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 6

BMP signaling is critical for the Itm2a+ P-SSC contribution to bone fracture healing.

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BMP signaling is critical for the Itm2a+ P-SSC contribution to bone frac...
(A and B) Bulk RNA-Seq data from Lin–Itm2a– and Lin–Itm2a+ periosteal cells isolated from 4-week-old WT mice. GO plot (A) and heatmap (B) show that BMP signaling was enriched in Lin–Itm2a+ periosteal cells. (C) Experimental design for tamoxifen induction, the bone fracture model, and tissue analysis of Itm2a-CreER Bmp2fl/fl and control mice. (D) Analysis by x-ray of fractured femurs in Itm2a-CreER Bmp2fl/fl mice and control mice at 7, 14, and 21 dpf. n = 4 mice per genotype per time point. (E) Callus index calculated from x-ray data showed callus formation at 7, 14, and 21 dpf in mutant and control mice. n = 4 mice per group. Data are presented as the mean ± SD (n = 4). (F) μ-CT (upper) and SO/fast green (SOFG) (lower) staining analysis of fractured femurs in Itm2a-CreER Bmp2fl/fl mice and control mice at 14 dpf. n = 4 mice per genotype. Scale bar: 100 μm. (G) Bone volume quantification showed callus formation at 14 dpf in mutant and control mice. n = 4 mice per group. Data are presented as the mean ± SD (n = 4). ***P < 0.001, by unpaired, 2-tailed t test. (H) Experimental design for tamoxifen induction, the bone fracture model, and tissue analysis of Itm2a-CreER R26-DTA and R26-DTA mice. (I) Confocal images of Itm2a+ cell (anti-Itm2a staining) at the periosteal region from R26-DTA (left) and Itm2a-CreER R26-DTA (right) mice. Scale bar: 50 μm. (J) μ-CT (upper) and SOFG (lower) staining analysis of fractured femurs in R26-DTA and Itm2a-CreER R26-DTA at 14 dpf. Scale bar: 100 μm. (K) Non-union percentage calculated from R26-DTA and Itm2a-CreER R26-DTA mice at 28 dpf. *P = 0.025, by Fisher’s exact test.