Fig. 3. TGFß3-dependent chondrogenesis causes COL2-containing cartilage matrix accumulation and induces cartilage-specific gene expression. (A) Cartilage-like particle formation with single positive EB cells in vitro. The sorted flk-1^– PDGFR⁺ cells (a-c) and flk-1⁺PDGFR^– cells (d-f) were individually subjected to the pellet culture in the presence of TGFß3. Particles formed in 17 days of culture were fixed with Gendre's fluid, paraffin-embedded, sectioned and stained with Toluidine blue (a,d). Two contiguous sections were also immunostained with AB765P (b,e) and 2B1.5 (c,f). Brown areas indicate the accumulation of COL1 (b,e) and COL2 (c,f). Immunostaining with rabbit IgG and mouse IgG did not yield any signals (data not shown). These results are representative of seven independent experiments. (B) Expression of cartilage-specific mRNAs during the TGFß-dependent generation of cartilage-like particles from single positive EB cells. The sorted flk-1^–PDGFR⁺ cells and flk-1⁺PDGFR^– cells were individually subjected to the TGFß3-containing pellet culture. On the day indicated, three aggregates were collected and RNAs were isolated for nested RT-PCR. No signals were observed without reverse transcription or when the first 21 cycles were done with a primer set for a different gene, indicating that the positive signals originated from the corresponding transcripts (data not shown). Essentially the same results were obtained from three independent experiments.