Histocompatible Primate Embryonic Stem Cells

描述（由申请人提供）：该项目的目标是产生关于采用体细胞核移植（SCNT）和直接重编程方法将灵长类动物体细胞重编程为多能状态的重要新见解，并使用表达谱分析、遗传和表观遗传分析以及体外和体内分化测定进行比较多能性评估。我们的主要假设是，使用这两种替代方法实验获得的灵长类动物多能细胞彼此等同，并且等同于从受精胚胎中分离的胚胎干细胞（ESC）。本申请的另一个目标是首次评估源自受精或SCNT胚胎的猴ESC和诱导多能（iPS）细胞在注射到发育中的胚胎中后产生嵌合体的潜力。为了实现这些目标，我们提出了以下具体目标：1）。通过体细胞的表观遗传和遗传重编程来创造猴子多能细胞。在实验1中，我们将通过SCNT从成年猴皮肤细胞中获得ESC，并测试我们的工作假设，即细胞质中关键多能因子-OCT 4，SOX 2，NANOG和CARM 1-的实验上调将增强重编程并增加SCNT胚胎发育和ESC分离的电流效率。在实验2中，我们将通过在多西环素诱导型启动子的控制下慢病毒转导编码关键重编程因子OCT 4、SOX 2、KLF 4、C-MYC、NANOG和LIN 28的基因，从相同的猴皮肤细胞产生猴iPS细胞。2）。检查新型多能细胞的多能性。我们的工作假设是，SCNT和直接重编程都可以支持体细胞完全重编程为多能状态。为了验证这一假设，在实验1中，我们将询问新细胞系的遗传、细胞遗传、表观遗传和转录谱。在实验2中，细胞系将在SCID小鼠的畸胎瘤中进行体内分化，并在体外定向分化为中胚层（心肌细胞）、外胚层（神经元表型）和内胚层（胰腺β细胞）。确定猴多能细胞促成嵌合体的潜力。我们假设，类似于他们的小鼠同行，灵长类胚胎干细胞和iPS细胞有潜力整合和参与嵌合后代的发展。为了验证这一假设，我们建议将表达GFP的猴ESC和iPS细胞注射到猴植入前胚胎中，并将所得嵌合胚胎转移到受体中以建立妊娠。随后将研究嵌合胎儿和足月后代的组织分布和生殖系定殖。

DESCRIPTION (provided by applicant): The goal of this project is to generate important new insights concerning reprogramming of primate somatic cells to the pluripotent state employing somatic cell nuclear transfer (SCNT) and direct reprogramming approaches and to conduct comparative pluripotency assessments using expression profiling, genetic and epigenetic analysis and in vitro and in vivo differentiation assays. Our main hypothesis is that primate pluripotent cells experimentally derived using these two alternative approaches are equivalent to each other and to embryonic stem cells (ESCs) isolated from fertilized embryos. Another goal of this application is to evaluate, for the first time, the potential of monkey ESCs derived from fertilized or SCNT embryos and induced pluripotent (iPS) cells to generate chimeras upon injection into developing embryos. To achieve these goals we propose the following specific aims: 1). to create monkey pluripotent cells by epigenetic and genetic reprogramming of somatic cells. In Experiment 1, we will derive ESCs by SCNT from adult monkey skin cells and test our working hypothesis that experimental upregulation of critical pluripotent factors - OCT4, SOX2, NANOG and CARM1- in cytoplasts will enhance reprogramming and increase the current efficiency of SCNT embryo development and ESC isolation. In Experiment 2, we will generate monkey iPS cells from the same monkey skin cells by lentiviral transduction of genes encoding critical reprogramming factors OCT4, SOX2, KLF4, C-MYC, NANOG and LIN28 under control of doxycyclin-inducible promoters. 2). Examine pluripotency of novel pluripotent cells. Our working hypothesis here is that both SCNT and direct reprogramming can support complete reprogramming of somatic cells to the pluripotent state. To test this assumption, in Experiment 1 we will interrogate genetic, cytogenetic, epigenetic and transcriptional profiles of novel cell lines. In Experiment 2, cell lines will be subjected to in vivo differentiation in teratomas in SCID mice and to in vitro directed differentiation into mesoderm (cardiomyocytes), ectoderm (neuronal phenotypes) and endoderm (pancreatic beta-cells).3). Determine the potential of monkey pluripotent cells to contribute to chimeras. We hypothesize that similar to their mouse counterparts, primate ESCs and iPS cells have the potential to integrate and participate in development of chimeric offspring. To test this hypothesis, we propose to inject GFP-expressing monkey ESCs and iPS cells into monkey preimplantation embryos and transfer the resultant chimeric embryos into recipients to establish pregnancies. Chimeric fetuses and full-term offspring will subsequently be studied for tissue distribution and germ line colonization.