Evaluation of a differentiation screening method using ES cells

为了建立ES细胞三维培养的分化筛选方法，将EBs在胶原凝胶和影响分化的化学物质的培养基中培养21天。这些化学物质包括细胞因子、EGF、FGF、IL-6、JL-12、TNF-α和多种维生素，如l -抗坏血酸、胆骨化醇和叶酸，以及BMP、Emdogain、MS-818、MS-430、4种肾上腺皮质激素和7种氨基酸。此外，还考察了EGF、FGF与MS-818、MS-430的联合作用。在支架的作用方面，考察了在海绵胶原中添加EGF、FGF、MS-818和BMP的效果。针对电子束施加外力的效果，研制了一种连续施加外力于细胞的装置。然后用胶原凝胶对EBs施加外力4小时。结果表明，IL-6、TNF-α、l -抗坏血酸、胆骨化醇和MS-430均能降低心肌搏动率，但与其他药物无显著差异。FGF诱导骨钙素的量是对照组的360%，BMP诱导量是对照组的310%，维生素D诱导量是对照组的180%。在综合效应方面，FGF与MS-818的联合诱导率为对照的420%，FGF与MS-430的联合诱导率为对照的280%。对于其他组合，没有观察到显著差异。IL-10和il - 12均为阴性。XPS也显示出类似结果的趋势。由于支架的作用，海绵胶原蛋白表现出类似凝胶的倾向。研究还表明，强外力对胚胎干细胞的分化并不总是有用的。本研究结果表明，化学物质对ES细胞分化的影响可以通过胶原三维培养法确定。

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.