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

项目摘要 该提案的主要目标是将a）与分化结果相关的机械见解与 通过在粘着斑（FA）处触发的细胞内信号传导事件的ECM接合，B）3D打印 ECM和ECM相关蛋白作为指导细胞分布与成熟的手段，从而使 制造厚的功能性心脏组织。该提案意义重大，因为它有可能产生 为患有急性和慢性心脏损伤的个人提供替代组织甚至心脏移植物。它 是第一个解决焦点粘附相对于表面粘附的表观均匀性的难题。 无数不同的ECM/整合素组合和相应的各种细胞行为， ECM接合。它通过提出FA的元件，即整联蛋白连接激酶（ILK）和 相关的磷酸酶，作为敏感的变阻器，可以增选产生所需的行为。这里 理想的行为是心脏分化，创新是利用优化的ECM制剂 作为生物墨水来创建3D心脏组织模拟物（3DCTM）， 有区别的类型。这一概念是可行的，因为奥格尔实验室长期研究了 与ECM参与相关的ECM和干细胞行为，McAlpine实验室专注于 关于3D打印功能材料的一系列应用，从生物到电子， 这些材料。这些小组还将与坎普实验室的专业知识进行交流， 产生诱导的心脏祖细胞（iCPC）以填充3DCTM，Provenzano实验室协助 与FA形成相关的分子机制，Garry实验室协助生物反应器 实施时，Zhang实验室将增加心血管生理学专业知识，Talkachova实验室将提供协助 利用光学成像评估3DCTM和ECM质谱核心设施的功能 用于调节ILK活性的组分和基因编辑工具。这些专业知识将汇集到 通过以下目标满足提案的主要目标：1）确定是否激活 局灶性粘连或costameres的整合素连接激酶（ILK）将整合素激活与β-连环蛋白激活偶联 通过GSK 3 β使与心肌细胞特化相关的基因能够表达，2）使用基于3D ECM的 用于鉴定支持内皮分化的ECM制剂和评估ILK的模型系统 依赖性，以及3）使用基于ECM的3D打印在空间上调节心脏细胞类型的分化， 模拟心脏组织这一概念的动机是基于广泛的文献检索，结果 从我们自己的实验;该方法的目的是确保结果的解释是 受到最小的偏见和提出的假设是真正的测试。

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.