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

项目摘要 成体干细胞在治疗许多疾病和损伤方面具有重要的治疗潜力。例如神经 祖细胞（NPC）目前正在20多个临床试验中研究用于多种适应症。 尽管它们具有重要的临床意义，但我们目前缺乏对生物学机制的理解， 在体外有效地扩增NPC，甚至作为神经球，同时保持它们的未分化，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，一旦 由于被封装，NPC通常会失去它们的干性和增殖能力。NPC干性的丧失也是 在整个衰老过程中和病理性疾病状态中观察到的， NPC自我更新和有偏分化。这些表型异常部分是由于复杂的 干细胞生态位的环境变化，包括细胞外基质生化改变， 生物力学性能因此，我们建议使用具有受控的细胞生长的3D体外水凝胶培养平台。 矩阵生物化学和生物力学将使人们能够探索以前无法验证的假设 周围细胞微环境影响NPC维持、扩增和 分化我们将使用一个蛋白质工程水凝胶家族来了解基质的影响 微环境对人iPSC衍生的NPC（hNPC）表型的影响。具体来说，我们将研究矩阵的作用 生物化学和生物力学特性对N-钙粘蛋白信号传导通路和下游的活化的影响 hNPC表型。在目标1中，我们调整弹性蛋白样蛋白（ELP）水凝胶内呈现的生化信号 展示N-钙粘蛋白模拟肽。我们假设细胞与人工N-钙粘蛋白的结合将 导致下游β-连环蛋白信号传导、干性维持和增强的对称性增殖 与神经球对照组相比。在目标2中，我们调整重组体呈现的生物力学线索， 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-细胞接触增加，诱导基于细胞的N-钙粘蛋白 信号传导、干性维持和增强的对称性增殖。在 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-钙粘蛋白的假说 ELP水凝胶力学改变了偏向hNPC分化的由外向内的信号转导。 这一过程的生物学机制将通过核结构（核纤层蛋白）的变化来探讨。 表达和核形态学）和表观遗传学（组蛋白修饰和染色体组织）。 进一步的机制的见解将探讨使用抑制剂和激动剂的关键机械转导信号 途径。我们设计的模块化水凝胶使我们能够探索特定基质线索 调节hNPC茎的维持和分化。鉴于这些细胞的巨大再生潜力， 我们的发现将为hNPC的临床扩增提供可靠的体外平台的设计。

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.