Engineering microscale hydrogel deposition to direct single stem cell differentiation

项目总结 成体干细胞具有广泛的再生受损组织的临床潜力。例如，间充质干细胞 干细胞(MSCs)已经在950多个临床试验中被研究，用于许多疾病的适应症。 然而，尽管它们具有重要的临床相关性，但目前还缺乏对 精确控制MSC功能，以获得可重复的治疗结果。已经使用了工程水凝胶 揭示骨髓间充质干细胞感知和响应基质生物物理线索的能力，这些线索随后影响 骨髓间充质干细胞的分化潜能。然而，将这些见解用于治疗目的一直是 具有挑战性，因为目前通过不受控制的混合将细胞群体与水凝胶相结合的方法 忽视提供给单个细胞的凝胶局部数量的异质性的重要性，导致 在单细胞水平上可变的和不清楚的细胞-材料相互作用。我们在此描述了一种高效的 在与凝胶无关的3D空间中控制单细胞周围微尺度水凝胶沉积的方法 构图和弹性。使用这种方法，我们的初步数据显示，MSCs的体积迅速扩大 当它们以更薄的凝胶形式附着在整合素配体上时。我们发现，将单个间充质干细胞包裹在薄凝胶中 即使在凝胶弹性较低的情况下，涂层也足以增强MSCs的成骨能力。我们将建造 根据这些结果，验证控制单个MSCs周围的局部凝胶沉积会影响 通过调节细胞体积扩张来调节膜张力和谱系规格。在目标1中，我们将确定 不同局部凝胶沉积对机械敏感离子调节体积减小的影响 通道及其对MSCs膜张力的影响。在目标2中，我们将确定局部凝胶的多样性 沉积影响单个MSC的命运和基于MSC的骨再生。我们预测存在一个 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MSCs的谱系特性。该项目是高度多学科的 它将结合生物材料、生物物理、遗传和体内方法的专业知识来 解决具体目标。这一结果将有助于将局部凝胶沉积定义为 干细胞的生长，从而影响干细胞的机制和命运。鉴于这些细胞的临床相关性， 我们的结果将为以MSC为基础的疗法的配方设计提供参考，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.