胚胎干细胞（hESCs）体外培养易无序分化，较难维持"干性"。研究表明：hESCs内细胞命运决定子Numb的表达及分布是影响hESCs分裂方式和"干性"的基础。申请者前期发现：我们研制的无血清替代物SSR较常规血清替代物KSR更能维持hESCs"干性"，经12次传代核型仍正常，伴Numb低表达和Notch高表达。提示SSR可能通过调节Numb/Notch影响hESCs"干性"。本项目拟在SSR体系中持续传代hESCs，观察其干性及Numb/Notch的表达变化；采用基因沉默及过表达技术，观察Numb、Notch及下游信号分子的变化，通过体外增殖分化及免疫缺陷鼠的体内研究，验证hESCs在体内外"干性"的维持，以期明确Numb/Notch通路在SSR培养体系中维持hESCs"干性"的机制。为体外培养扩增符合研究所需hESCs奠定实验基础，为研制、优化hESCs无血清培养体系提供借鉴及新思路。

Human embryonic stem cells (hESCs) are prone to differentiate when growing in serum-free media and difficult to maintain stemness in vitro. It was reported that the expression and asymmetric localization of cell-fate determinants Numb were the biological foundation of hESCs asymmetric mitosis, which plays a key role in maintaining the self-renewal of hESCs. We have previously developed a new self-made serum-free culture replacer(SSR) that is ideal for many stem cell culture. Our recent trial study further confirmed that SSR is better than the traditional knockout serum replacer (KSR) in maintaining the biological properties of hESCs. In SSR culture system, hESCs keep normal karyotype after being passaged for 12 times accompanied with lower expression of Numb and higher expression of Nocth1. These results strongly suggested that Numb/Notch signaling pathway could regulate the differentiation of hESCs. Therefore, the current proposal aims to further optimize the SSR for hESCs culture and probe the roles of Numb/Notch1 pathway in hESCs cultured in SSR system. To fulfill the goals, hESCs will be passaged continuously in SSR system and the change of Numb/Notch expression will be dynamically monitored; RNA interference and gene recombination techniques will be used to interfere with Numb or Notch, and the consequences of which will be investigated, as well as the localization of Numb protein in hESCs. We will also examine the downstream elements in Notch signaling system. Moreover, the proliferation and differentiation capacities of hESCs are to be investigated in vitro and in immunodeficient mouse. We believe this study will help elucidate the concrete mechanisms of Numb/Notch in maintaining the properties of hESCs as stem cells and provide experimental basis for obtaining large number hESCs for investigation and a new thought for developing or optimizing serum-free culture medium for hESCs.