"The Long-Term Expanded Ectomesenchymal Cells Are Still Dependent on FSB(A) SB431542-dependent maintenance of CD271 expression. Ectomesenchymal cells maintained under FSB (p10) were either passaged one to three times to FSB again (p11, p14), or to FGF2 (→F, Fp3) and then FACS analyzed.(B) Maintenance of sGAG production capacity. The ectomesenchymal cells maintained under FSB for 11–16 passages followed by FT treatment, and the freshly isolated chondrogenic paraxial mesoderm (PM) (Umeda et al., 2012) were subjected to pellet culture. The capacity for sGAG production is displayed as in Figure 4C. Negative control, hESCs; positive control, bovine articular cartilage (n = 3 technical repeats, mean ± SD).(C) ""Full-cartilage""-forming capacity remained in the FSB-expanded p13 ectomesenchymal cells derived from CD271hiCD73− H9 progeny. Toluidine Blue staining.(D) SB431542-dependent maintenance of chondrogenic activity. The p10 ectomesenchymal cells were passaged to either FSB for four passages (FSBp14) or F for two passages (→Fp2) and then subjected to FT treatment and 3D-pellet culture. The capacity for sGAG production is displayed as in (B). ∗p < 0.05 (n ≥ 3 independent cultures, one pellet/culture, mean ± SD).(E) Real-time RT-PCR analysis for investigating the effect of FSB on maintaining SOX9 expression (n = 3 technical repeats, mean ± SD)."
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