Regulation of cell reprogramming by matrix stiffness

项目摘要 细胞重编程代表着生物学的一大进步，并在 再生医学、疾病建模和药物筛选。在细胞重新编程过程中，细胞经历 导致细胞表型转换的表观遗传学变化。然而，生物物理因素是否以及如何 通过表观遗传修饰来调节细胞重编程还不是很清楚。我们发现， 生物物理因素，特别是细胞外基质(ECM)硬度，对表观遗传学有深远的影响 状态和成纤维细胞向诱导神经元(IN)细胞的转化，在 中等的细胞外基质硬度，由局部粘连和细胞骨架调节。此外，我们 发现肌动蛋白组装和转运到细胞核在表观遗传学中起着重要作用 调制。根据我们的初步数据，我们假设(1)生物物理线索，如ECM 僵硬调节FAs、肌动蛋白的组装/拆解、肌动蛋白的核运输，从而调节HAT的活性 调节表观遗传状态和细胞重编程过程，以及(2)中间僵硬水平是 最适合表观遗传重塑和细胞重新编程。为了检验我们的假设，我们提出了三个 具体目标：(1)研究矩阵刚性如何通过FA和肌动蛋白调节重编程 细胞骨架，(2)阐明基质硬度如何调节HAT和表观遗传状态以开启神经元 基因在重新编程过程中，以及(3)决定肌动蛋白核运输在基质僵硬中的作用- 在重新编程过程中HAT和表观遗传状态的调节。我们已经组建了一个多学科的 拥有机械生物学、细胞工程、高通量基因组和表观基因组学专业知识的团队 分析，和活细胞成像一起工作，研究潜在的生物物理和生物 机械装置。我们提出的研究将是第一批阐明细胞外基质硬度如何调节的研究之一。 细胞重编程的转录和表观遗传变化，以及细胞外基质僵硬如何调节焦点 粘连和细胞重新编程的细胞骨架。这个项目的发现将揭开新的 决定细胞命运的机制在细胞和组织工程中将有广泛的应用， 疾病建模和药物发现，为优化和开发药物提供了合理依据 用于体细胞重编程的新型生物材料。

Project Summary Cell reprogramming represents a major advancement in biology, and has wide applications in regenerative medicine, disease modeling and drug screening. During cell reprogramming, cells experience epigenetic changes that result in a cell phenotype switch. However, whether and how biophysical factors regulate cell reprogramming through epigenetic modifications are not well understood. We have found that biophysical factors, specifically extracellular matrix (ECM) stiffness, has profound effects on epigenetic state and the conversion of fibroblasts into induced neuronal (iN) cells, with the highest efficiency at an intermediate ECM stiffness, which is regulated by focal adhesions and the cytoskeleton. In addition, we have discovered that actin assembly and transport into nucleus plays an important role in epigenetic modulation. Based on our preliminary data, we hypothesize that (1) biophysical cues such as ECM stiffness regulates FAs, actin assembly/disassembly, nuclear transport of actin, and thus, HAT activity to modulate the epigenetic state and cell reprogramming process and (2) an intermediate level of stiffness is optimal for epigenetic remodeling and cell reprogramming. To test our hypothesis, we propose three Specific Aims: (1) Investigate how matrix stiffness regulates iN reprogramming through FAs and actin cytoskeleton, (2) Elucidate how matrix stiffness modulates HAT and the epigenetic state to turn on neuronal genes during iN reprogramming, and (3) Determine the role of actin nuclear transport in matrix stiffness- modulation of HAT and epigenetic state during iN reprogramming. We have assembled a multidisciplinary team with expertise on mechanobiology, cell engineering, high throughput genomic and epigenomic analysis, and live cell imaging to work together and investigate the underlying biophysical and biological mechanisms. Our proposed studies will be one of the first to elucidate how ECM stiffness regulates transcriptomic and epigenetic changes for cell reprogramming, and how ECM stiffness modulates focal adhesions and the cytoskeleton for cell reprogramming. Findings from this project will unravel new mechanisms of cell fate determination, which will have wide applications in cell and tissue engineering, disease modeling and drug discovery, and provide a rational basis for the optimization and development of novel biomaterials for somatic cell reprogramming.