Extracellular matrix regulation of differentiation via modulation of ILK: application to 3D bioprinting of cardiac tissue

项目总结 这一建议的主要目标是将机械的洞察力与区分结果与 通过在焦点粘连(FA)处触发的细胞内信号事件进行ECM参与，b)3D打印 细胞外基质和细胞外基质相关蛋白作为一种手段来指导细胞成熟时的分布，从而使 制造厚的、有功能的心脏组织。这项提议意义重大，因为它有可能产生 替代组织，甚至心脏移植，为患有急性和慢性心脏损伤的个人。它 是第一个解决焦点粘连相对于 无数不同的ECM/整合素组合以及相应的各种细胞行为 ECM接洽。它通过提出FA的元件，即整合素连接激酶(ILK)和 相关的磷酸酶，作为敏感的变阻器，可以被增选来产生所需的行为。在这里， 期望的行为是心脏分化，而创新是使用优化的ECM配方 作为生物墨水创建3D心脏组织模拟物(3DCTM)，能够指导多个细胞的细胞分布 分化类型。这一概念是可行的，因为奥格尔实验室长期以来一直在研究 与细胞外基质接触相关的细胞外基质和干细胞行为，麦克阿尔卑斯实验室关注 关于3D打印功能材料的一系列应用，从生物到电子和合并 这些材料。这些小组还将与坎普实验室的专业人员就其最近的 诱导心脏祖细胞(ICPC)的生成以填充3DCTM，普罗文扎诺实验室协助 利用与FA形成相关的分子机制，Garry实验室协助生物反应器 实施，张实验室增加心血管生理学专业知识，Talkachova实验室协助 用光学成像评估3DCTM的功能和ECM的质谱学核心设施 调节ILK活性的组件和基因编辑工具。共同努力，这些专业知识将被输送到 通过以下目标实现提案的主要目标：1)确定是否激活 粘连或胞囊的整合素连接的激酶(ILK)将整合素的激活与β-连环蛋白的激活偶联 通过Gsk3ECM实现与心肌细胞特性相关的基因表达，2)使用基于3DECM的β 识别支持内皮细胞分化的ECM配方并评估ILK的模型系统 依赖，以及3)使用基于ECM的3D打印在空间上调节心肌细胞类型的分化 心脏组织模仿。这一概念的动机是基于广泛的文献搜索和结果 来自我们自己的实验；该方法旨在确保对结果的解释是 受到最小偏差的约束，所提出的假设得到真正的检验。

PROJECT SUMMARY The primary objective of this proposal is to couple a) mechanistic insight relating differentiation outcomes to ECM engagement via intracellular signaling events triggered at the focal adhesion (FA), with b) 3D printing of ECM and ECM-associated proteins as a means to direct cell distribution with maturation and thereby enable fabrication of thick, functional cardiac tissue. The proposal is significant as it has the potential to generate replacement tissues and even heart grafts for individuals suffering from acute and chronic injury to the heart. It is the first of its kind to address the conundrum of the apparent uniformity of the focal adhesion relative to the myriad of different ECM/integrin combinations and the corresponding variety of cell behaviors that emerge from ECM engagement. It does so by proposing that elements of the FA, namely integrin linked kinase (ILK) and associated phosphatase, act as sensitive rheostats that can be co-opted to yield desired behavior. Here, the desired behavior is cardiac differentiation, and the innovation is the utilization of optimized ECM formulations as bioinks to create 3D cardiac tissue mimics (3DCTM) capable of directing cell distribution of multiple cell types with differentiation. This concept is feasible as the Ogle laboratory has long-studied the biochemistry of the ECM and stem cell behaviors associated with ECM engagement, and the McAlpine laboratory has focused on 3D printing of functional materials for a range of applications, from biological to electronic and the merger of these materials. These groups will also interface with expertise of the Kamp lab with respect to their recent generation of induced cardiac progenitor cells (iCPCs) to populate the 3DCTM, the Provenzano lab to assist with molecular mechanisms associated with FA formation, the Garry lab to assist with bioreactor implementation, the Zhang lab to add cardiovascular physiology expertise, and the Talkachova lab to assist with optical imaging to assess function of the 3DCTM and core facilities for mass spectrometry of ECM components and gene editing tools for modulating ILK activity. Together, this expertise will be funneled toward meeting the primary objective of the proposal via the following aims: 1) determine whether activation of integrin-linked kinase (ILK) of focal adhesions or costameres couples integrin activation to β-catenin activation via GSK3β to enable expression of genes associated with cardiomyocyte specification, 2) use 3D ECM-based model systems to identify ECM formulations supportive of endothelial differentiation and assess ILK dependence, and 3) use ECM-based 3D printing to modulate differentiation of cardiac cell types spatially in a cardiac tissue mimic. The motivation for this concept was based on extensive literature search, and results from our own experimentation; the approach was designed to insure that the interpretations of the results are subject to minimal bias and the hypotheses posed are truly tested.