Stem Cell Derivation from Germ Cells

描述(申请人提供)：原始生殖细胞(PGCs)是配子的胚胎前体，正确发育是成年生育的先决条件。PGCs在胚胎和胎儿中不能正确地存活、增殖或分化，可能会导致成年动物的不孕。在某些情况下，正常的PGC分化受到干扰，PGC形成多能干细胞，称为胚胎癌细胞(EC)。这些继而形成睾丸肿瘤、畸胎瘤或畸胎癌，这是年轻人最常见的癌症形式。目前对引导正常PGC分化的分子机制以及在睾丸癌变过程中受到干扰的分子机制知之甚少。在体外，在Kit配体(KL)和白血病抑制因子(LIF)的存在下培养的PGCs可以像在体内一样长时间地增殖。但是，当加入碱性成纤维细胞生长因子(bFGF或FGF2)时，PGCs继续增殖，并产生被称为胚胎生殖细胞(EG细胞)的多能干细胞。这一过程模拟了体内睾丸肿瘤的形成过程，在此过程中，PGCs产生了多能EC细胞。虽然EC和EG细胞是多能的，但我们的初步数据显示，PGCs不能形成任何其他类型的细胞，因此被认为是无能的。因此，将PGC转换为EC或EG细胞代表了从零能力状态到多能力状态的转换。在体外用碱性成纤维细胞生长因子控制PGC活性的能力为研究哺乳动物发育潜力的控制提供了一个独特的系统。我们设计了一种新的逆转录病毒基因转移系统来剖析和操纵KL、LIF和bFGF激活的PGCs信号通路，以确定这些通路在干细胞发育中的相对重要性。了解控制PGC转化为多能干细胞的分子机制将填补我们在发育潜能调节、睾丸肿瘤形成和生殖系发育方面的知识空白，并产生关于干细胞生理学的一般信息。该提案的具体目的是：i)确定bFGF在改变PGCs发育潜力中的作用，ii)确定bFGF在干细胞发育中的作用方式，iii)确定bFGF在体内多能干细胞形成中的作用，以及iv)确定bFGF暴露后PGCs中上调的基因。

DESCRIPTION (provided by applicant): Correct development of Primordial Germ Cells (PGCs), the embryonic precursors of the gametes, is a prerequisite for adult fertility. Failure of PGCs to survive, proliferate or differentiate correctly in the embryo and fetus can result in sterility in the adult animal. In some cases normal PGC differentiation is perturbed and PGCs form pluripotent stem cells, termed Embryonal Carcinoma (EC) cells. These in turn form testicular tumors, teratomas or teratocarcinomas, which are the most common form of cancer in young men. Little is known about the molecular mechanisms responsible for guiding normal PGC differentiation and which are perturbed during testicular carcinogenesis. In vitro, PGCs cultured in the presence of Kit ligand (KL) and leukemia inhibitory factor (LIF) proliferate as long as they would in vivo. But when basic Fibroblast growth factor (bFGF or FGF2) is added, PGCs continue to proliferate and give rise to pluripotent stem cells termed Embryonic Germ (EG) cells. This process mimics the formation of testicular tumors in vivo in which PGCs give rise to pluripotent EC cells. Although EC and EG cells are pluripotent, our preliminary data show that PGCs cannot form any other cell type and are therefore considered nullipotent. Thus the conversion of a PGC into an EC or EG cell represents a conversion from a nullipotent to a pluripotent state. The ability to manipulate PGC potency in vitro with bFGF provides a unique system with which to study the control of developmental potency in mammals. We designed a novel retroviral gene transfer system to dissect and manipulate the signaling pathways activated in PGCs by KL, LIF and bFGF to determine the relative importance of these pathways in stem cell development. Understanding the molecular mechanisms controlling the conversion of a PGC into a pluripotent stem cell will fill gaps in our knowledge of developmental potency regulation, testicular tumor formation and germline development as well as generating general information about stem cell physiology. The Specific Aims of the proposal are: i) To determine the role of bFGF in altering developmental potential of PGCs, ii) To define the mode of action of bFGF in stem cell development, iii) To define the role of bFGF in pluripotent stem cell formation in vivo and iv) To identify genes up-regulated in PGCs following exposure to bFGF.