Histocompatible Primate Embryonic Stem Cells

描述(申请人提供)：该项目的目标是利用体细胞核移植(SCNT)和直接重编程方法产生关于灵长类体细胞重编程到多能性状态的重要新见解，并使用表达谱、遗传和表观遗传学分析以及体外和体内分化分析进行相对多能性评估。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多能细胞彼此等价，并且相当于从受精胚胎中分离出来的胚胎干细胞(ESCs)。这项应用的另一个目标是首次评估来自受精或SCNT胚胎的猴子ESCs的潜力，并诱导多能(IPS)细胞在注射到发育中的胚胎时产生嵌合体。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新编程来创造猴子的多能细胞。在实验1中，我们将通过SCNT从成年猴子皮肤细胞中分离出ESCs，并验证我们的工作假设，即在细胞质中上调关键的多潜能因子OCT4、SOX2、NANOG和CARM1将增强重新编程，并提高SCNT胚胎发育和ESC分离的当前效率。在实验2中，我们将在多西环素诱导启动子的控制下，通过慢病毒转导编码关键重编程因子OCT4、SOX2、KLF4、C-MYC、NANOG和LIN28的基因，从相同的猴子皮肤细胞中产生猴iPS细胞。2)。检测新的多能细胞的多能性。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到多能性状态。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、细胞遗传学、表观遗传学和转录图谱。在实验2中，将对SCID小鼠的畸胎瘤细胞系进行体内分化，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2细胞)。确定猴子多能细胞对嵌合体的贡献潜力。我们推测，与小鼠类似，灵长类ESCs和iPS细胞具有整合和参与嵌合子代发育的潜力。为了验证这一假设，我们建议将表达GFP的猴子ESCs和iPS细胞注射到猴子的植入前胚胎中，并将产生的嵌合胚胎移植到受体体内，以建立怀孕。随后将研究嵌合胎儿和足月后代的组织分布和生殖系定植。公共卫生相关性：在这项应用中，我们将研究在非人类灵长类动物模型中通过两种替代方法获得的自体(患者匹配的)干细胞的潜力。这些实验的结果反过来将允许生产有用的临床前猴子模型，用于测试涉及自体干细胞的治疗应用，以治疗广泛的退行性疾病。

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 2-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. PUBLIC HEALTH RELEVANCE: In this application we will study the potential of autologous (patient-matched) stem cells derived by two alternative approaches in the nonhuman primate model. The outcomes of these experiments, in turn, will allow the production of useful preclinical monkey models for testing therapeutic applications involving autologous stem cells for the treatment of wide range of degenerative diseases.