Figure 3: Quantitative analysis of the maintenance of ES/iPS cells in an undifferentiated state by an AP-positive colony forming assay. (A) ES/iPS cells were cultured on the various feeder layers for 10 d in LIF-free medium. Then, ES/iPS cells were re-plated onto a MEF feeder layer and cultured in medium containing 1 × 103 U/ml LIF for 2 d. The control experiment (MEF+LIF) was performed by culturing ES/iPS cells on a MEF feeder layer in medium containing 1 × 103 U/ml LIF. (B) Effect of the physical interaction of ES/iPS cells with feeder cells using magnetic culture. Magnetically-labeled ES/iPS cells were cultured on the various feeder layers in LIF-free medium under an applied magnetic force for 10 d. The control experiment (MEF+LIF) was performed by culturing magnetically-labeled ES/iPS cells on a MEF feeder layer in medium containing 1 × 103 U/ml LIF under an applied magnetic force for 10 d. ES/iPS cells were then re-plated onto a MEF feeder layer and cultured in medium containing 1 × 103 U/ml LIF for 2 d. *P<0.05 vs the respective data shown in Figure 3A. (C) Effect of a ROCK inhibitor on ES/iPS cell culture on the various feeder layers. ES/iPS cells were cultured on the various feeder layers in LIF-free medium containing a ROCK inhibitor (Y-27632) for 10 d. ES/iPS cells were then re-plated onto a MEF feeder layer and cultured in medium containing 1 × 103 U/ml LIF for 2 d. The control experiment (MEF+LIF) was performed by culturing ES/iPS cells on a MEF feeder layer in medium containing 1 × 103 U/ml LIF and Y-27632 for 10 d. *P<0.05 vs the respective data shown in Figure 3A. (D) Effect of a Rho activator on ES/iPS cells cultured on the various feeder layers. ES/iPS cells were cultured on the various feeder layers in LIF-free medium containing a Rho activator (LPA) for 10 d. ES/iPS cells were then re-plated onto a MEF feeder layer and cultured in medium containing 1 × 103 U/ml LIF for 2 d. The control experiment (MEF+LIF) was performed by culturing ES/iPS cells on a MEF feeder layer in medium containing 1 × 103 U/ml LIF and LPA for 10 d. *P<0.05 vs the respective data shown in Figure 3A. Experiments were performed in triplicate, and data are the means ± SD. Black column, 129/Sv; white column, H-1; gray column, iPS-MEFNg- 20D-17.