The interplay between mesenchymal stem cell secretome and fibroblast differentiation in the confined microenvironment of idiopathic pulmonary fibrosis

项目摘要/摘要 特发性肺纤维化(IPF)是一种以辨证分型为特征的慢性肺部疾病 成纤维细胞转化为收缩的肌成纤维细胞，从而产生过多的细胞外基质(ECM)。没有 IPF治疗有效，确诊后中位生存期约为3年。增加了 基质沉积是IPF的一个特征，它增加了肺组织的硬度，从而促进了成纤维细胞 分化为肌成纤维细胞并进一步促进疾病的发展。随之而来的基质沉积增加 增加细胞经历的限制程度，但限制在成纤维细胞分化中的作用 是未知的。骨髓间充质干细胞(MSC)注射目前被认为是治疗特发性肺间质纤维化的潜在方法。 在临床试验中。然而，一些研究表明，MSC治疗会恶化结果，产生相互矛盾的结果。 研究表明，保护作用是由于MSC分泌的因子，这些因子通常是从 MSCs在标准组织培养塑料上进行二维培养。已经证明，操纵MSC 微环境改变了MSC分泌组，但限制对MSC分泌组的影响尚不清楚。 我们假设a)成纤维细胞经历的越来越多的限制将鼓励它们分化为 和b)MSCs经历的更多的限制将增加它们对 成纤维细胞，抑制肌成纤维细胞分化。为了研究这一假设，我们提出了两个具体目标： 1)评价基质组成和限制程度对成纤维细胞向肌成纤维细胞转化的影响 以及细胞力学在此过程中所起的作用，以及2)评估MSC分泌的效果 不同限制程度对成纤维细胞向肌成纤维细胞分化及基质沉积的影响 由成纤维细胞和肌成纤维细胞组成。对于目标1)成纤维细胞将在限制设备中培养，他们的 分化为肌成纤维细胞将通过α-SMA免疫荧光染色和基因和 肌成纤维细胞标志物的蛋白质表达分析。牵引力与染色体 缩合作用将作为分化机制中的潜在参与者进行研究。对于AIM 2)，MSCs将成为 在封闭装置内培养并收集其分泌因子。这些分泌因子将被应用于 成纤维细胞、成纤维细胞向肌成纤维细胞的分化将再次被描述。细胞外小泡将会是 被调查为MSC分泌物的治疗效果的潜在贡献者。因此，我们的目标是确定 限制和配体呈递在肺成纤维细胞和肌成纤维细胞力学中的作用及其机制 对MSC分泌物的反应。成功完成这些目标将提高对森林论坛进展的理解 改进骨髓间充质干细胞培养方法，用于特发性肺纤维化的治疗。总的来说，这项研究将加强 了解人类肺部生物学，推进翻译研究，减少人类疾病。这 培训奖学金将由马里兰大学费舍尔生物工程系提供便利。

PROJECT SUMMARY/ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung characterized by the differentiation of resident fibroblasts into contractile myofibroblasts that deposit excessive extracellular matrix (ECM). There are no effective treatments for IPF, and the median survival time after diagnosis is approximately 3 years. Increased matrix deposition is a hallmark of IPF that increases the stiffness of lung tissue, thereby encouraging fibroblast differentiation into myofibroblasts and furthering disease progression. Increased matrix deposition concomitantly increases the degree of confinement experienced by cells, yet the role of confinement in fibroblast differentiation is unknown. Mesenchymal stem cell (MSC) injection is currently being targeted as a potential therapeutic for IPF in clinical trials. However, some studies indicate that MSC therapy worsens outcome, yielding conflicting results. Studies have suggested that protective effects are due to MSC secreted factors, which are often collected from MSCs cultured in 2D on standard tissue culture plastic. It has been shown that manipulating the MSC microenvironment alters the MSC secretome, yet the effect of confinement on the MSC secretome is unknown. We hypothesize that a) increasing confinement experienced by fibroblasts will encourage their differentiation into myofibroblasts, and b) increasing confinement experienced by MSCs will increase their protective effects on fibroblasts, inhibiting myofibroblast differentiation. To investigate this hypothesis, we propose two Specific Aims: 1) Evaluate the effect of matrix composition and degree of confinement on fibroblast to myofibroblast differentiation, and the role cell mechanics play in this process, and 2) Evaluate the effect of MSC secreted factors in various degrees of confinement on differentiation of fibroblasts to myofibroblasts and matrix deposition by fibroblasts and myofibroblasts. For aim 1) fibroblasts will be cultured within confining devices and their differentiation into myofibroblasts will be characterized via α-SMA immunofluorescence staining and gene and protein expression analysis of characteristic myofibroblast markers. Traction forces and chromosome condensation will be investigated as potential players in the differentiation mechanism. For aim 2) MSCs will be cultured within confining devices and their secreted factors collected. These secreted factors will be applied to fibroblasts, and fibroblast to myofiroblast differentiation will again be characterized. Extracellular vesicles will be investigated as a potential contributor to the therapeutic effects of MSC secretions. Thus, we aim to determine the role of confinement and ligand presentation in lung fibroblast and myofibroblast mechanics, and their response to MSC secretions. Successful completion of these aims will improve understanding of IPF progression and improve methods of MSC culture for use in IPF treatments. Broadly, this research will enhance understanding of human pulmonary biology, advance translational research, and reduce human disease. This training fellowship will be facilitated by the University of Maryland Fischell Department of Bioengineering.