Stimulation of skeletal stem cells in the growth plate promotes linear bone growth
Dana Trompet, Anastasiia D. Kurenkova, Baoyi Zhou, Lei Li, Ostap Dregval, Anna P. Usanova, Tsz Long Chu, Alexandra Are, Andrei A. Nedorubov, Maria Kasper, Andrei S. Chagin
Dana Trompet, Anastasiia D. Kurenkova, Baoyi Zhou, Lei Li, Ostap Dregval, Anna P. Usanova, Tsz Long Chu, Alexandra Are, Andrei A. Nedorubov, Maria Kasper, Andrei S. Chagin
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Research Article Bone biology Stem cells

Stimulation of skeletal stem cells in the growth plate promotes linear bone growth

Abstract

Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (epiphyseal skeletal stem cells, epSSCs), but their function in connection with linear bone growth remains unknown. Here, we explore the possibility that modulating the number of epSSCs can correct differences in leg length. First, we examined regulation of the number and activity of epSSCs by Hedgehog (Hh) signaling. Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched1 (Ptch1) ablation in Pthrp-creER Ptch1fl/fl tdTomato mice promoted proliferation of epSSCs and clonal enlargement. Transient intra-articular administration of SAG also elevated the number of epSSCs. When SAG-containing beads were implanted into the femoral secondary ossification center of 1 leg of rats, this leg was significantly longer 1 month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced 2 and 6 months after implantation. We conclude that Hh signaling activates growth plate epSSCs, which effectively leads to increased longitudinal growth of bones. This opens therapeutic possibilities for the treatment of differences in leg length.

Authors

Dana Trompet, Anastasiia D. Kurenkova, Baoyi Zhou, Lei Li, Ostap Dregval, Anna P. Usanova, Tsz Long Chu, Alexandra Are, Andrei A. Nedorubov, Maria Kasper, Andrei S. Chagin

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Figure 1

The effects of treatment with SAG, a pharmacological activator of Hh signaling, on the behavior of epSSCs are dependent on age.

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The effects of treatment with SAG, a pharmacological activator of Hh sig...
(A) Experiments involve intraperitoneal injections of SAG or vehicle (DMSO) into Pthrp-creER R26R-tdTomato mice before (B–I) and after (J–Q) stem cell niche formation. (B) Representative images of tdTomato+ cells in Pthrp-creER R26R-tdTomato mice treated with DMSO (n = 4 mice) or SAG (n = 5) from P6 to P18 and clone size (C) quantified on confocal Z-stacks of thick sections (total 606 cells in 353 clones for DMSO and 637 cells in 465 clones for SAG). The percentage of clones containing single cells (D), doublets plus triplets (E), and 4 or more cells (F). Ki67 staining (G) indicates decreased cell proliferation in the uppermost 50 μm of the growth plate in SAG-treated mice (H) and Tomato+ cells within this region (I) (n = 3/3 mice control/experiment). (J) Representative images of tdTomato+ cells in Pthrp-creER R26R-tdTomato mice treated with DMSO (n = 6) or SAG (n = 5) from P25 to P38 and clone size (K) quantified on confocal Z-stacks of thick sections (351 cells in 206 clones for DMSO and 596 cells in 348 clones for SAG). Arrowheads indicate dyads. The percentage of clones containing single cells (L), doublets plus triplets (M), and 4 or more cells (N). Ki67 staining (O) indicates increased cell proliferation in the uppermost 50 μm of the growth plate in SAG-treated mice (P) and Tomato+ cells within this region (Q) (n = 4/5 mice control/experiment). Scale bars: 100 μm, and insets in G and O are 165 × 165 mm from the original images. Means ± SD. #P < 0.1, *P < 0.05, **P < 0.01, as determined by 2-tailed unpaired t test or the Kolmogorov-Smirnov test. SOC, secondary ossification center; GP, growth plate; RZ, resting zone; dashed lines depict the border between SOC and GP in B and J and the uppermost 50 μm of the growth plate in G and O.