Single-cell transcriptomics identifies chondrocyte differentiation dynamics in vivo and in vitro

Developing in vitro chondrocytes that replicate in vivo development would benefit musculoskeletal disease modeling and regenerative medicine. Although current methodologies have made progress, challenges such as off-target differentiation can result in heterogeneous cell states. Furthermore, the lack of comparison with human embryonic tissue precludes detailed evaluation of in vitro cells. Here, we perform single-cell RNA sequencing (scRNA-seq) of embryonic long bones and combine this with public data to form an atlas of endochondral ossification. We use this to evaluate published in vitro chondrogenesis protocols that use human cell lines, finding variability in cells produced by each. We apply single-nuclear RNA sequencing (snRNA-seq) to our human embryonic stem cell chondrogenesis protocol and perform trajectory alignment with in vivo data to shed light on off-target differentiation in vitro. Using this information, we inhibit FOXO1, a transcription factor active in embryonic osteoblasts and in vitro cells, to increase chondrocyte transcripts in vitro. This work offers a framework for improving in vitro chondrogenesis using developmental data.