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

特发性肺纤维化受限微环境中间充质干细胞分泌组与成纤维细胞分化之间的相互作用

基本信息

批准号：
9918156
负责人：
Mary Doolin
金额：
$ 1.32万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2020-08-14
项目状态：
已结题

项目摘要

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.

项目概要/摘要特发性肺纤维化（IPF）是一种慢性肺部疾病，其特征是常驻肺纤维化的分化。成纤维细胞转变为收缩性肌成纤维细胞，沉积过量的细胞外基质（ECM）。没有 IPF的有效治疗方法，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，它会增加肺组织的硬度，从而促进成纤维细胞分化为肌成纤维细胞并进一步促进疾病进展。基质沉积同时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未知。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中。然而，一些研究表明间充质干细胞治疗会使结果恶化，产生相互矛盾的结果。研究表明，保护作用是由于间充质干细胞分泌的因子造成的，这些因子通常是从在标准组织培养塑料上进行 2D 培养的 MSC。研究表明，操纵 MSC 微环境改变 MSC 分泌组，但限制对 MSC 分泌组的影响尚不清楚。我们假设 a) 成纤维细胞经历的限制增加将促进它们分化为肌成纤维细胞，b) 间充质干细胞经历的限制增加将增强其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) 评估基质成分和对成纤维细胞对肌成纤维细胞的限制程度的影响分化，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因素对成纤维细胞向肌成纤维细胞分化和基质沉积的影响由成纤维细胞和肌成纤维细胞。为了实现目标 1)，成纤维细胞将在限制装置内培养，并且它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特征性肌成纤维细胞标志物的蛋白质表达分析。牵引力和染色体凝结将作为分化机制中的潜在参与者进行研究。对于目标 2) MSC 将是在限制装置内培养并收集其分泌因子。这些分泌因子将被应用到成纤维细胞以及成纤维细胞向肌成纤维细胞的分化将再次得到表征。细胞外囊泡将被研究为 MSC 分泌物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。总的来说，这项研究将增强了解人类肺部生物学，推进转化研究，减少人类疾病。这马里兰大学费谢尔生物工程系将提供培训奖学金。