Optimizing techniques for tracking transplanted stem cells in vivo
Optimizing techniques for tracking transplanted stem cells in vivo
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DOI：
10.1634/stemcells.2005-0149
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发表时间：
2005-10-01
期刊：
影响因子：
5.2
通讯作者：
Blau, HM
中科院分区：
文献类型：
--
作者：
Brazelton, TR;Blau, HM
骨髓来源的细胞（BMDCs）的潜力，以促进非造血组织产生了相当大的争论，在最近几年。争议的原因包括用于跟踪BMDC植入的技术的差异，不适当的组织制备，缺乏适当的阳性和阴性对照，以及对如何正确收集和解释来自落射荧光显微镜和共聚焦显微镜的图像的基本误解。我们的实验室是第一个使用骨髓移植的转基因小鼠组成型表达增强型绿色荧光蛋白（GFP）的研究能力的BMDC产生非造血组织类型，一个系统，现在被广泛使用。在我们6年的经验，使用GFP，以及β-半乳糖苷酶和Y染色体，在体内跟踪BMDC，我们已经确定了许多困难，并开发了技术来解决这些问题。我们讨论了这些方法中的几种，特别是，我们描述了比率分析技术，用于改善检测来自转基因骨髓的移植细胞。最后，为了帮助解决报告的差异与BMDCs有助于骨骼肌纤维的频率，我们证明了骨骼肌中的高度自发荧光肌纤维的模式是明显不同的GFP表达肌纤维，并描述如何明确的结论，可以从这些数据。
The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data.