In Vivo Assessment of Embryonic Stem Cell Teratoma Prevention

描述（由申请人提供）：在这些细胞的广泛治疗应用成为可能之前，评估分化胚胎干细胞群形成畸胎瘤潜力的能力代表了迫切的需求。凯斯西储大学和BioInVision公司的研究人员。将共同创建平台技术，用于验证现有的胚胎干细胞分离和纯化模式（如抗体标记和荧光激活细胞分选），以及未来的纯化模式。生物平台由胚胎干细胞组成，胚胎干细胞在未分化时报告一种荧光标记物（红色），而在内皮谱系分化时报告另一种标记物（绿色）。将通过接种绿色细胞、在长期培养物中培养这些细胞并确定红色未分化细胞的任何外观，在体外评价分选。为了评估体内细胞，我们将使用BioInVision独特的冷冻成像技术，该技术可以检测小鼠中的单个荧光细胞。简单地说，冷冻成像系统包括一个大的阶段，电动低温恒温器与特殊功能的成像;一个修改，明场/荧光显微镜;和一个机器人xyz成像系统定位器，所有这些都是完全自动化的控制系统，以允许切片和平铺成像的整个鼠标。基本实验范例将包括分选分化的（绿色）细胞并通过尾部注射植入它们。几天后，冷冻成像将用于识别畸胎瘤和/或有可能形成畸胎瘤的未分化（红）细胞。冷冻成像平台相对于其他成像方法（如生物发光，MRI和PET）具有明显的优势，因为它不仅能够检测大的明显畸胎瘤，而且还能够发现未来可能形成畸胎瘤的单细胞。 公共卫生相关性：该提案与公共卫生问题密切相关，因为其长期目标是提供一种方法，在胚胎干细胞用于治疗之前评估这些细胞的选择和纯化策略的有效性。畸胎瘤形成的能力是阻止胚胎干细胞在临床上用于糖尿病、帕金森病和脊髓损伤等疾病的主要障碍。

DESCRIPTION (provided by applicant): The capacity to evaluate the teratoma forming potential of differentiated embryonic stem cell populations represents an urgent need before widespread therapeutic application of these cells is possible. Researchers at Case Western Reserve University and BioInVision, Inc. will join together to create platform technologies for validating existing embryonic stem cell separation and purification paradigms (such as antibody labeling and Fluorescent Activated Cell Sorting), as well as future purification paradigms. The biologic platform consists of embryonic stem cells which report one fluorescent marker (red) when undifferentiated, and another marker (green) when differentiated down the endothelial lineage. Sorting will be evaluated in vitro by plating green cells, growing those cells in long-term culture, and determining any appearance of red undifferentiated cells. To assess cell in vivo, we will use BioInVision's unique cryo-imaging technology which can detect single fluorescent cells in a mouse. Briefly, the cryo-imaging system consists of a large stage, motorized cryostat with special features for imaging; a modified, bright-field/fluorescence microscope; and a robotic xyz imaging system positioner, all of which are fully automated by a control system to allow sectioning and tiled imaging of an entire mouse. The basic experimental paradigm will consist of sorting differentiated (green) cells and implanting them via tail injection. Days later, cryo-imaging will be used to identify teratomas and/or undifferentiated (red) cells with the potential of forming a teratoma. The cryo-imaging platform represents a distinct advantage over other imaging methods, such as bioluminescence, MRI, and PET, in that it is capable of detecting not only large overt teratomas, but also finding single cells which may have the capacity to form teratomas in the future. PUBLIC HEALTH RELEVANCE: This proposal is critically concerned with the issue of public health in that its long-term goal is to provide a means for evaluating the effectiveness of embryonic stem cell selection and purification strategies prior to such cells being used therapeutically. The capacity for teratoma formation is the main barrier preventing use of embryonic stem cells clinically, for diseases such as diabetes and Parkinson's Disease and spinal cord injury.