Fig. 4: Basigin overexpression alters human skeletal lineage dynamics that impair endothelial function.

a Single cell transcriptomic analysis of tissue grafts from mouse SSCs either exposed to placebo, GC, human PTH or GC+human PTH (hPTH 1-34) displayed as UMAP showing high expression of Basigin in SSPCs. SSPCs, skeletal stem and progenitor cells. Left bottom: Violin plot showing increased expression of Bsg in GC group across all cells. b Representative image of tube formation assay by VeraVec endothelial cells exposed to control supernatant or supernatant of human SSCs overexpressing Basigin. c Quantification of ImageJ-based tube formation analysis. n = 6 independent samples from two independent experiments. d Representative brightfield images of the endothelial scratch assay (left) and its quantification (right). n = 6 independent samples from three independent experiments. e Measurement of reactive oxygen species in cultured endothelial cells after 24 h supernatant exposure. Left: representative fluorescence images. Right: Quantification of fluorescence signal. n = 6. All experiments with supernatant from at least two donors and two independent experiments. f Colony-forming unit ability of primary human SSCs either overexpressing control vector or Basigin. CFU-Fs stained by Crystal violet. n = 6 independent replicates from cells of two biologically independent donors. g In vitro osteogenesis (Alizarin Red S stain) by hSSCs of the same groups. n = 3 independent replicates from cells of one biological donor. h In vitro chondrogenesis (Alcian Blue stain) assays of the same groups. n = 6 independent replicates from cells of two biologically independent donors. i Schematic of subcutaneous transplant approach. Created in BioRender. Ambrosi, T. (2025) https://BioRender.com/4wl26h3. j Immunohistochemistry of Basigin (green) expression in SSC-generated grafts with corresponding quantification. n = 3 biologically independent donor cells in three biologically independent mice per group. k Representative Alizarin Red S staining of sectioned grafts and quantification of mineralized tissue in grafts containing transplanted human SSCs as assessed by Alizarin Red S staining. n = 6 sections from three biologically independent grafts. l Representative TRAP staining of sectioned grafts and quantification of TRAP-positive area of grafts. n = 6 sections from three biologically independent grafts. m Representative immunohistochemistry staining for Endomucin and DAPI of sectioned graft. Small insert shows Endomucin staining without DAPI. All data shown as mean ± SEM. Statistical testing between Placebo and other groups by two-sided unpaired Student’s t-test. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001. Scale bars, 50 µm.