Embryonic Stem Cell Research Career Enhancement

胚胎干细胞研究职业提升

• 批准号: 6806938
• 负责人: Kenneth S. Zuckerman
• 金额: $ 23.07万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6806938
• 关键词: biological signal transduction; bone morphogenetic proteins; cell biology; cell differentiation; human embryonic stem cell line; pluripotent stem cells; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): The purpose of this proposal is for Dr. Zuckerman to obtain intensive training in embryonic stem (ES) cell biology. He will train in the laboratory of Dr. Martin Pera in the Institute for Reproduction and Development at Monash University in Melbourne, Australia. Dr. Pera is a pioneer in human ES cell technology and a contributer of nearly half of the human ES cell lines that are approved by NIH and are currently available. All of the training and experiments done under this grant will utilize these NIH-approved ES cell lines (ES01- ES06). Training will include technical training in isolation, identification, and maintenance of ES cells in culture, techniques for inducing ES cell differentiation into cells of desired lineages, and methods of extrinsic DNA transfer into ES cells. There will be extensive guided and self-driven reading of ES cell and related scientific and ethical literature. There will be frequent discussions with Dr. Pera, weekly lab meetings for discussion of recent results, and frequent seminars on topics relevant to ES cell biology. There is a rich stem cell research environment in Melbourne. Hematopoietic stem cell biology pioneer Don Metcalf and his extended group are at the Walter and Eliza Hall Institute, and Paul Simmons and his group at the Peter MacCallum Cancer Institute work on stem cell plasticity. Until recently, human ES cells could only be grown and maintained in the undifferentiated pluripotent state over a mouse embryo fibroblast feeder cell layer. Bone Morphogenic Protein (BMP)-2 is produced by the ES cells and/or early differentiated progeny, especially under conditions of stress. BMP-2 drives differentiation of ES cells into endodermal cells. Gremlin, a DAN family member, which is upregulated by BMP-2, has been shown recently to be a relatively specific antagonist of BMP-2 and BMP-4. In preliminary studies, Dr. Pera found that feeder cells in ES cell cultures produce gremlin transcripts. We will test the hypothesis that gremlin, by interfering with BMP-2 function, may play a large role in the ability of the feeder cells to maintain ES cells in the undifferentiated state. We will examine the ability of gremlin to prevent ES cell differentiation in cultures with no feeder cells and the ability of "impotent" feeder cells to support ES cell self-renewal and growth after being transfected with gremlin cDNA and overexpressing gremlin protein. We also will determine whether inhibition of gremlin production abolishes the ability of a functional feeder cell layer to maintain ES cells in the undifferentiated state.

描述（由申请人提供）： 该提案的目的是让 Zuckerman 博士获得胚胎干 (ES) 细胞生物学方面的强化培训。他将在澳大利亚墨尔本莫纳什大学生殖与发育研究所马丁·佩拉博士的实验室接受培训。 Pera 博士是人类 ES 细胞技术的先驱，也是近一半经 NIH 批准且目前可用的人类 ES 细胞系的贡献者。在此资助下进行的所有训练和实验都将利用这些 NIH 批准的 ES 细胞系 (ES01-ES06)。培训将包括培养中 ES 细胞的分离、鉴定和维持方面的技术培训，诱导 ES 细胞分化为所需谱系细胞的技术，以及将外在 DNA 转移到 ES 细胞中的方法。将对 ES 细胞以及相关的科学和伦理文献进行广泛的引导和自我驱动阅读。我们将与 Pera 博士进行频繁的讨论，每周举行一次实验室会议来讨论最近的结果，并经常举办与 ES 细胞生物学相关主题的研讨会。墨尔本拥有丰富的干细胞研究环境。造血干细胞生物学先驱唐·梅特卡夫（Don Metcalf）和他的扩展小组在沃尔特和伊丽莎霍尔研究所（Walter and Eliza Hall Institute）工作，保罗·西蒙斯（Paul Simmons）和他在彼得·麦卡勒姆癌症研究所（Peter MacCallum Cancer Institute）的小组致力于干细胞可塑性。直到最近，人类 ES 细胞只能在小鼠胚胎成纤维细胞饲养细胞层上生长并维持未分化的多能状态。骨形态发生蛋白 (BMP)-2 由 ES 细胞和/或早期分化的后代产生，特别是在应激条件下。 BMP-2 驱动 ES 细胞分化为内胚层细胞。 Gremlin 是 DAN 家族成员，受 BMP-2 上调，最近被证明是 BMP-2 和 BMP-4 的相对特异性拮抗剂。在初步研究中，Pera 博士发现 ES 细胞培养物中的饲养细胞产生 gremlin 转录本。我们将检验这样的假设：gremlin 通过干扰 BMP-2 功能，可能在饲养细胞维持 ES 细胞处于未分化状态的能力中发挥重要作用。我们将检查 gremlin 在没有饲养细胞的培养物中阻止 ES 细胞分化的能力，以及“无能”饲养细胞在转染 gremlin cDNA 并过表达 gremlin 蛋白后支持 ES 细胞自我更新和生长的能力。我们还将确定抑制 gremlin 产生是否会消除功能性饲养细胞层维持 ES 细胞处于未分化状态的能力。