IMAGING NHES CELS IN VITRO, IN CHIMERAE, AND AFTER TRANSFER

NHES 细胞在体外、嵌合体内和转移后的成像

• 批准号: 7716538
• 负责人: GERALD SCHATTEN
• 金额: $ 62.33万
• 依托单位: MAGEE-WOMEN'S RES INST AND FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7716538
• 关键词: Allogenic; Allografting; Autologous Transplantation; Back; Cells; Chimera organism; Cloning; Collaborations; DNA Methylation; Derivation procedure; Development; Embryo; Embryo Transfer; Epigenetic Process; Female; Fertilization in Vitro; Fetal Development; Fetus; Generations; Genes; Genomic Imprinting; Germ Layers; Germ Lines; Hematopoietic stem cells; Heterogeneity; Image; Immune; In Vitro; Inflammation; Injection of therapeutic agent; Inner Cell Mass; Intracytoplasmic Sperm Injections; Invasive; Knowledge; Label; Macaca mulatta; Microscopy; Monkeys; Mus; Necrosis; Neurons; Normalcy; Oocytes; Outcome; Partner in relationship; Placenta; Pregnancy; Primates; Safety; Site; Somatic Cell; Staging; Stem cell transplant; Teratoma; Testing; Therapeutic; Time; Tissues; Transgenes; Transplantation; Undifferentiated; Vascularization; Week; blastocyst; day; embryo cell; embryo/fetus; embryonic stem cell; fetal; human embryonic stem cell; immunosuppressed; implantation; imprint; in utero; in utero transplantation; in vivo; male; natural Blastocyst Implantation; nonhuman primate; nuclear transfer; pluripotency; skin allograft; stable cell line; stem

Project I investigates embryonic stem cell pluripotency directly through the generation of chimeric primate blastocysts, fetuses and offspring with nonhuman primate ES cells (nhpES) - including nhpES derived from blastocysts generated from nuclear transfer (NTnhpES). Through sophisticated non-invasive imaging, this project responsibly answers crucial questions about: primate ES pluripotency during development (aim I); primate ES cell stability and utility after NT (aim II); genomic imprinting in NHPs to solve clinically urgent concerns regarding ART epigenetic consequences; and primate ES fate after transplantation (aim IV). Nine questions are posed in four Specific Aims: Aim I. To dynamically image nhpES cell contributions in developing primate chimerae: 1.1. Will nhpES cells contribute to chimeric blastocysts, fetuses and healthy offspring? 1.2.With tetraploid embryos (4N), will ES cells contribute primarily to the inner cell mass in chimeric blastocysts, fetus and offspring? Primate chimera are generated in four ways, and the fate of each chimera is followed in vitro during preimplantation development, determining the cellular contributions of the ES and tetraploids to the expanded blastocyst stage after differentially labeling ES or embryos with GFP-transgenes, as well as in utero during fetal development and in the offspring. Aim II. Are ES cells derived after nuclear transfer (NTnhpES cells) developmentally restricted? Two questions are posed: 2.1. Are NTnhpES cell lines stable? 2.2 Will NTnhpES cells contribute to chimeric fetuses and offspring? Aim III. Do Genomic Imprints in NHP Embryos, Fetuses, Placentae, Amniotic Cells and Offspring Differ between ART and NT-Chimerae versus Natural Matings? 3.1. What is the level of DNA methylation in embryos, fetuses, placentae, and offspring after ART, NT, or natural matings? 3.2. What are the parental expressions of selected imprinted genes during NHP preimplantation development? 3.3. What is the DNA methylation status of specific genes in rhesus preimplantation embryos? Aim IV. Dynamic imaging of ES cell fates after transplantation. 4.1. Are NT-derived nhp-ESCs tolerated when transplanted back into the female from which both the somatic cell and the oocyte were obtained? 4.2 Are allogenic ES cells rejected like allografts or do their pre-implantation origins confer immunological privilege? This aim will investigate the tolerance of nhpES in allografts and autografts. Taken together, this project will provide crucial information regarding ES cell pluripotency in nonhuman primates and evaluate the safety of stem cell transplantation in nonhuman primates.

项目I直接通过产生嵌合灵长类囊胚来研究胚胎干细胞的多能性， 具有非人灵长类胚胎干细胞(NhpES)的胎儿和后代--包括来自胚泡的nhpES 来自核移植(NTnhpES)。通过复杂的非侵入性成像，该项目负责任地回答 关键问题：灵长类胚胎在发育过程中的多能性(目标I)；灵长类胚胎干细胞的稳定性和在 NT(AIM II)；在NHP中进行基因组印记，以解决临床上对ART表观遗传后果的迫切关注； 和灵长类动物移植后的命运(目标IV)。在四个具体目标中提出了九个问题：目标一至 动态成像nhpES细胞在灵长类嵌合体发育中的作用：1.1。NhpES细胞是否会对 嵌合胚泡、胎儿和健康后代？1.2对于四倍体胚胎(4N)，ES细胞是否有贡献 主要是嵌合胚泡、胎儿和后代中的内细胞团？灵长类嵌合体产生于四个 方法和每个嵌合体的命运在体外被跟踪，在植入前发育期间，决定细胞 ES和四倍体在ES或胚胎差异标记后对扩张囊胚期的作用 GFP-转基因，以及在胎儿发育期间和后代的子宫内。目的II.胚胎干细胞是不是在 核移植(NTnhpES细胞)发育受限？提出了两个问题：2.1。是NTnhpES细胞吗？ 品系稳定？2.2 NTnhpES细胞会对嵌合胎儿和后代做出贡献吗？AIM III.基因组印记在 NHP胚胎、胎儿、胎盘、羊膜细胞和后代在ART和NT-Chimerae之间存在差异 天然交配？3.1.接受抗逆转录病毒治疗后，胚胎、胎儿、胎盘和后代的DNA甲基化水平是多少？ NT，还是自然配对？3.2.选择的印记基因在NHP过程中的父母表达是什么 植入前发育？3.3.恒河猴着床前特定基因的DNA甲基化状态如何 胚胎？目的IV.移植后ES细胞命运的动态成像。4.1.是否容忍NT来源的NHP-ESCs 当移植回同时获得体细胞和卵母细胞的雌性体内时？4.2 同种异体ES细胞像同种异体移植一样被排斥，还是它们的植入前来源授予免疫特免权？这一目标 将调查nhpes在同种异体移植物和自体移植物中的耐受性。综上所述，该项目将提供至关重要的 非人灵长类中ES细胞多能性的信息和干细胞移植的安全性评估 非人灵长类动物。