Fourier Transform Infrared Microspectroscopy as a Tool for Embryonic Stem Cell Studies
Fourier Transform Infrared Microspectroscopy as a Tool for Embryonic Stem Cell Studies
复制标题
傅里叶变换红外显微光谱作为胚胎干细胞研究的工具
作者：
D. Ami;P. Mereghetti;A. Natalello;S. Doglia
胚胎干细胞（ES）是一种自我更新的多能细胞，起源于哺乳动物囊胚的内部细胞群（Smith, 2001）。它们潜在地产生每一种细胞类型的独特能力不断吸引着不同研究领域的兴趣。事实上，胚胎干细胞是研究细胞分化分子机制的有力工具，在细胞治疗、组织工程、再生医学和药物筛选方面具有重要应用（Trounson, 2006; Vats et al., 2005）。所有这些应用都需要快速和敏感的分析，通过识别分化状态的特定标记来评估分化过程。迄今为止，胚胎干细胞的分化主要通过免疫组织化学、基因表达分析、分化细胞的功能分析和流式细胞术等生化方法进行监测，即使提供全面的细胞特征也耗时、昂贵且通常需要复杂的样品处理。由于这些原因，开发干细胞研究的新方法是非常可取的。在过去的几十年里，光谱学方法被应用于完整细胞的研究，特别是振动光谱学被证明是表征复杂生物系统的强大技术（Heraud & Tobin, 2009）。特别是，傅里叶变换红外（FTIR）和拉曼光谱是非侵入性和无标记的振动（微）光谱，可以获得完整细胞、组织和整个生物体的分子组成和结构信息（Schulze等人，2010；Tanthanuch等人，2010;Chan & Lieu, 2009； Walsh等人，2009；Ami等人，2004；Choo等人，1996），在一次测量中提供独特的分子指纹。通过这种方式，可以快速表征生物系统中同时发生的不同过程，这对于标准生化方法来说是一项不容易的任务。由于使用红外显微镜与FTIR光谱仪相结合，从选定的样品区域收集吸收光谱成为可能。有趣的是，这些技术，由于其快速的时间分辨率，已经成功地用于快照和“冻结”复杂系统中的分子事件（Miller & Dumas, 2010; Hamm, 2009）。
Embryonic stem (ES) cells are self-renewing and pluripotent cells that arise from the inner cell mass of the mammalian blastocyst (Smith, 2001). Their unique capability of potentially generating every cell type continuously attracts the interest of different fields of research. Indeed, ES cells represent a powerful tool for the study of the molecular mechanisms of cell differentiation with important applications in cell therapies, tissue engineering, regenerative medicine and pharmaceutical screening (Trounson, 2006; Vats et al., 2005). All these applications require rapid and sensitive assays to evaluate the differentiation process through the identification of specific markers of the differentiation status. To date, ES cell differentiation is mainly monitored by biochemical methods such as immunohistochemistry, gene expression analysis, functional assays of the differentiating cells and flow cytometry, that even if providing a comprehensive characterization of cells are time consuming, expensive and often require a complex sample handling. For these reasons, the development of new approaches for stem cell studies is highly desirable. In the last decades, optical spectroscopy approaches were applied to the study of intact cells and in particular vibrational spectroscopies revealed to be powerful techniques for the characterization of complex biological systems (Heraud & Tobin, 2009). In particular, Fourier transform infrared (FTIR) and Raman are non invasive and label-free vibrational (micro)spectroscopies that allow to obtain information on the molecular composition and structure of intact cells, tissues and whole organisms (Schulze et al., 2010 ; Tanthanuch et al., 2010; Chan & Lieu, 2009; Walsh et al., 2009; Ami et al., 2004; Choo et al., 1996), providing a unique molecular fingerprint within a single measurement. In this way, it is possible to characterize rapidly different processes that take place simultaneously in biological systems, a non easy task for the standard biochemical approaches. Thanks to the use of an infrared microscope coupled to a FTIR spectrometer, it becomes possible to collect the absorption spectrum from a selected sample area. Interestingly, these techniques, thank to their fast – time resolution, have been successfully used to snapshot and “freeze” molecular events in complex systems (Miller & Dumas, 2010; Hamm, 2009).