Identifizierung und funktionelle Untersuchung von humanen adulten spermatogonalen und germalen Stammzellen

我们的体外和体内实验表明，我们能够从浓缩的精原细胞中培养出成人多能生殖系来源的干细胞(HaGSCs)(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。HAGSCs在体外可分化为三种胚层的细胞，并形成畸胎瘤。这是第一批来自成人来源的多能干细胞，适合在人类身上使用，同时在伦理上没有争议。然而，目前用于分离人和/或小鼠成体精原干细胞和hGSCs的表面标志物很少，包括c-Kit、Thy-1、α6-整合素、β1-整合素、CD9和GFRa1，其中大部分只在小鼠身上进行了试验。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的细胞损失，并且不仅富含hSSCs，而且还富含参与精子发生的其他细胞。在拟议的项目中，我们计划开发新的方法来分离和鉴定高纯度的hSSCs和haGSCs，利用我们庞大的干细胞特异性抗体库，这些抗体库有望更有效地分离这些细胞。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hSSCs的培养和选择方法，以便从小切片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.