Cartilage subpopulation in PPB
The other clusters were developing in the direction toward chondrocytes. Clusters 0, 1, 3, 7, 8, 10, and 11 (5 806 cells in total) exhibited high expression of TWIST1 , ANK3 , HTRA1 , and BMP4 , which are markers of osteogenesis (Fig.3A, Supporting Information Figure S3). Subsets of chondrocytes were distinguished based on further markers. A mature minor subset (cluster 7, 617 cells) was defined by chondrocyte markers that emerge in late differentiation, such asITM2A , MGP , and OGN . Cluster 10 (402 cells) was characterized by DKK2 and TNMD , which was possibly also at the terminal stage of their trajectory with the same status as cluster 7. Another major stem cell subset, clusters 1, 3, 8, and 11, expressed decisive genes in early chondrogenesis, including SOX9 andSOX9 -coordinated factor-like PAX1 , PAX9 and they maybe were a group of prechondrocytes. Notably, these cartilages were more naïve in their differentiation compared with the muscle subpopulation. Additionally, the characteristic cartilage structure was not observed pathologically and the tumor was negative for immunohistochemical staining of S100 protein. GO analysis of these clusters validated our presumed results. These cells participated in development of the skeletal system and functions such as maintenance and rebuilding of the extracellular matrix17 (Fig.3B).