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. 2023 Jul 21;11:39. doi: 10.1038/s41413-023-00272-x

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Cell subclustering and pseudotemporal trajectory analysis reveal a hierarchy of osteochondrogenic progenitors in relation to Tppp3 expression. a UMAP visualization of mesenchymal and pericyte subclusters. Three subclusters were found among mesenchymal cells: Cluster 0 (Tppp3^- Acan⁺), Cluster 1 (Tppp3⁺ Acan^-) and Cluster 3 (Tppp3⁺ Acan⁺). b Expression of mesenchymal (Pdgfra), pericyte (Pdgfrb, Mcam, Acta2) and tenocyte-like cells (Scx) and osteochondrogenic markers (Runx2, Acan) in each cell subcluster. c Pseudotime trajectory analysis of the four subclusters. d Expression of pericyte and mesenchymal markers across pseudotime. e Expression of Tppp3 across pseudotimes at all timepoints or individually at each time point. f Expression of tenocyte (Scx, Tnmd), chondrocyte (Acan) and osteoblast-related genes (Runx2) across pseudotime. g Heatmap of the pseudotime trajectory of pericytes (left), mesenchymal cells (right), and their common osteochondrogenic branch (center). h GO term analysis of clusters identified with pseudotime analysis, including pericytes, mesenchymal cells, and osteochondrogenic cells. i Heatmap showing the expression of secreted factors that regulate heterotopic ossification in the four cell subclusters. n = 4 biological replicates per time point. Animals and 1 460 (0 d), 6 317 (7 d) and 3 484 (21 d) mesenchymal cells per timepoint for scRNA-seq