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

Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (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 signaling (Hh). Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched (Ptch1) ablation in Pthrp-CreER;Ptch1 fl/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 (SOC) of one leg of rats, this leg was significantly longer one month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced two 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 new therapeutic possibilities for the treatment of differences in leg length Overall design: To activate hedgehog signaling, mice were injected i.p. with SAG (n=4) or DMSO (dimethyl sulfoxide) as a control vehicle (n=4) once a day from postnatal day 30 (P30) till P36. Mice were sacrificed at P38. For each mouse, cells from two femoral growth plates were isolated by FACS-sorting with stem cell markers: genetic PTHRP-mCherry and CD73 antibody staining.