Novel assay for functional capacities of pluripotent stem cells

描述(申请人提供)：多能干细胞能够分化为所有身体组织，并能够通过无限增殖进行自我更新。多能干细胞最初是在组织培养中通过培养小鼠胚胎的内细胞团(ICM)获得的。这种小鼠胚胎干细胞(MESCs)及其分化产生的组织的功能能力传统上是通过形成嵌合体来检测的。这是通过将mESCs引入小鼠早期胚胎，然后将胚胎移植到寄养雌性动物的子宫中怀孕和出生来实现的。这类研究证明了mESCs的多能性，因为干细胞对所有器官和组织都有贡献，包括生殖系。然而，用于确定人类多能干细胞分化能力的方法根本不能说明从它们衍生的组织的功能能力。因此，到目前为止，几乎没有任何关于人类多能干细胞来源的细胞或组织的功能能力的信息。例如，嵌合体研究表明，mESCs不仅能够在所有身体组织中完全正常功能，而且在体外生长的mESCs在返回到发育中的胚胎后不会增加异常生长(即肿瘤)或发育不稳定的风险。这一见解对移植来自人类胚胎干细胞的组织的可比风险没有提供信息，因为最近已表明这些组织具有与微胚胎干细胞显著不同的生物学性质。这些信息需要开发一种新的多能干细胞功能能力的分析方法。拟议的研究涉及多能小鼠和人类干细胞，这些干细胞经过基因改造，整合了编码结构性表达的绿色荧光蛋白(GFP)报告基因和有条件表达的红色荧光蛋白(RFP)报告基因的转基因。多能干细胞将被诱导分化为中胚层或神经外胚层，这些分化的后代将被移植到植入后小鼠胚胎的中胚层或神经外胚层，以测试其在组织或器官环境中的功能能力。然后，胚胎将在旋转的管子中培养1至3天，直到形成早期器官雏形。注射细胞的所有后代都将被绿色荧光蛋白标记；分化为特定靶组织的细胞将被绿色荧光蛋白标记。标记细胞的分子和生理特性将表明干细胞来源的分化细胞是否具有证实其正常的关键特征。将使用类似的方法来确定关键环境毒物的影响，特别是C6H6和甲基汞，这两种毒物分别对血液和神经系统具有发育毒性。

DESCRIPTION (provided by applicant): Pluripotent stem cells are capable of differentiating into all body tissues and are able to renew themselves through unlimited proliferation. Pluripotent stem cells were first derived by growing the inner cell mass (ICM) of mouse embryos in tissue culture. The functional capacity of such mouse embryonic stem cells (mESCs) and the tissues generated by their differentiation has traditionally been assayed by formation of chimeras. This was achieved by introducing mESCs to an early mouse embryo and then transferring the embryo to the uterus of a foster female for gestation and birth. Such studies demonstrated the pluripotency of mESCs, as the stem cells contributed to all organs and tissues, including the germline. However, the method used to determine the differentiative capacity of human pluripotent stem cells tells nothing whatsoever about the functional capacity of the tissues derived from them. Consequently, there is hardly any information available to date about the functional capacities of cells or tissues derived from human pluripotent stem cells. For example, chimera studies show not only that mESCs are capable of fully normal function in all body tissues but also that growing mESCs in vitro does not increase the risk of abnormal growth (i.e., tumors) or developmental instability once they are returned to a developing embryo. This insight in not informative about the comparable risks of transplanting tissues derived from human ESCs, because these have recently been shown to have a substantially different biological nature from mESCs. Such information requires development of a novel assay for functional capacities of pluripotent stem cells. The proposed studies involve pluripotent mouse and human stem cells that have been genetically altered to incorporate transgenes encoding a constitutively expressed green fluorescent protein (GFP) reporter gene as well as a conditionally expressed red fluorescent protein (RFP) reporter gene. The pluripotent stem cells will be induced to differentiate into mesoderm or neuroectoderm, and these differentiated progeny will be transplanted to the mesodermal or neuroectodermal layers of post-implantation mouse embryos to test their functional capacity in a tissue or organ context. Embryos will then be cultured for 1 to 3 days in rotating tubes until early organs rudiments form. All descendants of the injected cells will be GFP-labeled; cells that differentiate into the specific target tissue wil be RFP labeled. The molecular and physiological properties of labeled cells will indicate whether the stem cell-derived differentiated cells share key features that confirm their normality. A similr approach will be used to determine the impact of key environmental toxicants, specifically C6H6 and MeHg, which are developmental toxicants for the blood and nervous systems, respectively.