Morphology and characteristics of ES-derived chondrocytes after 32 days of culture. (A, B) Determination of proteoglycans in EBs with alcian blue in chondrocyte cultures induced with TGFβ1 [2 ng/ml] and BMP-2 [10 ng/ml] from d3–5 of culture and with BMP-2 [10 ng/ml], ascorbic acid [50 μg/ml] and insulin [1 μg/ml] from day 5 onwards (A) compared to control cultures (B). Bar = 8.3 μm. (C, D) Analysis of cartilage-specific matrix proteins in 32 day old EBs induced with the same supplements as in (A, B) by means of immunohistochemistry. Staining with anti-collagen type II (C) and anti-cartilage proteoglycan (D), respectively, both visualized by a secondary AlexaFluor 488 conjugated antibody. Bar = 106 μm. (E) Concentration-dependent effect of BMP-2 on chondrocyte-specific gene expression in 32-day old EBs. Values of cultures supplemented with 10 ng/ml BMP-2 are shown compared to 2 ng/ml as used by Kramer et al. , which was set as 1. Values represent means of three independent experiments ± standard deviation, obtained by quantitative RT-PCR analysis. **P < 0.01; ***P < 0.001. (F) Proteoglycan content of EB extracts on day 32. BMP-2 directs increased proteoglycan synthesis in both 2 ng/ml and 10 ng/ml. *P < 0.1; **P < 0.01. Mean ± standard deviation, n = 3.