Embryonic Stem Cell Research Career Enhancement

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的莫纳什大学生殖与发育研究所的马丁·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NIH批准的近一半人类ES细胞系的贡献者。所有的训练和实验都将使用这些NIH批准的ES细胞系（ES 01-ES 06）。培训将包括分离、鉴定和维持培养中的ES细胞的技术培训，诱导ES细胞分化为所需谱系细胞的技术，以及将外源DNA转移到ES细胞中的方法。将有广泛的指导和自我驱动的阅读胚胎干细胞和相关的科学和伦理文献。Pera，每周一次的实验室会议，讨论最近的结果，并经常讨论与ES细胞生物学相关的主题。墨尔本有丰富的干细胞研究环境。造血干细胞生物学先驱Don Metcalf和他的扩展小组在Walter和Eliza Hall研究所，Paul Simmons和他的小组在Peter MacCallum癌症研究所从事干细胞可塑性研究。直到最近，人ES细胞只能在小鼠胚胎成纤维细胞饲养细胞层上生长并保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家族的成员，其被BMP-2上调，最近已被证明是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，即gremlin通过干扰BMP-2的功能，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。我们将研究gremlin在没有饲养细胞的培养物中阻止ES细胞分化的能力，以及用gremlin cDNA转染并过表达gremlin蛋白后“无能”饲养细胞支持ES细胞自我更新和生长的能力。我们还将确定是否抑制gremlin生产废除功能性饲养细胞层的能力，以维持ES细胞在未分化状态。

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.