Heterotopic ossification (HO) is pathological bone formation characterized by ossification within muscle, tendons, or other soft tissues. However, the cells of origin and mechanisms involved in the pathogenesis of HO remain elusive. Here we show that deletion of suppressor of fused (Sufu) in cathepsin K–Cre–expressing (Ctsk-Cre–expressing) cells resulted in spontaneous and progressive ligament, tendon, and periarticular ossification. Lineage tracing studies and cell functional analysis demonstrated that Ctsk-Cre could label a subpopulation of tendon-derived progenitor cells (TDPCs) marked by the tendon marker Scleraxis (Scx). Ctsk⁺Scx⁺ TDPCs are enriched for tendon stem cell markers and show the highest self-renewal capacity and differentiation potential. Sufu deficiency caused enhanced chondrogenic and osteogenic differentiation of Ctsk-Cre–expressing tendon-derived cells via upregulation of Hedgehog (Hh) signaling. Furthermore, pharmacological intervention in Hh signaling using JQ1 suppressed the development of HO. Thus, our results show that Ctsk-Cre labels a subpopulation of TDPCs contributing to HO and that their cell-fate changes are driven by activation of Hh signaling.

Bone osteogenic sarcoma has a poor prognosis, as the exact cell of origin and the signaling pathways underlying tumor formation remain undefined. Here, we report an osteogenic tumor mouse model based on the conditional knockout of liver kinase b1 (Lkb1, also known as Stk11) in Cathepsin K–Cre–expressing (Ctsk-Cre–expressing) cells. Lineage-tracing studies demonstrated that Ctsk-Cre could label a population of periosteal cells. The cells functioned as mesenchymal progenitors with regard to markers and functional properties. LKB1 deficiency increased proliferation and osteoblast differentiation of Ctsk⁺ periosteal cells, while downregulation of mTORC1 activity, using a Raptor genetic mouse model or mTORC1 inhibitor treatment, ameliorated tumor progression of Ctsk-Cre Lkb1^fllfl mice. Xenograft mouse models using human osteosarcoma cell lines also demonstrated that LKB1 deficiency promoted tumor formation, while mTOR inhibition suppressed xenograft tumor growth. In summary, we identified periosteum-derived Ctsk-Cre–expressing cells as a cell of origin for osteogenic tumor and suggested the LKB1/mTORC1 pathway as a promising target for treatment of osteogenic tumor.

Bone fracture is repaired predominantly through endochondral ossification. However, the regulation of endochondral ossification by key factors during fracture healing remains largely enigmatic. Here, we identify histone modification enzyme LSD1 as a critical factor regulating endochondral ossification during bone regeneration. Loss of LSD1 in Prx1 lineage cells severely impaired bone fracture healing. Mechanistically, LSD1 tightly controls retinoic acid signaling through regulation of Aldh1a2 expression level. The increased retinoic acid signaling in LSD1-deficient mice suppressed SOX9 expression and impeded the cartilaginous callus formation during fracture repair. The discovery that LSD1 can regulate endochondral ossification during fracture healing will benefit the understanding of bone regeneration and have implications for regenerative medicine.

The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A ( Itm2a ) enriched in SSCs using single-cell transcriptomics. Itm2a ⁺ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a ⁺ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a ⁺ cells rarely expressed CD34 or Osx , but expressed periosteal markers such as Ctsk , CD51 , PDGFRA , Sca1 , and Gli1 . Itm2a ⁺ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 ( Bmp2 ) deficiency or ablation of Itm2a ⁺ P-SSCs resulted in defects in fracture healing. ITM2A ⁺ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.

Annulus fibrosus (AF) is an important part of the intervertebral disc (IVD) and its injury leads to back pain and impaired mobility. The stem/progenitor cells are essential for the maturation and repair of the AF, however, the identity of AF stem/progenitor cells remain elusive. In this study, we sorted cells from the murine IVDs and performed the single-cell RNA sequencing. Using single-cell transcriptomics, genetic lineage tracing, in vitro stem cell experiment, ablation models and cell transplantation, we elucidate the role of AF progenitor cells in maturation and injury. On the basis of single-cell RNA-sequencing (scRNA-seq) analysis of the intervertebral disc, we found that the transcription factor Scleraxis (Scx) can specifically label a progenitor cell population of the outer AF. By lineage tracing assay, Scx-lineage AF cells proliferate mainly prior to sexual maturity, but barely proliferate after age of 8 weeks. The Scx-expressing AF cells are enriched for stem/progenitor cell markers and show a higher proliferative capacity and differentiation potential than the Scx⁻ cells. The ablation of Scx-expressing AF cells impairs the maturation of AF. The Scx⁺ AF cells are enriched for TGFβ signaling. Transplantation of Scx-lineage cells to injured AF with Connective tissue growth factor (CTGF) improved the AF healing. Scleraxis-expressing progenitor cells are critical for the maturation of AF and demonstrate therapeutic potential for AF regeneration. These findings expand the important role of stem cells in maturation and repair and provide new strategy for cellular therapy of AF repair.