Fig. 2. Genes characteristically expressed during chondrocyte hypertrophy and calcification were found to be differentially expressed during ES cell differentiation in vitro. Genes encoding extracellular matrix proteins of cartilage tissue such as collagen II (A), collagen X (B), osteocalcin (C) and the osteoblast-specific transcription factor Cbfa-1 (D) were expressed in EBs from different cultivation stages from 2 (5+2 d) up to 30 days (5+30 d) after plating as demonstrated by RT-PCR. The primers used to amplify collagen II transcripts detected two splice variants (A), the juvenile variant A and the adult variant B. The transcriptional levels relative to those of the house-keeping gene HPRT are presented. Mean values±s.e.m. derived from independent experiments (n=4) are shown.