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

项目总结/摘要 特发性肺纤维化（IPF）是一种慢性肺部疾病，其特征在于居民肺纤维化的分化， 成纤维细胞转化为收缩性肌成纤维细胞，其存款过量的细胞外基质（ECM）。没有 IPF的有效治疗，诊断后的中位生存时间约为3年。增加 基质沉积是IPF的一个标志，它增加肺组织的硬度，从而促进成纤维细胞 分化成肌成纤维细胞并进一步促进疾病进展。同时增加基质沉积 增加了细胞所经历的限制程度，但限制在成纤维细胞分化中的作用 不明间充质干细胞（MSC）注射目前被视为IPF的潜在治疗方法 在临床试验阶段然而，一些研究表明，MSC治疗的结果，产生矛盾的结果。 研究表明，保护作用是由于MSC分泌的因子，这些因子通常从MSC中收集。 MSC在标准组织培养塑料上以2D培养。已经表明，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）成纤维细胞所经历的越来越多的限制将促进它们分化为 B）MSC经历的限制增加将增加它们对肌成纤维细胞的保护作用， 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与肌成纤维细胞的影响 分化，以及细胞力学在此过程中发挥的作用，以及2）评估MSC分泌的效果 不同程度限制成纤维细胞向肌成纤维细胞分化和基质沉积的因素 成纤维细胞和肌成纤维细胞。对于目的1）成纤维细胞将在限制装置内培养，并且其 分化成肌成纤维细胞将通过α-SMA免疫荧光染色和基因和 特征性肌成纤维细胞标志物的蛋白质表达分析。牵引力与染色体 将研究冷凝作为分化机制中的潜在参与者。对于目标2）MSC将是 在封闭装置中培养并收集它们的分泌因子。这些分泌的因子将应用于 成纤维细胞和成纤维细胞到成肌纤维细胞的分化将再次被表征。细胞外囊泡将被 作为MSC分泌物的治疗效果的潜在贡献者进行研究。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作用，及其 对MSC分泌物的反应。成功完成这些目标将提高对IPF进展的理解 并改进用于IPF治疗的MSC培养方法。从广义上讲，这项研究将提高 了解人类肺部生物学，推进转化研究，减少人类疾病。这 培训奖学金将由马里兰州大学财政部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.