Identifizierung und funktionelle Untersuchung von humanen adulten spermatogonalen und germalen Stammzellen

我们的体外和体内实验表明，我们能够从富集的精原细胞中培养成人多能生殖系来源的干细胞（haGSCs） （Conrad et al., 2008）。这些haGSCs具有类似于多能胚胎干细胞（hES）的表达谱。haGSCs可以在体外分化为所有三种胚层的细胞，它们也可以形成畸胎瘤。这是第一个来自成人来源的多能干细胞，适合用于人类，同时在伦理上没有争议。然而，目前仅有极少数表面标记物可用于人类和/或小鼠成年精原干细胞（hSSCs/ mscs）和haGSCs的预期分离，包括c-kit、Thy-1、α6-整合素、β1-整合素、CD9和GFRa1，其中大多数仅在小鼠中进行了测试。此外，这些标记对精原细胞的选择性不高。目前使用的方法会导致大量细胞损失，不仅会使hsc富集，还会使参与精子发生的其他细胞富集。在这个项目中，我们计划开发新的方法来分离和表征高纯度的hsc和haGSCs，利用我们庞大的干细胞特异性抗体库，这些抗体是更有效地分离这些细胞的有希望的候选物。最后，我们将使用单细胞分选建立具有胚胎干细胞样生长行为的克隆。中心目标是优化hsc的培养和选择方法，以从小活检中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.