Microfabrication-based modulation of embryonic stem cell differentiation
Microfabrication-based modulation of embryonic stem cell differentiation
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DOI：
10.1039/b704739h
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发表时间：
2007-01-01
期刊：
影响因子：
6.1
通讯作者：
Yarmush, Martin L.
中科院分区：
文献类型：
--
作者：
Park, Jaesung;Cho, Cheul H.;Yarmush, Martin L.
胚胎干细胞在分化过程中形成自发聚集体，聚集体中的细胞间通讯在分化过程中起着重要作用。由于缺乏可靠的方法来产生均匀的聚集体大小，ES细胞的受控分化方案的开发受到阻碍。常规技术，如悬滴和悬浮培养，不允许精确控制ES细胞聚集体的大小。为了克服这个问题，我们微制造的粘性支架，使小鼠ES（mES）细胞聚集体的特定尺寸范围从100 μ m到500 μ m的直径。利用这种技术，我们研究了初始聚集体大小对ES细胞分化的影响。诱导分化20天后，我们使用基因和蛋白质表达测定以及生化功能分析了干细胞群。值得注意的是，我们发现胚层分化取决于ES细胞聚集体的初始大小。在测试的ES细胞聚集体大小中，直径为300 μ m的聚集体显示出与使用“悬滴”技术制备的胚状体类似的三个胚层的分化概况。与较大的（500 μ m）聚集体相比，较小的（100 μ m）聚集体显示外胚层标记物的表达增加，而与100 μ m聚集体相比，500 μ m聚集体显示中胚层和内胚层标记物的表达增加。这些结果表明，聚集体的初始大小是ES细胞分化的一个重要因素，并且可以影响胚层选择以及分化的程度。
Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 mu m to 500 mu m in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 mu m diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop'' technique. The smaller (100 mu m) aggregates showed the increased expression of ectodermal markers compared to the larger (500 mu m) aggregates, while the 500 mu m aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 mu m aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.