胚胎干细胞表面的仿生聚合物修饰诱导其定向分化的研究

Embryonic stem cells (ESCs) are pluripotent cells capable of differentiating into any somatic cell type. ESCs have emerged as one of the most promising renewable sources applied in the field of cell therapy and tissue transplantation for the treatment of diseases. Therefore, discovering a great strategy for efficiently promoting ESCs into specific cell types in short term is the key of cell replacement therapy. Natural polysaccharides with biological activities have been extensively regarded as important regulators of ESCs behaviors. However, the development of the influence on ESCs behaviors has been restricted due to the heterogeneity of natural polysaccharides (structure, species diversity, etc). In this project, we draw up a strategy of preparing different types of biomimetic polysaccharides with various precisely controlled structures and molecular weights utilizing highly-efficient polymerization, combining with cell surface engineering to insert the synthesized polymers into cell membranes, as if coating a layer of well-defined biomimetic polysaccharides, based on which, the synthetic methodology of functional polysaccharides analogues will be investigated systematically, along with the optimization of polysaccharides’ anchoring mode. Furthermore, we will explore the effects of the structures, molecular weights and any other factors of exogenous biomimetic polysaccharides inserted into cell membranes on ESCs, deeply analyzing the inherent mechanism of direct differentiation of ESCs induced by biomimetic polysaccharides, and reveal the structural features of polymers capable of efficiently promoting ESCs differentiate into specific cell types in short term. The outcome of this project will not only contribute to illustrate the mechanism of stem cells behaviors influenced by polysaccharides, but also provide reliable theory guidance for the cell therapy.

胚胎干细胞（ESCs）能在一定条件下分化成所有类型的体细胞，在细胞治疗和组织移植方面具有广阔的应用前景，而发展一种在短期内诱导ESCs定向高效分化的方法则是实现其应用的关键。天然聚糖是一类能够调控ESCs分化行为的重要物质，但是由于其结构多样性和复杂性，限制了人们研究该类物质的种类和结构等因素对ESCs行为的影响规律。本项目拟通过可控聚合方法制备系列结构和分子量精确可控的聚糖类似物，并结合细胞表面工程将聚合物插入到ESCs膜。将系统研究聚糖类似物的合成方法学，优化聚合物在细胞膜上的锚定方式，探索引入到细胞膜的外源性功能聚糖类似物的结构及分子量等因素对ESCs行为的影响规律，深入剖析聚糖类似物影响ESCs定向分化的内在机制，揭示短期内能诱导ESCs定向高效分化成特定细胞类型的分子结构特点。该研究不但将有助于理解天然聚糖影响干细胞行为的作用机制，还将为基于ESCs的细胞疗法提供可靠的理论指导。

本项目按计划成功完成了“胚胎干细胞表面的仿生聚合物修饰诱导其定向分化的研究”的研究目标。在糖聚物的结构设计和对胚胎干细胞以及其他细胞功能调控方面开展了系统的研究工作，具体如下：1）制备了一系列具有特定结构的生物活性糖聚合物，通过将糖单元、带有磺酸基团的单元以及其他功能性单元进行拆分和重组，利用可逆加成断裂链转移（RAFT）聚合，通过简单地改变单体的投料比以及聚合反应时间来调控糖聚物的组成以及分子量。2）采用RAFT聚合和聚合物后修饰（PPM）相结合的方式，制备了末端修饰磷脂基团的天然糖胺聚糖的类似物、多胆固醇基团接枝的糖聚物分子以及能与跨膜蛋白Halo-tag对接的氯代烷烃修饰的生物活性分子，为满足不同的细胞膜改性需求，提供多元化的手段。3）通过倒置荧光成像、流式细胞技术、荧光相干光谱等技术对细胞表面引入的聚合物进行定量表征，验证了不同锚定基团官能化的聚合物对细胞膜表面改性的可行性。4）深入研究了糖聚物改性诱导胚胎干细胞分化，以及内皮细胞、肿瘤细胞和免疫细胞增殖、分化以及迁移等生物学行为的影响。本项目系统性的研究了外源性功能糖聚物的结构及分子量等因素对细胞生物学行为的影响规律。该研究不但有助于理解天然聚糖影响细胞行为的作用机制，还将为基于胚胎干细胞的细胞疗法提供可靠的理论指导。