Department of Biomedical Engineering, University of Wisconsin-Madison, 2-114 Engineering Centers Building, 1550 Engineering Drive, Madison, Wisconsin 53706, USA.
Integr Biol (Camb). 2013 Jul 24;5(7):993-1003. doi: 10.1039/c3ib20286k. Epub 2013 Jun 13.
Embryoid bodies (EBs) are large (>100 μm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 μm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.
胚状体（EBs）是由干细胞、分化细胞和细胞外基质（ECM）蛋白组成的大型（>100μm）3D 微组织，大致再现了早期胚胎发育。EBs 被广泛用作体外模型系统，用于研究干细胞分化以及促进组织发育的复杂物理和化学相互作用。尽管从 EBs 中已经了解了很多分化知识，但有效探测和正确分析这些 3D 微组织的实际和技术困难限制了它们的实用性和进一步应用。我们描述了我们实验室开发的多光子流动细胞术（MPFC）技术平台的进展，该平台可根据大小和荧光报告基因对大量完整的 EBs 进行检测和分选。通过在 MPFC 软件中实现图像处理算法，现在可以实时和同时测量大小和荧光强度。我们将该平台应用于从诱导多能干细胞（miPS）中产生的 EB 群体的纯化，这些 EB 群体由于大小或 NKX2-5 的表达而表现出增强的心肌细胞分化潜力，NKX2-5 是一种预示着前体细胞的同源域蛋白。EB 形成后不久进行纯化的大 EB（330-400μm，直径）在稍后的时间点形成心肌细胞的潜力明显高于中或小 EB。此外，EB 形成后不久表达 NKX2-5 的 EB 更有可能在稍后的时间点形成搏动区域，这表明心肌细胞分化。总的来说，这些研究强调了 MPFC 基于分选时表现出的首选特征或预示未来特征或功能能力的特征来纯化 EBs 和类似微组织的能力。
