Engineering microscale hydrogel deposition to direct single stem cell differentiation

项目摘要 成体干细胞具有再生受损组织的广泛临床潜力。比如说， 已经在超过950个临床试验中研究了间充质干细胞（MSC）用于 许多疾病的迹象。然而，尽管它们具有显著的临床相关性， 缺乏精确控制MSC功能的机制理解， 治疗结果。工程水凝胶已被用于揭示MSC的能力， 感知并响应基质生物物理线索，从而影响分化 MSC的潜力然而，将这些见解用于治疗目的已经被 具有挑战性，因为目前的方法通过将细胞群与水凝胶接触， 不受控制的混合忽略了凝胶局部量不均匀的重要性 呈现给单个细胞，导致细胞-材料相互作用的变化和不清楚， 单细胞水平。我们在此描述了一种控制微尺度水凝胶的高效方法， 在3D空间中的单个细胞周围的沉积与凝胶组成和弹性无关。 使用这种方法，我们的初步数据显示，当MSC在细胞中表达时， 在较薄的凝胶中粘附于整联蛋白配体。我们发现，将单个MSC封装在薄的 凝胶涂层足以增强MSC的成骨潜能，即使当凝胶弹性 低我们将在这些结果的基础上检验控制局部凝胶沉积的假设， 单个MSC周围的细胞通过调节细胞增殖影响膜张力和谱系特化 体积膨胀在目标1中，我们将确定不同局部凝胶沉积对 通过调节机械敏感性离子通道减少调节体积及其对 MSC的膜张力。在目标2中，我们将确定不同的局部凝胶沉积如何影响 单一MSC命运和基于MSC的骨再生。我们预测， 当凝胶沉积变薄时选择性激活的转录程序， 从而独立于凝胶弹性影响MSC的谱系特化。该项目 高度多学科的，因为它将采用生物材料， 生物物理学、遗传学和体内方法来解决具体目标。这些结果将帮助 将局部凝胶沉积定义为干细胞生长的重要决定因素，从而影响 干细胞机制和命运考虑到这些细胞的临床相关性，我们的结果将为我们提供信息。 用于改善再生结果的基于MSC的治疗剂的配方设计。

PROJECT SUMMARY Adult stem cells hold broad-ranging clinical potential to regenerate injured tissues. For instance, mesenchymal stem cells (MSCs) have been investigated in over 950 clinical trials for use in many disease indications. Despite their significant clinical relevance, however, there is currently lack of the mechanistic understanding to precisely control MSC functions for reproducible therapeutic outcomes. Engineered hydrogels have been used to reveal the ability of MSCs to sense and respond to matrix biophysical cues, which subsequently impact the differentiation potential of MSCs. However, leveraging these insights for therapeutic purposes has been challenging, since current approaches to interface a cell population with a hydrogel by uncontrolled mixing overlook the significance of heterogeneity in the local amount of the gel presented to individual cells, leading to variable and unclear cell-material interactions at the single cell level. We describe herein a highly efficient approach to control microscale hydrogel deposition around single cells in a 3D space independently of gel composition and elasticity. Using this approach, our preliminary data show that MSCs rapidly expand in volume when they adhere to an integrin ligand in thinner gels. We show that encapsulating single MSCs in a thin gel coating is sufficient to enhance the osteogenic potential of MSCs even when gel elasticity is low. We will build upon these results to test the hypothesis that controlling local gel deposition around single MSCs impacts membrane tension and lineage specification by regulating cell volume expansion. In Aim 1, we will determine the effect of varying local gel deposition on regulatory volume decrease by modulating mechanosensitive ion channels and its impact on membrane tension of MSCs. In Aim 2, we will determine how varied local gel deposition impacts single MSC fate and MSC-based bone regeneration. We predict that there exists a transcriptional program that is selectively activated when the gel deposition becomes thinner, thereby impacting lineage specification of MSCs independently of gel elasticity. The project is highly multidisciplinary in that it will employ a combination of expertise in biomaterials, biophysical, genetic, and in vivo approaches to address the specific aims. The results will help to define local gel deposition as an important determinant of stem cell growth, thereby impacting stem cell mechanics and fate. Given the clinical relevance of these cells, our results will inform formulation design of MSC-based therapeutics for improved regenerative outcomes.