Mechanopriming for cell engineering

用于细胞工程的机械引发剂

• 批准号: 10737574
• 负责人: Song Li
• 金额: $ 50.84万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737574
• 关键词: 3-Dimensional; Autologous; Biology; Biomedical Technology; Biophysics; Biosensor; Brain; Cell Nucleus; Cell Reprogramming; Cell physiology; Cells; Chromatin; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Cues; DNA; DNA Methylation; Disease; Disease model; Dissociation; Drug Screening; Epigenetic Process; Fibroblasts; Gene Activation; Gene Expression; Genes; Genome; Genomics; Heterochromatin; Histones; Individual; Injury; Maps; Mechanics; Mediating; Methods; Microfluidic Microchips; Microfluidics; Molecular Conformation; Nerve Tissue; Neurons; Neurosciences; Nuclear; Nuclear Lamina; Patients; Peripheral; Process; Protocols documentation; Publications; Regenerative Medicine; Regulation; Role; Site; Somatic Cell; Source; Spinal Cord; Technology; Testing; Three-dimensional analysis; Tissue Engineering; Translations; Work; cell growth; cellular engineering; chromatin remodeling; design; drug discovery; epigenetic profiling; epigenetic regulation; epigenomics; fabrication; genome-wide; improved; innovation; insight; microdevice; migration; multidisciplinary; multiple omics; nervous system disorder; permissiveness; response; scale up; transcription factor; transcriptomic profiling

Project Summary Cell reprogramming represents a major advancement in biology, and has wide applications in regenerative medicine, disease modeling and drug screening. Somatic cells such as fibroblasts can be directly converted into induced neuronal (iN) cells via the forced expression of three transcription factors: Ascl1, Brn2 and Myt1l (BAM). However, a major challenge of cell reprogramming, especially iN reprogramming, is the low reprogramming efficiency, which has limited the translation of this technology for biomedical applications. Biophysical factors from the microenvironment have been shown to regulate many aspects of cell functions such as cell growth, migration and differentiation. Recently, we have shown that mechanical deformation of cell nucleus through microfluidic channels can promote open chromatin structure and enhance cell reprogramming, yet the underlying mechanisms are not well understood. Based on our recent findings, we hypothesize that a mechanopriming process such as mechanical squeezing of cell nucleus can induce NL reorganization and a permissive chromatin state to facilitate the activation of neuronal genes in the heterochromatin of fibroblasts, which promotes iN reprogramming and CRISPR- mediated gene editing/activation. To test our hypothesis, we propose three Specific Aims: (1) To investigate how nuclear deformation modulates the epigenetic state to enhance iN reprogramming; (2) To determine the role of nuclear lamina in mediating nuclear deformation-induced LAD dissociation, epigenetic changes and iN reprogramming; (3) To investigate the enhancement of CRISPR-mediated neuronal gene activation by mechanical squeezing. We have assembled a multidisciplinary team with expertise on cell engineering, microdevice fabrication, high-throughput genomic and epigenomic analysis, neuroscience, biosensors and CRISPR gene editing to work together and investigate the mechanical regulation of epigenetic state and cell reprogramming. We propose to optimize a high-throughput microfluidic device, further investigate the causative mechanisms and profile the genome-wide site-specific epigenetic changes induced by nuclear deformation, which can provide a rational basis for the design of site-specific gene editing for cell engineering. Accomplishment of this project will advance our understanding of how biophysical factors regulate cell reprogramming and the epigenetic state, and unravel new mechanisms of cell fate determination, which will have wide applications in gene editing, cell and tissue engineering, disease modeling and drug discovery.

项目摘要 细胞重编程代表了生物学的一个重大进步，并且在生物学中具有广泛的应用。 再生医学、疾病建模和药物筛选。体细胞如成纤维细胞可以是 通过三种转录因子的强制表达直接转化为诱导的神经元（iN）细胞： Ascl 1、Brn 2和Myt 11（BAM）。然而，细胞重编程的一个主要挑战，特别是iN， 重编程，是低的重编程效率，这限制了该技术的翻译 用于生物医学应用。来自微环境的生物物理因素已被证明可以调节 细胞功能的许多方面，如细胞生长、迁移和分化。最近，我们发现 细胞核通过微流体通道的机械变形可以促进染色质开放， 结构和增强细胞重编程，但根本的机制还没有得到很好的理解。基于 根据我们最近的研究结果，我们假设机械预充过程，如机械挤压， 细胞核可以诱导NL重组和允许的染色质状态，以促进激活 成纤维细胞异染色质中的神经元基因，促进iN重编程和CRISPR- 介导的基因编辑/激活。为了验证我们的假设，我们提出了三个具体目标：（1） 研究核变形如何调节表观遗传状态以增强iN重编程;（2） 确定核纤层在介导核变形诱导的LAD解离中的作用， 表观遗传学变化和iN重编程;（3）研究CRISPR介导的 通过机械挤压激活神经元基因。我们组建了一个多学科团队， 细胞工程、微器件制造、高通量基因组和表观基因组分析方面的专业知识， 神经科学，生物传感器和CRISPR基因编辑共同努力，研究机械 调节表观遗传状态和细胞重编程。我们建议优化高通量 微流控装置，进一步研究致病机制，并分析全基因组位点特异性 核变形引起的表观遗传学变化，这可以为核变形的设计提供合理依据。 用于细胞工程的位点特异性基因编辑该项目的完成将促进我们的 了解生物物理因素如何调节细胞重编程和表观遗传状态， 揭示了细胞命运决定的新机制，这将在基因编辑，细胞 以及组织工程、疾病建模和药物发现。