Evaluation of a differentiation screening method using ES cells

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To develop a differentiation screening method using 3D culture with ES cells, EBs were cultured for 21 days, in a culture media of collagen gel and chemicals affecting differentiation. The chemicals consisted of cytokines, EGF, FGF, IL-6, JL-12, TNF-α and mLIF vitamins such as L-ascorbic acid, cholecalciferol and folic acid, and BMP, Emdogain, MS-818, MS-430, four adrenocortical hormones and seven amino acids. In addition, the combined effects of EGF, FGF and MS-818, MS-430 were also examined. Regarding the effects of scaffolds, the effects of adding EGF, FGF, MS-818 and BMP to collagen sponge were examined. As for the effects of appliying an external force to EBs, a device to continuously apply external force to cells was manufactured. Then external force was applied to EBs in collagen gel for four hours. The results demonstrated that the myocardial pulsation rate decreased with IL-6, TNF-α, L-ascorbic acid, cholecalciferol or MS-430, but there was no significant difference observed with other chemicals. Regarding the amount of osteocalcin, FGF induced 360% of that in the control, BMP induced 310% in the control and vitamin D induced 180% that of that in the control. Regarding the combined effects, the combination of FGF and MS-818 induced 420% and the combination of FGF and MS-430 induced 280% of that in the control. For other combinations, there were no significant differences observed. Both IL-10 and 12 were negative. XPS also showed a tendency toward similarresults.Due to the effects of the scaffolds, collagen sponge showed a tendency similar to gel. It was also shown that a strong external force was not always useful in the differentiation of ES cells.The results of this study have shown that the effects of chemical substances on ES cell differentiation can be determined with the 3D culture method by collagen.