Itm2a expression marks periosteal skeletal stem cells that contribute to bone fracture healing
Wenhui Xing, … , Bo O. Zhou, Weiguo Zou
Wenhui Xing, … , Bo O. Zhou, Weiguo Zou
Published September 3, 2024
Citation Information: J Clin Invest. 2024;134(17):e176528. https://doi.org/10.1172/JCI176528.
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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 3

Itm2a-CreER labels P-SSCs.

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Itm2a-CreER labels P-SSCs. (A) Strategy used to construct Itm2a-CreER-m...
(A) Strategy used to construct Itm2a-CreER-mCherry mice. KI, knockin. (B) Representative confocal images of Itm2a lineage cells (ZsGreen+) and Itm2a expression cell (mCherry+) distribution in periosteal (PO) and bone marrow (BM) regions in cells from Itm2a-CreER R26-Ai6 mice that had been tamoxifen treated at 4 weeks of age. Mice were analyzed 2 days after the treatment. Scale bars: 100 μm. (C) Representative confocal imaging of the osteoblast marker OPN and Itm2a lineage cells (ZsGreen+ cells) in femur sections from Itm2a-CreER R26-Ai6 mice that had been tamoxifen treated at 4 weeks of age. Mice were analyzed 2 days after the treatment. n = 3 mice per condition from 3 independent experiments. Scale bars: 50 μm. (D) Representative confocal images of the stem cell marker CD200 and Itm2a lineage cells (ZsGreen+ ) in femur sections from mice that had been tamoxifen treated at 4 weeks of age. Mice were analyzed 2 days after the treatment. n = 3 mice per condition from 3 independent experiments. Scale bars: 50 μm. (E) Flow cytometric analysis of SSCs in ZsGreen+ cells from long bone digests from mice that had been treated with tamoxifen at 4 weeks of age. Mice were analyzed 2 days after the treatment. n = 4.