Novel assay for functional capacities of pluripotent stem cells

描述（由申请人提供）：多能干细胞能够分化成所有身体组织，并能够通过无限增殖自我更新。多能干细胞最初是通过在组织培养中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嵌合体的形成来检测的。这是通过将mESCs导入早期小鼠胚胎，然后将胚胎移植到寄养雌性子宫进行妊娠和分娩来实现的。这些研究证明了间充质干细胞的多能性，因为干细胞有助于所有器官和组织，包括种系。然而，用于测定人类多能干细胞分化能力的方法并不能说明由它们衍生的组织的功能能力。因此，迄今为止几乎没有任何关于人类多能干细胞衍生的细胞或组织的功能能力的信息。例如，嵌合体研究表明，间充质干细胞不仅能够在所有身体组织中发挥完全正常的功能，而且体外培养的间充质干细胞一旦回到发育中的胚胎中，也不会增加异常生长（即肿瘤）或发育不稳定的风险。这一发现并不能提供关于人类ESCs来源的移植组织的可比风险的信息，因为这些组织最近被证明与mESCs具有本质不同的生物学性质。这些信息要求开发一种新的多能干细胞功能测定方法。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，纳入了编码组成表达的绿色荧光蛋白（GFP）报告基因的转基因，以及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。然后胚胎将在旋转试管中培养1至3天，直到早期器官雏形形成。所有注射细胞的后代将被gfp标记；分化为特定靶组织的细胞将被RFP标记。标记细胞的分子和生理特性将表明干细胞衍生的分化细胞是否具有确认其正常的关键特征。类似的方法将用于确定关键环境毒物的影响，特别是C6H6和甲基汞，它们分别是血液和神经系统的发育毒物。

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.