Fig. 3. Feeder cell antagonism of BMP action. (A) The effect of feeder cell density on the response of human ES cells to BMP-2. ES cells were plated onto feeder cells prepared at a density of either 6.6x104/cm² (high density) or 1.3x104/cm² (low density) and were grown for 5 days with or without treatment with 50 ng/ml BMP-2. The wells were then fixed and stained with monoclonal antibody GCTM-2 followed by detection with anti-mouse immunoglobulin conjugated to alkaline phosphatase; red staining against blue counterstain indicates activity. (B) RT-PCR analysis for transcripts of three BMP antagonists in ES cells, differentiating cultures of ES cells and mouse embryo fibroblasts. Gremlin transcripts are strongly expressed in mouse embryo fibroblasts.