Fig. 6: ITGA9 enabled reporter-independent isolation of SOX9⁺ scl-progenitors.

A RNA-seq analysis revealed multiple surface markers enriched in SOX9⁺ scl-progenitors. Transcriptomic analysis was done by comparing SOX9⁺ scl-progenitors vs SOX9^- cells sorted from Day-6 sclerotomal cells from the traditional method (n = 3 for each group). Statistics: Exact test based on the negative binomial distribution (two-sided) by edgeR. B Confirmation of differential gene expression between SOX9⁺ scl-progenitors and SOX9^- cells by RT-qPCR. For RT-qPCR, error bars (mean ± sd) represented data from six biological replicates; for RNA-seq, error bars (mean ± sd) represented data from three biological replicates. C Confirmation of SOX9 and ITGA9 co-expression in differentiated sclerotomal cells by flow cytometry. Isotype antibody served as the control. The proportion of SOX9⁺ cells among the ITGA9⁺ population was shown on the right, which was gated according to undifferentiated hPSCs. D Confirmation of SOX9 and ITGA9 co-expression by immunostaining. ITGA9⁺ cells were sorted and stained with anti-SOX9. Scale bars: 50 μm. E Sclerotome-specific genes were highly enriched in ITGA9⁺ cells. Error bars:mean ± sd, n = 6 samples. F ITGA9⁺ cells retained osteochondral competence. Cell differentiation was induced the same as in Supplementary Fig. 2G. Scale bars: 200 μm (Alizarin red); 100 μm (spheroids). G Negative immunostaining of hypertrophic and osteogenic markers indicated no ossification. COL X: hypertrophic chondrocyte marker. COL I: the marker for fibrous and bone tissues. The mouse femur was used as the positive control. Scale bars: 100 μm. H, I Sclerotome-specific genes were also enriched in ITGA9⁺ cells derived by the current method. Error bars:mean ± sd, n = 6 samples. ACTB served as the housekeeping control. Statistics: Student t-test (two-tailed), by SPSS v26.0. ***p < 0.001, ****p < 0.0001. p = 0.0000021 (ITGA9); p = 0.00000045 (SOX9); p = 0.0000086 (PAX1); p = 0.0002 (PAX9); p = 0.0000084 (TWIST1); p = 0.0000062 (NKX3-2). Source data are provided as a Source Data file.