then use to evaluate a series of publishedin vitrochondrogenesis protocols, finding substantial variability in cell states produced by each. We apply single-nuclear RNA sequencing to one protocol to enable direct comparison betweenin vitroandin vivo,and perform trajectory alignment between the two to reveal differentiation dynamics at the single-cell level, shedding new light on off-target differentiationin vitro. Using this information, we inhibit the activity of FOXO1, a transcription factor predicted to be active in embryonic bone development and in chondrogenic cellsin vitro, and increase chondrocyte transcriptsin vitro.This study therefore presents a new framework for evaluating tissue engineering protocols, using single-cell data from human development to drive improvement and bring the prospect of true engineered cartilage closer to reality.