Fig. 1: Derivation of expandable SOX9+ sclerotomal progenitors from hPSCs.
From: Recapitulation of endochondral ossification by hPSC-derived SOX9+ sclerotomal progenitors
A The strategy for stepwise induction of SOX9+ sclerotomal progenitors (scl-progenitors) (top), mimicking the developmental process (bottom). B RT-qPCR confirmed sclerotome-specific gene expression in SOX9+ Scl-progenitors. Error bars: mean ± sd), n = 6 samples. ACTB (beta-actin) was used as the housekeeping control. ND: not detected in 40 cycles. C SOX9 and TWIST1 immunostaining in SOX9+ scl-progenitors at day 4. Nuclear staining: DAPI. Scale bars: 200 μm. D Western blotting on day-4 SOX9+ scl-progenitors confirmed expression of SOX9 and TWIST1. E Flow cytometry analysis revealed near uniform derivation of SOX9+ scl-progenitors. Traditional and optimized methods were compared. Undifferentiated SOX9-tdTomato hPSCs served the negative control. F The multi-batch analysis confirmed the consistent performance of the optimized differentiation method. Independent batches were analyzed by flow cytometry. The box (extending from 25th to 75th percentiles with median in the middle) and whiskers (minima to maxima) represent data from independent batches of differentiation (n = 9 for optimized method; n = 10 for traditional method). Statistics: Student’s t-test (two-tailed), by SPSS v26.0. ***p < 0.001 (p = 0.0002). G Experimental strategy for expanding SOX9+ scl-progenitors in vitro. H Continuous expansion of SOX9+ scl-progenitors for ~2 months in the defined medium. I SOX9+ scl-progenitors exhibited further maturation during in vitro expansion. RT-qPCR analysis showed increased expression of sclerotome-specific genes in the expanded cells. Error bars: mean ± sd), n = 6 samples. N.D., not detected in 40 cycles. Statistics: Student t-test (two-tailed), by SPSS v26.0. *p < 0.05 (p = 0.0127), **p < 0.01 (p = 0.0048 for PAX1 and p = 0.0024 for TWIST1); ****p < 0.0001 (p = 0.000038). J Immunostaining of SOX9 and TWIST1 confirmed the maintenance of cell identity in expanded scl-progenitors. Nuclear staining: DAPI. Scale bars: 200μm. K, L Flow cytometry analysis confirmed the maintenance of SOX9+ scl-progenitor’s identity at different passages. K The percentage of SOX9+ cells at different passages. L Repeatability test of three independent samples of expanded scl-progenitors at different passages by flow cytometry. M The expansion medium was specific for supporting SOX9+ scl-progenitors. SOX9+ scl-progenitor and SOX9- cells were sorted and compared in the expansion medium for multiple passages. Source data are provided as a Source Data file.
