Long-term cultures of ES-D3 cells can be established in the absence of LIF. ES-D3 cells were seeded (300 cells/mm2) onto LN-511-, LN-111-, Col-I-, Col-IV- or FN-coated tissue culture wells (3.5 cm Ø) and grown in the presence or absence of 10 ng/ml of LIF, as indicated. Upon confluency the cells were trypsinized, counted and reseeded at 300 cells/mm2. Cell doubling index was calculated for each time point as average number of divisions by a cell per 24 hours. The graph represents data from 3–4 independent experiments performed in triplicates except for cells grown on Col-I in the absence of LIF where only 2 out of 6 samples contained enough cells to be reseeded after P2 due to cell loss during culture. P-values were calculated using student’s t-test and are marked with an asterisk (* < 0.05; ** <0.005).