Wilson Kitchener, Yu Jin, Lee Andrew, Wu Joseph C
Department of Radiology, Stanford University School of Medicine, USA.
J Vis Exp. 2008 May 2(14):740. doi: 10.3791/740.
The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.
人类胚胎干细胞（hESCs）的发现极大地增加了对再生医学感兴趣的医学科学家可用的工具。然而，将hESCs以及从hESCs分化而来的细胞直接注射到活生物体中，迄今为止受到显著的细胞死亡、畸胎瘤形成和宿主免疫排斥的阻碍。了解移植后hESCs在体内的行为需要新的成像技术来纵向监测hESCs的定位、增殖和活力。分子成像为研究人员提供了一种高通量、低成本且灵敏的方法，用于在数天、数周甚至数月内追踪体内细胞增殖。这一进展显著增进了对活体内hESCs植入、增殖和畸胎瘤形成的时空动力学的理解。分子成像的一项重大进展是将非侵入性报告基因检测从分子和细胞生物学扩展到体内多模态成像平台。这些报告基因在利用宿主细胞转录机制的工程启动子和增强子的控制下，通过多种载体和非载体方法导入细胞。一旦进入细胞，报告基因可以组成性转录，也可以仅在特定的生物学或细胞条件下转录，这取决于所使用的启动子类型。然后，使用诸如D - 荧光素等信号生成探针，通过灵敏的非侵入性仪器（如CCD相机）检测报告基因转录和翻译成生物活性蛋白的情况。为了避免荧光成像中在体内追踪干细胞所需的激发光，生物发光报告基因成像系统仅需要外源性施用的探针来诱导发光。源自萤火虫Photinus pyralis的萤火虫荧光素酶编码一种催化D - 荧光素转化为光学活性代谢物氧化荧光素的酶。然后可以用外部CCD相机监测光学活性。在其染色体DNA中携带报告构建体的稳定转导细胞将把报告构建体DNA传递给子细胞，从而能够纵向监测hESCs在体内的存活和增殖。此外，由于信号产生需要报告基因产物的表达，只有存活的亲代和子代细胞才会产生生物发光信号；凋亡或死亡的细胞则不会。在本视频中，将描述使用生物发光成像追踪干细胞增殖和畸胎瘤形成所需的具体材料和方法。
