Bio-MAPS: BioMolecular-Array Patterns for Precision Differentiation of Intestinal Stem Cells

在生产组织工程和再生医学的产品时，控制细胞分化是至关重要的。在体内，细胞分化是由组织中的生化和机械特征模式决定的。已经提出了许多技术来模拟这些图案，但它们的精确度和重复性需要改进。在这项努力中，研究小组的目标是建立一种新的技术，以建立指导肠道干细胞分化的生化模式，这对了解胃肠功能和疾病非常重要。分化的细胞将被收获并用作构建肠壁模型的积木。这一努力的结果将改变我们对组织生物化学如何决定肠道细胞行为的理解。这反过来将提供一个独特的工具箱，为制造“再生医学准备”的细胞制定新的“生物材料配方”。为了扩大影响，这项研究的技术成果将被纳入学术课程，通过比较医学研究所提供新的教程，并为生物技术专业人员创建关于细胞生产的特别课程。研究小组还将与美国制造业网络中的NIIMBL研究所的研究和行业利益相关者联系，以评估他们的技术在治疗细胞和组织工程中的使用。组织工程师最感兴趣的是干细胞的精确分化，干细胞被用作构建有机物和微生理模型的构建块，用于研究发育生物学、病理学和药物发现。该项目的细胞分化材料方法引入了生物分子阵列模式(Bio-MAP)，即通过配体介导(非共价)吸附在聚合物基质中整合的细胞信号因子的组合3D梯度。这一概念将被证明用于多潜能肠道干细胞的精确分化。因此，该项目将采用由胶原(Col)、BMP-2、WNT-3A和锯齿状配体(刺激Notch途径)组成的生物分子调色板，其在肠隐窝和绒毛中的交叉梯度被认为是指导细胞分化的。将开发单梯度Bio-map(恒定胶原蛋白加梯度的层粘连蛋白、BMP2或WNT3)，以将Bio-map结构(即组合因子的浓度和梯度)与人ISC及后续谱系的黏附、增殖和分化相关联，并确定保持干细胞表型的设计规则和模式，而不是直接分化为分泌(粘液)谱系或可吸收的肠细胞。该项目将开发复杂梯度生物图谱(COL、BMP-2、WNT-3和锯齿状配体)，其中一致或相反的信号因子的组合被用作Notch结合配体--这里是锯齿状配体(JLL)--的三级呈现的背景，以确定协同模式，以指导分化走向定量可预测的表型和基因类型结果。互补的分析和细胞学技术将结合起来阐明选定信号因子及其几何显示的基本协同机制。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Controlling cell differentiation is critical when manufacturing products for tissue engineering and regenerative medicine. In the body, cell differentiation is governed by patterns of biochemical and mechanical features in tissues. Numerous techniques have been proposed to mimic these patterns, but their precision and reproducibility need improvement. In this effort, the research team aims to establish a new technology to build biochemical patterns directing the differentiation of intestinal stem cells, which are important for understanding gastrointestinal function and disease. Differentiated cells will be harvested and used as building blocks to construct models of the gut wall. The results of this effort will transform our understanding of how tissue biochemistry determines cell behaviors in the intestine. This will in turn provide a unique toolbox to formulate new “biomaterial recipes” for manufacturing cells that are “regenerative medicine-ready." To broaden impact, the technological outcomes of this research will be integrated in academic curricula, new tutorials via the Comparative Medicine Institute, and create ad hoc courses on cell production for biotech professionals. The research team will also connect with research and industrial stakeholders at NIIMBL, an institute in the Manufacturing USA network, to evaluate the use of their technology for the engineering of therapeutic cells and tissues.Of paramount interest to tissue engineers is the precise differentiation of stem cells that are utilized as building blocks to construct organoids and microphysiological models for investigating developmental biology, pathology, and drug discovery. The project's approach to cell differentiation materials introduces Bio-Molecular-Array Patterns (Bio-MAPs), i.e., combined 3D gradients of cell signaling factors integrated within polymer matrices via ligand-mediated (non-covalent) adsorption. This concept will be demonstrated for the precision differentiation of multi-potent intestinal stem cells. Accordingly, the project will adopt a biomolecular palette comprising collagen (COL), BMP-2, Wnt-3A, and Jagged-like ligands (which stimulate the Notch pathway), whose intersecting gradients in the intestinal crypt and villus are known to direct cell differentiation. Single-gradient Bio-MAPs (constant collagen plus gradients of laminin, BMP2, or Wnt3) will be developed to correlate Bio-MAP architecture (i.e., concentration and gradients of combined factors) to the adhesion, proliferation, and differentiation of human ISC and subsequent lineages and to identify design rules and patterns that maintain stem-like phenotypes vs. direct differentiation towards secretory (mucus-producing) lineages or absorptive enterocytes. The project will develop complex-gradient Bio-MAPs (COL, BMP-2,Wnt-3, and Jagged-like ligands), where combinations of coherent or opposing signaling factors are utilized as backgrounds against tertiary presentation of Notch-binding ligands - herein, Jagged-like ligand (JLL) - to identify synergistic patterns that direct the differentiation towards quantitatively predictable phenotypic and genotypic outcomes. Complementary analytical and cytological techniques will be coupled to elucidate fundamental synergistic mechanisms of the select signaling factors and their geometric display.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.