Tomov Martin L, Olmsted Zachary T, Paluh Janet L
SUNY Polytechnic Institute, Colleges of Nanoscale Science and Engineering, Nanobioscience, Nanofab East, 257 Fuller Road, Albany, New York, 12203, USA.
Macromol Biosci. 2015 Jul;15(7):892-900. doi: 10.1002/mabi.201500051. Epub 2015 Mar 25.
In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm EBs are monocystic versus 500 μm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.
在多能干细胞分化过程中，胚状体（EBs）在谱系特化中提供了一个三维[3D]多细胞前体。EBs的内部结构尚未得到充分表征，但预计是分化的一个重要参数。在这里，我们使用定制的SU-8模具来生成聚二甲基硅氧烷的透明光刻模板阵列（LTA-PDMS），用于高通量分析人类胚胎干细胞（hESC）EB的形成和内部结构。比较了通过使用单细胞、二维细胞团或三维早期聚集体在直径为200和500μm的微阵列孔中形成的EBs。我们观察到，200μm的EBs是单囊的，而500μm的EBs是多囊的，包含大、中、小尺寸的囊肿。在500μm EBs的贴壁分化过程中，多囊特性会损害从3D到2D的转变，从而产生不均匀的单层细胞。我们的研究结果表明，EB核心结构具有大小依赖性特征，这会影响其在早期分化过程中的结构和细胞群体均匀性。
