Isolation and Characterization of Testis Stem Cells

描述（申请人提供）：我们的长期目标是研究精原干细胞的生物学，A/单个精原细胞。人们对A/单精原细胞非常感兴趣，因为它们对于理解细胞自我更新与分化的基本机制，对于不孕症的治疗，对于避孕药的开发以及对于睾丸癌，特别是精原细胞瘤的病因学的理解具有重要意义。然而，对这些细胞的生物学研究受到严重阻碍，因为它们数量少，没有独特的标记存在，并且很难从其他更成熟的精原细胞中物理分离它们。我们最近开发了一种方法，使我们能够分离出a /单个精原细胞的纯种群，我们已经开始在体外研究它们的行为。利用微阵列技术，我们还分析了A/单精原细胞在胶质细胞系来源的神经营养因子（GDNF）存在下的转录程序，GDNF是一种由支持细胞产生的生长因子。在第一个目的中，我们将分离单个精原细胞并通过生殖细胞移植评估其克隆潜能。在第二个目标中，我们将确定哪些信号通路是由GDNF诱导的，导致单个精原细胞自我更新或分化。在第三个目标中，我们将研究GDNF如何与其他生长因子/受体相互作用以促进自我更新或分化。

DESCRIPTION (provided by applicant): Our long-term goal is to study the biology of spermatogonial stem cells, the A/single spermatogonia. There is great interest in the A/single spermatogonia because of their importance for understanding basic mechanisms of cell self-renewal versus differentiation, for the treatment of infertility, for the development of contraceptives and for the understanding of the etiology of testicular cancer, in particular seminoma. However, studies on the biology of these cells have been severely hampered because their number is low, no unique marker exists and it has been difficult to physically separate them from the other more mature spermatogonia. We recently developed a method that allows us to isolate pure populations of A/single spermatogonia, and we have started to study their behavior in vitro. Using microarrays, we also have analyzed the transcriptional program of the A/single spermatogonia in the presence of glial cell line-derived neurotrophic factor (GDNF), a growth factor produced by Sertoli cells. In the first aim, we will isolate Asingle spermatogonia and evaluate their clonogenic potential by germ cell transplantations. In the second aim, we will determine which signaling pathways are induced by GDNF, leading to Asingle spermatogonia self-renewal or differentiation. In the third aim, we will study how GDNF interacts with other growth factors/receptors to promote self-renewal or differentiation. In summary, this line of work will expand our knowledge of the functional characteristics of the A/single spermatogonia and give us better insight into the molecular mechanisms that drive the first steps of spermatogenesis. In particular, the role of the growth factor GDNF in regulating self-renewal and/or differentiation will be elucidated.