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

在制造组织工程和再生医学产品时，控制细胞分化是至关重要的。在体内，细胞分化受组织中生化和机械特征的模式支配。已经提出了许多技术来模拟这些模式，但它们的精度和可重复性需要改进。在这项工作中，研究小组的目标是建立一种新的技术来构建指导肠道干细胞分化的生化模式，这对理解胃肠道功能和疾病很重要。分化的细胞将被收集并用作构建肠壁模型的基石。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。这将反过来提供一个独特的工具箱来制定新的“生物材料配方”，用于制造“可再生医学”的细胞。为了扩大影响，这项研究的技术成果将被整合到学术课程中，通过比较医学研究所的新教程，并为生物技术专业人员创建关于细胞生产的特别课程。该研究团队还将与NIIMBL（美国制造业网络中的一个研究所）的研究和工业利益相关者联系，以评估其技术在治疗性细胞和组织工程中的应用。组织工程师最感兴趣的是干细胞的精确分化，干细胞被用作构建类器官和微生理模型的基石，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-MAPs），即通过配体介导的（非共价）吸附将细胞信号因子的3D梯度整合到聚合物基质中。这一概念将为多能肠干细胞的精确分化提供依据。因此，该项目将采用由胶原蛋白（COL）、BMP-2、Wnt-3A和锯齿状配体（刺激Notch通路）组成的生物分子调色板，这些配体在肠隐窝和绒毛中的交叉梯度已知可指导细胞分化。将开发单梯度Bio-MAP（恒定胶原+层粘连蛋白、BMP2或Wnt3梯度），以将Bio-MAP结构（即组合因子的浓度和梯度）与人类ISC及其后续谱系的粘附、增殖和分化联系起来，并确定维持茎样表型与直接向分泌（产生粘液）谱系或吸收性肠细胞分化的设计规则和模式。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），利用相干或相反信号因子的组合作为背景，对抗缺口结合配体（这里称为锯齿状配体（JLL））的三级呈现，以确定协同模式，指导分化向定量可预测的表型和基因型结果发展。互补的分析和细胞学技术将结合阐明选择的信号因子及其几何显示的基本协同机制。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.