Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation.
Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation.
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大颗粒多光子流式细胞术纯化完整的胚状体，表现出增强的心肌细胞分化潜力。
DOI：
10.1039/c3ib20286k
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Ogle,BM
中科院分区：
文献类型：
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作者：
Buschke,DG;Vivekanandan,A;Squirrell,JM;Rueden,CT;Eliceiri,KW;Ogle,BM
类胚体是由干细胞、分化细胞和细胞外基质蛋白组成的大型(100μm)三维显微组织，大致概括了早期胚胎发育。胚胎干细胞作为体外模型系统被广泛用于研究干细胞分化和促进组织发育的复杂的物理和化学相互作用。尽管人们已经了解了许多与EBS的区别，但有效地探测和正确分析这些3D显微组织的实际和技术困难限制了它们的实用和进一步应用。我们描述了我们实验室开发的技术平台的进步，多光子流式细胞术(MPFC)，基于大小和荧光记者检测和分类大量完整的EBS。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尺寸和荧光强度。我们应用这个平台来纯化从小鼠诱导的多能干细胞(MIPS)产生的EBS群体，这些细胞由于NKX2-5的大小或表达而显示出增强的心肌细胞分化潜力，NKX2-5是一种指示心肌前细胞的同源结构域蛋白。在EB形成后立即纯化的大EBS(直径330-400μm)在以后的时间点上显示出比中、小EBS更高的形成心肌细胞的潜力。此外，在EB形成后不久表达NKX2-5的EB更有可能在以后的时间点形成搏动区，这表明心肌细胞正在分化。总而言之，这些研究强调了MPFC根据分选时表现出的首选特征或指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。
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 asin vitromodel 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.