Novel assay for functional capacities of pluripotent stem cells

多能干细胞功能能力的新测定

• 批准号: 8526391
• 负责人: Roger Arnold Pedersen
• 金额: $ 13.3万
• 依托单位: UNIVERSITY OF CAMBRIDGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-10 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8526391
• 关键词: Activities of Daily Living; Affect; Biological; Biological Assay; Birth; Blood; Cell Therapy; Cells; Chimera organism; Derivation procedure; Development; Embryo; Embryo Transfer; Environment; Epiblast; Evaluation; Female; Fostering; Germ Layers; Green Fluorescent Proteins; Growth; Hematopoietic; Human; Immunodeficient Mouse; In Vitro; Inner Cell Mass; Label; Life; Mesoderm; Methods; Modeling; Molecular; Morphologic artifacts; Mus; Nature; Nervous system structure; Neuroectoderm; Normal tissue morphology; Normalcy; Organ; Phenotype; Physiological; Pluripotent Stem Cells; Population; Pregnancy; Property; Reporter Genes; Risk; Scientist; Staging; Stem cell transplant; Stem cells; Stress; Testing; Therapeutic Uses; Tissues; Toxic Environmental Substances; Transgenes; Transplantation; Transplanted tissue; Tube; Uterus; base; embryo culture; embryo tissue; embryonic stem cell; gastrulation; human embryonic stem cell; human stem cells; implantation; in utero; in vitro Assay; insight; novel; novel strategies; pluripotency; preimplantation; progenitor; red fluorescent protein; research study; response; tissue culture; toxicant; tumor

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.

描述（由申请人提供）：多能干细胞能够分化成所有身体组织，并且能够通过无限增殖来更新自身。多能干细胞首先是通过在组织培养物中培养小鼠胚胎的内细胞团（ICM）而获得的。传统上通过嵌合体的形成来测定此类小鼠胚胎干细胞（mESC）及其分化产生的组织的功能能力。这是通过将 mESC 引入早期小鼠胚胎中，然后将胚胎转移到寄养雌性的子宫中进行妊娠和分娩来实现的。此类研究证明了 mESC 的多能性，因为干细胞对所有器官和组织（包括种系）都有贡献。然而，用于确定人类多能干细胞分化能力的方法并不能说明源自它们的组织的功能能力。因此，迄今为止几乎没有任何关于源自人类多能干细胞的细胞或组织的功能能力的信息。例如，嵌合体研究不仅表明 mESC 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 mESC 在返回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。这种见解并没有提供有关移植源自人类 ESC 的组织的可比较风险的信息，因为这些组织最近被证明与 mESC 具有显着不同的生物学性质。这些信息需要开发一种新的检测多能干细胞功能的方法。拟议的研究涉及经过基因改造的多能小鼠和人类干细胞，以纳入编码组成型表达的绿色荧光蛋白（GFP）报告基因以及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，以测试它们在组织或器官环境中的功能能力。然后胚胎将在旋转管中培养 1 至 3 天，直到早期器官雏形形成。注射细胞的所有后代都将被 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性的关键特征。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。