Evaluation of a differentiation screening method using ES cells

为了建立一种ES细胞三维培养的分化筛选方法，将EBS在含有胶原胶和影响分化的化学物质的培养液中培养21d。这些化学物质包括细胞因子、表皮生长因子、成纤维细胞生长因子、IL-6、JL-12、肿瘤坏死因子-α和mLIF维生素，如L抗坏血酸、胆钙素和叶酸，以及骨形态发生蛋白、Emdogain、MS-818、MS-430、4种肾上腺皮质激素和7种氨基酸。此外，还观察了表皮生长因子、成纤维细胞生长因子与MS-818、MS-430的联合作用。在支架的作用方面，考察了在胶原海绵中添加EGF、成纤维细胞生长因子、MS-818和BMP的效果。至于对EBS施加外力的影响，制作了一种对细胞连续施加外力的装置。然后在胶原凝胶中对EBS施加外力4小时。结果表明，IL-6、肿瘤坏死因子-α、L-抗坏血酸、胆钙素或MS-430均可使心肌搏动频率减慢，而与其他药物无明显差异。在骨钙素含量方面，成纤维细胞生长因子诱导的骨钙素是对照组的360%，BMP诱导的是对照组的310%，维生素D诱导的是对照组的180%。在联合作用方面，成纤维细胞生长因子和MS-818的联合作用是对照组的420%，而成纤维细胞生长因子和MS-430的联合作用是对照组的280%。对于其他组合，没有观察到显著的差异。IL-10和12均为阴性。XPS也显示出类似的趋势，由于支架的作用，胶原海绵呈现出与凝胶相似的趋势。本研究的结果表明，化学物质对ES细胞分化的影响可以用胶原蛋白三维培养的方法来确定。

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.