Itm2a expression marks periosteal skeletal stem cells that contribute to bone fracture healing
Wenhui Xing, Heng Feng, Bo Jiang, Bo Gao, Jiping Liu, Zaiqi Xie, Yazhuo Zhang, Xuye Hu, Jun Sun, Matthew B. Greenblatt, Bo O. Zhou, Weiguo Zou
Wenhui Xing, Heng Feng, Bo Jiang, Bo Gao, Jiping Liu, Zaiqi Xie, Yazhuo Zhang, Xuye Hu, Jun Sun, Matthew B. Greenblatt, Bo O. Zhou, Weiguo Zou
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Research Article Bone biology

Itm2a expression marks periosteal skeletal stem cells that contribute to bone fracture healing

Abstract

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.

Authors

Wenhui Xing, Heng Feng, Bo Jiang, Bo Gao, Jiping Liu, Zaiqi Xie, Yazhuo Zhang, Xuye Hu, Jun Sun, Matthew B. Greenblatt, Bo O. Zhou, Weiguo Zou

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Figure 4

Itm2a+ P-SSCs generate osteoblasts, chondrocytes, and marrow stromal cells upon injury.

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Itm2a+ P-SSCs generate osteoblasts, chondrocytes, and marrow stromal cel...
(A) Experimental design for tamoxifen (Tam) induction, the bone fracture model, and tissue analysis. (B) Representative confocal images of Itm2a lineage cells (ZsGreen+) at the uninjured periosteum (left) and femoral fracture day-3 callus (right) in sections from Itm2a-CreER R26-Ai6 mice. Scale bar: 100 μm. (C) Representative 7 dpf confocal images of Itm2a lineage cells (ZsGreen+) colocalized with chondrocytes (COL2A1) at the fractured femur site in sections from Itm2a-CreER R26-Ai6 mice. Scale bars: 100 μm. (D) Representative 14 dpf confocal images of Itm2a lineage cells (ZsGreen+) colocalized with osteoblasts (OPN) at the fractured femur site in sections from Itm2a-CreER R26-Ai6 mice. Scale bars: 100 μm. (E) Representative 14 dpf confocal images of Itm2a lineage cells (ZsGreen+ ) colocalized with bone marrow stromal cells (LepR) at the fractured femur site in sections from Itm2a-CreER R26-Ai6 mice. Scale bars: 50 μm. (F) Percentage of Itm2a lineage cells (ZsGreen+) among COL2A1+ chondrocytes, OPN+ osteoblasts and LepR+ bone marrow stromal cells calculated from C, D, and E, respectively. n = 3 mice per condition. Data are presented as the mean ± SD. (G) Experimental design for tamoxifen induction, bone drill model, and tissue analysis. (H) Representative confocal images of femoral fracture sites in sections from Itm2a-CreER R26-Ai6 mice at 14 dpf after tamoxifen treatment. n = 4 mice per condition from 4 independent experiments. Scale bars: 100 μm. (I) Experimental design for tamoxifen induction, bone scratch model, and tissue analysis. (J) Representative confocal images of injury sites in tibia sections from Itm2a-CreER R26-Ai6 mice at 7 dpf and 14 dpf after tamoxifen administration. Scale bars: 100 μm. n = 4 mice per condition from 4 independent experiments.