Identifizierung und funktionelle Untersuchung von humanen adulten spermatogonalen und germalen Stammzellen

我们的体外和体内实验已经表明，我们能够从富集的精原细胞产生人成体多能生殖系来源的干细胞（haGSC）的培养物（Conrad等人，2008年）。这些haGSC具有类似于多能胚胎干细胞（hES）的表达谱。haGSC可以在体外分化为所有三个胚层细胞，它们也形成畸胎瘤。这是第一个来自成人来源的多能干细胞，适用于人类，同时在伦理上没有争议。然而，目前，只有非常少的表面标志物可用于人和/或鼠成体精原干细胞（hSSC/mSSC）和haGSC的前瞻性分离，包括c-kit、Thy-1、α6-整联蛋白、β1-整联蛋白、CD 9和GFRa 1，其中大多数仅在小鼠中进行了测试。此外，这些标记物对精原细胞的选择性不高。目前采用的方法导致大量细胞损失，并且不仅富集hSSC，而且也富集参与精子发生的其他细胞。在拟议的项目中，我们计划开发用于高度纯化的hSSC和haGSC的分离和表征的新方法，利用我们的大型干细胞特异性抗体库，这些抗体是更有效分离这些细胞的有希望的候选者。最后，我们将使用单细胞分选来建立具有ES细胞样生长行为的克隆。一个中心目标是优化用于产生hSSC的培养和选择方法，以从小活检获得足够的细胞数量用于治疗应用。

Our in vitro and in vivo experiments have shown that we are able to generate cultures of human adult pluripotent germline-derived stem cells (haGSCs) from enriched spermatogonia (Conrad et al., 2008). These haGSCs possess expression profiles which resemble those of pluripotent embryonic stem cells (hES). haGSCs can be differentiated in vitro into cell of all three germ layers, and they also form teratomas. These are the first pluripotent stem cells from an adult source which are suitable for use in humans while at the same time being ethically non-controversial. At present, however, only a very few surface markers are available for the prospective isolation of human and/or murine adult spermatogonial stem cells (hSSCs/mSSCs) and haGSCs, including c-kit, Thy-1, α6-integrin, β1-integrin, CD9 and GFRa1, most of which have only been tested in the mouse. In addition, these markers are not highly selective for spermatogonia. Currently employed methods cause substantial loss of cells and enrich not only hSSCs, but other cells involved in spermatogenesis as well. In the proposed project, we plan to develop new methods for the isolation and characterization of highly purified hSSCs and haGSCs, taking advantage of our large library of stem cell-specific antibodies which are promising candidates for a more efficient isolation of these cells. Finally, we will use single cell sorting to establish clones with ES cell-like growth behavior. A central goal is to optimize the culture and selection methods for the generation of hSSCs to obtain sufficient cell numbers from small biopsies for therapeutic applications.