Analysis of Mitochondrial Dimensions and Cristae Structure in Pluripotent Stem Cells Using Transmission Electron Microscopy

Q2 Biochemistry, Genetics and Molecular Biology

Current Protocols in Stem Cell Biology Pub Date : 2018-10-10 DOI:10.1002/cpsc.67

I. C. Tobias, R. Khazaee, D. H. Betts

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引用次数: 10

Abstract

Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.

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多能干细胞线粒体尺寸和嵴结构的透射电镜分析

利用胚胎干细胞(ESCs)和诱导多能干细胞(iPSCs)建模的线粒体结构和功能的动态改变调节着细胞命运的决定，并成为多种年龄相关疾病和遗传疾病的基础。透射电子显微镜(TEM)可以获得线粒体的高分辨率显微照片，但线粒体的超微结构在样品处理过程中容易扭曲。本单元描述了一种保存线粒体膜结构的方法，用于TEM样品制备。该程序可用于评估线粒体在分化、重编程或ESC代谢实验操作过程中的超微结构变化。我们提供了全面的方案:(1)制备用于TEM的ESC培养物;(2)单个ESCs的薄片提取;(3)线粒体和嵴的对比染色和形态计量学分析。本单元还描述了低细胞数样品的替代程序和形态测量图像分析的支持协议。总的来说，这些方案允许观察和定量分析ESCs中的线粒体。©2018 by John Wiley &儿子,Inc。

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来源期刊

Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.