Engineered biomaterials to modulate cell-cell signaling for the robust expansion of stem cells

项目总结 成体干细胞在治疗许多疾病和损伤方面具有巨大的治疗潜力。例如，神经性 祖细胞(NPC)目前正在进行20多项临床试验，以用于各种适应症。 尽管它们与临床有重要的相关性，但我们目前缺乏对 在体外高效扩增神经干细胞，即使作为神经球也是如此，同时保持其未分化、再生 茎表型。最近，3D矩阵已经成为干细胞扩增的工具；不幸的是，曾经 被封装后，NPC通常会失去茎的特性和增殖能力。鼻咽癌茎的丧失也是 在整个衰老过程和病理疾病状态下观察到的整个衰老过程中 鼻咽癌的自我更新和偏向分化。这些表型异常部分是由复杂性引起的。 干细胞生态位的环境变化，包括改变的细胞外基质，生化和 生物力学特性。因此，我们建议使用3D体外水凝胶培养平台，并控制 基质生物化学和生物力学将使以前无法验证的假说的探索成为可能 周围细胞微环境影响鼻咽癌维持、扩增和转移的机制 差异化。我们将使用一系列蛋白质工程水凝胶来了解基质的影响 微环境对人iPSC源性鼻咽癌(HNPC)表型的影响。具体来说，我们将研究矩阵的作用 N-钙粘素信号通路及其下游激活的生化和生物力学特性 HNPC表型。在目标1中，我们调整了弹性蛋白样蛋白(ELP)水凝胶中呈现的生化信号 以展示N-钙粘附素模拟肽。我们假设细胞与人造N-钙粘附素的结合将 导致下游-连环蛋白信号传递、茎干保持和增强的对称增殖 与神经球对照组相比。在目标2中，我们调整了由重组人提供的生物力学线索 ELP水凝胶通过粘弹性应力松弛实现动态基质重塑。我们假设 动态基质重塑将导致细胞间接触增加，诱导基于细胞的N-钙粘附素 与神经球对照相比，信号、茎的维持和增强的对称增殖。在……里面 目的3，我们评估了通过控制特定基质材料的性质来调节N-钙粘附素的假设 提呈和ELP水凝胶机制改变了由外向内的信号转导，从而偏向hNPC分化。 这一过程背后的生物学机制将通过改变核结构(层粘连蛋白)来探索 表观遗传学(组蛋白修饰和染色体组织)。 将使用关键机械转导信号的抑制剂和激动剂来探索进一步的机械洞察力。 小路。我们设计的模块化水凝胶使我们能够探索特定基质提示的机制 调节hNPC的茎维持和分化。鉴于这些细胞的巨大再生潜力， 我们的发现将为设计一个强大的hNPC临床扩增的体外平台提供依据。

PROJECT SUMMARY Adult stem cells hold significant therapeutic potential to treat many diseases and injuries. For example, neural progenitor cells (NPCs) are currently being investigated in over 20 clinical trials for use in a variety of indications. Despite their significant clinical relevance, we currently lack the biological mechanistic understanding to efficiently expand NPCs in vitro, even as neurospheres, while maintaining their undifferentiated, regenerative stem phenotype. Recently, 3D matrices have emerged as a tool for stem cell expansion; unfortunately, once encapsulated, NPCs commonly lose their stemness and ability to proliferate. Loss of NPC stemness is also observed in vivo throughout the aging process and in pathological disease states causing diminished ability for NPC self-renewal and biased differentiation. These phenotypic abnormalities are due in part to complex environmental changes in the stem cell niche including altered extracellular matrix biochemical and biomechanical properties. Therefore, we propose the use of a 3D in vitro hydrogel culture platform with controlled matrix biochemistry and biomechanics that will enable the exploration of previously untestable hypotheses on the mechanisms by which the surrounding cell microenvironment influences NPC maintenance, expansion, and differentiation. We will use a family of protein-engineered hydrogels to understand the impact of the matrix microenvironment on human iPSC-derived NPC (hNPC) phenotype. Specifically, we will study the role of matrix biochemical and biomechanical properties on activation of the N-cadherin signaling pathway and downstream hNPC phenotype. In Aim 1, we tune the biochemical cues presented within elastin-like protein (ELP) hydrogels to display a N-cadherin-mimetic peptide. We hypothesize that cell engagement with the artificial N-cadherin will result in downstream -catenin signaling, stemness maintenance, and enhanced symmetric proliferation compared to neurosphere controls. In Aim 2, we tune the biomechanical cues presented by the recombinant ELP hydrogels to enable dynamic matrix remodeling through viscoelastic stress relaxation. We hypothesize that dynamic matrix remodeling will result in increased cell-cell contacts, induction of cellular-based N-cadherin signaling, stemness maintenance, and enhanced symmetric proliferation compared to neurosphere controls. In Aim 3, we evaluate the hypothesis that control of specific matrix material properties to tune N-cadherin presentation and ELP hydrogel mechanics alters outside-in signal transduction that biases hNPC differentiation. The biological mechanisms underlying this process will be explored via changes in nuclear architecture (lamin expression and nuclear morphology) and epigenetics (histone modification and chromosomal organization). Further mechanistic insight will be explored using inhibitors and agonists of key mechanotransduction signaling pathways. Our engineered, modular hydrogels allow us to explore the mechanisms by which specific matrix cues regulate hNPC stem maintenance and differentiation. Given the immense regenerative potential of these cells, our findings will inform the design of a robust in vitro platform for the clinical expansion of hNPCs.