Improving Endoderm Specification with Hybrid Materials and Growth Factors

描述(申请人提供)：在过去的十年里，干细胞生物学和材料科学领域的独特交叉产生了许多关于干细胞与其周围生态位之间相互作用的关键观察结果。除了研究良好的生长因子外，细胞外基质的特定内在特性似乎足以启动干细胞分化，例如，更坚硬的底物产生成肌样和成骨细胞样的细胞类型。然而，这些材料通常只表现出单一的固有基质属性，或者它们缺乏适当的纤维结构，与生长因子的结合，或者在发育过程中在基质中发现的时空变化。因此，使用这些合成材料的工程化组织的用途在某种程度上受到了限制。考虑到确定性内胚层来源的组织在空间和时间上的相对复杂性，例如消化道、肝脏等，更谨慎的初始方法可能是通过与最终内胚层的产生最密切相关的内在基质属性来更好地模拟生态位。利用与生理相关的生长因子信号和基质特性的组合，将监测胚胎干细胞(ESCs)的内胚层规格，以确定这些信号或“设计标准”中的哪一组最有效。然后，我们将把我们对这些标准的理解整合到两种“智能”的自然-合成混合生物材料中：1)硫代化的透明质酸和纤维连接蛋白基质，与聚乙二醇二丙烯酸酯的时间敏感交联相结合，以产生这些固有特性的时间变化；2)互穿聚合物-纤维连接蛋白网络，以创建具有特定固有基质特性的空间梯度和特征。与目前的生物材料相比，这两种材料都将创造更复杂的微环境，并更好地模拟内胚层生态位，这可能会促进胚胎干细胞向内胚层的分化。力映射光谱等新技术将有助于我们对这些材料的生物物理和生化特性进行表征，并将反馈到材料设计中，以创建最适合ESCS的自然-合成混合基质。这些见解将为支架提供第一套明确的设计标准，并开发一种创造未来治疗生物材料的过程。 与公共卫生相关：超过3000万美国人患有某种形式的特定内胚层衍生器官的慢性功能障碍，例如消化道、肾脏、肝脏等，鉴于缺乏捐赠器官，开发长期器官替代策略的必要性至关重要。工程化组织已被提出作为应对这一危机的一种手段，即胚胎干细胞将被培养成组织，然后移植到体内以缓解功能障碍。然而，以前的尝试在开发这种组织方面取得的成功有限，可能是因为它们没有解决周围环境的内在属性，众所周知，周围环境的内在属性会将干细胞引导到特定类型的成人组织中。

DESCRIPTION (provided by applicant): Over the past decade, the unique intersection of the fields of stem cell biology and material science have produced a number of key observations about the interaction between stem cells and their surrounding niche. In addition to well-studied growth factors, specific intrinsic properties of the extracellular matrix appear to be sufficient to initiate stem cell differentiation, e.g. more rigid substrates produce myoblast- and osteoblasts-like cell types. However, these materials typically display only a single intrinsic matrix property or they lack the appropriate fibrillar structure, combination with growth factors, or spatiotemporal variations found in matrix during development. As a result, the utility of engineered tissues using these synthetic materials have been somewhat limited. Given the relative spatial and temporal complexity of definitive endoderm-derived tissues, e.g. digestive tract, liver, etc., a more prudent initial approach may be to better mimic the niche via the intrinsic matrix properties that are most germane to the production of definitive endoderm. Using physiologically-relevant combinations of growth factor signals and matrix properties, embryonic stem cells (ESCs) will be monitored for endoderm specification to determine which set of these cues or "design criteria" is most effective. We will then integrate our understanding of these criteria into two "smart," natural-synthetic hybrid biomaterials: 1) a thiolated hyaluronic acid and fibronectin matrix coupled to time-sensitive crosslinking from poly(ethylene glycol)-diacrylate to yield temporal variations of these intrinsic properties and 2) an interpenetrating polymer- fibronectin network to create spatial gradients and features having specific intrinsic matrix properties. Both materials will create dramatically more complex microenvironments compared to current biomaterials as well as better mimic the endoderm niche, which may improve ESC-to-endoderm differentiation. Novel techniques, such as force mapping spectroscopy, will aid in our characterization of these materials' biophysical and biochemical properties and will feedback into material design to create natural-synthetic hybrid substrates that are best-suited for ESCs. These insights will provide both a first set of clear-cut design criteria for the scaffolds as well as develop a process to create future therapeutic biomaterials. PUBLIC HEALTH RELEVANCE: Over 30 million Americans suffer from some form of chronic dysfunction of a definitive endoderm-derived organ, e.g. digestive tract, kidney, liver, etc., and given a lack of donor organs, the need to develop long-term organ replacement strategies is vital. Engineered tissues have been proposed as a means of dealing with this crisis, where embryonic stem cells would be grown into a tissue and subsequently implanted in the body to alleviate the dysfunction. Previous attempts, however, have had limited success in developing such tissues, likely since they do not address the intrinsic properties of the surrounding environment, which are known to direct stem cells into specific types of adult tissues.