Fig. 2: Recapitulation of endochondral ossification by SOX9⁺ scl-progenitors.

A Experimental strategy to recapitulate endochondral ossification using SOX9⁺ scl-progenitors. B 4-week grafts showed hypertrophic chondrocytes and initial calcification. i Graft overview with calcified hypertrophic region outlined (black dotted line). ii human origin confirmed by anti-KU80. iii-iv cartilaginous tissues visualized by Safranin O and COL II staining. v hypertrophy indicated by COL X immunostaining. vi calcified region marked by Alizarin red. Scale bars: 1 mm. C RT-qPCR indicated that the day-12 early chondrocytes lacked COL10A1 expression. Error bars: mean ± sd (n = 6 samples). Statistics: Student’s t-test (two-tailed), one-way ANOVA (SPSS v26.0). NS, not significant (p = 0.5715). ****p < 0.0001 (p = 0.00000000020). D 8-week grafts showed vascular invasion and multiple ossification centers. i blood vessel network on graft surface. ii-iii anti-KU80 confirmed human origin. iv multiple calcified regions and ossification centers revealed by Alizarin red. v Safranin O staining revealed multi-directional growth-plate-like tissues surrounding ossification centers. Scale bars: 1 mm (i, ii and iv), 500 μm (iii, v). E Representative growth plate-like tissues: anti-COL II (proliferating zone), anti-COL X (hypertrophic zone). Scale bars: 200 μm. F Columnar chondrocytes in growth plate-like tissues (8-week grafts). RZ: resting zone; PZ: proliferating zone; HZ: hypertrophic zone. Scale bars: 100 μm. G Masson’s trichrome staining revealed Bone-like tissues (8-week grafts). Scale bars: 200 μm. H Complete bone with a medullary cavity at 16 weeks. i bone formation and vascularization. ii-iv H&E staining showed bone marrow cavity formation with adipocytes, stromal, and hematopoietic cells. TB, trabecular bone. v-vi anti-COL I and anti-human VIM confirmed bone formation and its human origin. Arrowheads, COL I, and human VIM colocalization. vii-ix adipocytes were stained by Oil red and anti-SCD1. Anti-KU80 confirmed the human origin of some adipocytes. x host hematopoietic cells stained by anti-mouse CD45. Scale bars: 1 mm (i), 500 μm (ii, v), 50 μm (iii, vii-x), 20 μm (iv, vi). I Vascular architecture and mesenchymal stroma in 16-week grafts. i anti-COL I, and anti-EMCN revealed trabecular bone intertwined by sinusoidal vessels. ii host mesenchymal stromal cells verified by anti-LEPR. iii-iv niche cytokines were stained by anti-CXCL12 and anti-SCF. v EMCN⁺vascular endothelial cells enfolded by KU80⁺ human cells. Scale bars: 100 μm. Source data are provided as a Source Data file.