Fig. 3: Chondrogenic induction of ExpLBM cells using two- and three-dimensional culture conditions.

a, Flow cytometry analysis of SOX9 expression in ExpLBM cells derived from various hPSCs. b, Two-dimensional chondrogenic induction (2DCI) protocol for ExpLBM cells. c, Alcian blue staining after the 2DCI-based differentiation of ExpLBM cells. After culturing PRRX1^3′tdTomato-reporter line-derived ExpLBM cells with step 1 medium for 6 d, the cells were treated with step 2 medium for 6 d. The cells were then fixed and stained with Alcian blue (n = 3 biologically independent experiments). d, Analysis of chondrocyte marker genes during the 2DCI protocol. Total RNA was extracted at the indicated time points and the gene expression levels were determined by RT–qPCR analysis. All values were normalized to those of ACTB mRNA (n = 3 biologically independent experiments). ND, not detected. c,d, **P < 0.01; ***P < 0.001; NS, not significant. Statistical significance was determined using a two-sided unpaired Student’s t-test (c) or one-way ANOVA with correction for multiple testing using the Bonferroni method (d). e, Three-dimensional chondrogenic induction (3DCI) protocol for ExpLBM cells. f, Images of hPSCs-derived particles using the 3DCI protocol. g, Histological analysis of particles generated using the 3DCI protocol. Particles were generated from ExpLBM cells derived from each hPSC, fixed and then stained with haematoxylin–eosin (HE), Alcian blue or Safranin O, or subjected to immunohistological analysis using antibodies against SOX9, COL2, COL1 or COLX. f,g, Data for day 42 of step 3. h, Subcutaneous transplantation of cartilaginous particles in NOD-SCID mice. Cartilaginous particles (414C2-derived; step 3 treatment, 21 d) were subcutaneously transplanted into NOD-SCID mice and their engraftment was assessed by HE, Alcian blue or Safranin O staining at 8 weeks after transplantation. c,f–h, Representative images are shown.