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。例如， 干细胞（MSC）已经在超过950个临床试验中被研究用于许多疾病适应症。 然而，尽管它们具有显著的临床相关性，但目前缺乏对 精确控制MSC功能，以获得可重现的治疗效果。工程水凝胶已经被用于 揭示了MSC感知和响应基质生物物理线索的能力，这随后影响了MSC的功能。 MSC的分化潜能。然而，将这些见解用于治疗目的已经被 具有挑战性，因为目前通过不受控制的混合使细胞群与水凝胶界面的方法 忽略了呈现给单个细胞的凝胶的局部量的异质性的重要性，导致 单细胞水平上的细胞-物质相互作用可变且不清楚。我们在此描述了一种高效的 在3D空间中控制单细胞周围的微尺度水凝胶沉积的方法 组成和弹性。使用这种方法，我们的初步数据显示，MSC的体积迅速扩大， 当它们在较薄的凝胶中粘附于整联蛋白配体时。我们发现，将单个MSC封装在薄凝胶中， 即使当凝胶弹性低时，涂层也足以增强MSC的成骨潜力。我们将建立 根据这些结果来检验控制单个MSC周围的局部凝胶沉积影响 膜张力和谱系规范通过调节细胞体积扩张。在目标1中，我们将确定 通过调节机械敏感离子改变局部凝胶沉积对调节性容积减小的影响 通道及其对MSCs膜张力的影响。在目标2中，我们将确定如何改变局部凝胶 沉积影响单个MSC的命运和基于MSC的骨再生。我们预测， 当凝胶沉积变得更薄时，选择性地激活转录程序，从而 独立于凝胶弹性影响MSC的谱系特化。该项目是高度多学科的， 它将采用生物材料，生物物理，遗传和体内方法的专业知识相结合， 解决具体目标。这些结果将有助于将局部凝胶沉积定义为一个重要的决定因素， 干细胞生长，从而影响干细胞力学和命运。考虑到这些细胞的临床相关性， 我们的结果将为基于MSC的治疗剂的配方设计提供信息，以改善再生结果。

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.