Engineered matrix microarrays to enhance the regenerative potential of iPSC-derived endothelial cells

摘要 外周动脉疾病(PAD)影响着800万美国人，导致疼痛、坏疽和肢体 截肢。目前的治疗方法有限。我们之前证明了人类诱导的多能干细胞 细胞衍生内皮细胞(IPSC-ECs)可以改善PAD动物模型的血液灌注量；然而， 它们的血管生成潜力仍然有限。虽然许多单变量(单变量)矩阵研究 强调了基于基质的信号对内皮细胞生存和功能的重要性，但很少有人关注 在模拟自然界复杂性的多变量材料中理解这些过程 细胞外基质(ECM)。为了解决这一局限性，我们开发了一种工程化的组合家族 具有独立可调的生化和生物力学信号的细胞外基质(EECM)，包括僵硬 和高通量的应力松弛速率，矩阵阵列研究IPSC-EC存活和血管生成 潜力。在目标1中，我们检验了多变量分析将导致最优识别的假设 增强IPSC-ECs再生能力并发现以前未知的串扰的eECM 在不同的矩阵提示之间。使用自然衍生的ECM组件的矩阵阵列的初步工作 确定了几个以前未知的矩阵线索之间的协同和拮抗相互作用。 我们在这些令人兴奋的结果的基础上，创建了一个新的阵列平台，用于模块化的组合筛选 EECM专为临床翻译而设计。EECM是一种可注射水凝胶，由重组 工程模拟基质蛋白与聚乙二醇的动态共价交联 化学(DCC)。基质生化线索通过蛋白质工程进行修改，而凝胶硬度和 应力松弛速率分别通过交联物的数目和动力学独立地进行调节。一个 279个独特的、组合的eECM的体外阵列将被筛选以检测IPSC-EC的活性、表型和 功能。多因素数学分析也将对每个eECM变量的相对重要性进行排名 作为导致协同增强的相互作用效应。结果将使用常规组织进行验证 培养化验。在目标2中，我们测试了IPSC-ECs在促血管生成的多变量eECM上将 有一个独特的、机械的签名。整合素介导的信号通路将被定量评估 将观察到的血管生成反应与机械性途径相关联，并通过得失证实- 功能障碍研究。RNA测序将揭示参与这一过程的新途径和驱动基因， 最终展示了多变量eECM促血管生成作用的分子特征。 在目标3中，我们在最优的eECM中对IPSC-ECs的治疗潜力进行了体内验证 在PAD的小鼠模型中。对照包括接种于eECM中的带有单变量细胞结合配体的细胞或非 最佳机械性能，或在生理盐水中输送细胞。细胞存活将通过生物发光来跟踪 成像：激光多谱勒光谱和组织学将决定血管再生。

ABSTRACT Peripheral arterial disease (PAD) affects 8 million Americans and results in pain, gangrene, and limb amputation. Current treatments are limited. We previously demonstrated that human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can improve blood perfusion in animal models of PAD; however, their angiogenic potential remains limited. While many single-variable (univariate) matrix studies have emphasized the importance of matrix-based cues for endothelial cell survival and function, few have focused on understanding these processes in multivariate materials, which mimic the complexity of the natural extracellular matrix (ECM). To address this limitation, we develop a combinatorial family of engineered ECMs (eECMs) with independently tunable biochemical and biomechanical cues, including stiffness and stress relaxation rate for high-throughput, matrix array studies of iPSC-EC survival and angiogenic potential. In Aim 1, we test the hypothesis that multivariate analysis will lead to the identification of optimal eECMs that enhance the regenerative capacity of iPSC-ECs and uncover previously unknown cross-talk between distinct matrix cues. Preliminary work using matrix arrays of naturally derived ECM components identified several previously unknown synergistic and antagonistic interactions between matrix cues. We build on these exciting results by creating a new array platform for combinatorial screening of modular eECMs designed for clinical translation. The eECM is an injectable hydrogel composed of recombinantly engineered matrix-mimetic proteins and polyethylene glycol crosslinked using dynamic covalent chemistry (DCC). Matrix biochemical cues are modified through protein engineering, while gel stiffness and stress relaxation rate are independently tuned through the number and kinetics of crosslinks, respectively. An in vitro array of 279 unique, combinatorial eECMs will be screened for iPSC-EC viability, phenotype, and function. Multi-factorial mathematical analyses will rank the relative importance of each eECM variable, as well as interaction effects that lead to synergistic enhancement. Results will be validated using conventional tissue culture assays. In Aim 2, we test the hypothesis that iPSC-ECs on pro-angiogenic, multivariate eECMs will have a distinctive, mechanistic signature. Integrin-mediated signaling pathways will be quantitatively assessed to correlate observed angiogenic responses to mechanistic pathways, and confirmed through gain- and loss- of-function studies. RNA sequencing will reveal new pathways and driver genes mediating the process, ultimately demonstrating a molecular signature characteristic of pro-angiogenic effects of multivariate eECMs. In Aim 3, we perform in vivo validation of the therapeutic potential of iPSC-ECs within the optimal eECM in a murine model of PAD. Controls include cells seeded in eECM with univariate cell-binding ligands or non- optimal mechanical properties, or cells delivered in saline. Cell survival will be tracked by bioluminescence imaging; laser Doppler spectroscopy and histology will determine vascular regeneration.