Enhance myogenic transdifferentiation efficiency using engineering approaches

利用工程方法提高生肌转分化效率

• 批准号: 10647491
• 负责人: Wei Shen
• 金额: $ 19.66万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647491
• 关键词: Aging; Biological; Cell model; Cells; Chromatin; Disease; Disease model; Engineering; Enhancers; Epigenetic Process; Exhibits; Future; Genes; Genetic Transcription; Goals; Heterogeneity; Human; MicroRNAs; Modeling; Molecular; Muscle; Muscle Cells; Muscle Fibers; Myopathy; Nature; Pathway interactions; Patients; Pattern; Phenotype; Physiological; Process; Reproducibility; Risk; Somatic Cell; Surface; Technology; Teratoma; Testing; Therapeutic; cost; differential expression; direct application; genome-wide analysis; high throughput screening; improved; matrigel; new technology; novel; rational design; three dimensional cell culture; transcription factor; transdifferentiation

Project Summary Myogenic direct reprogramming from non-muscle somatic cells has become an important strategy to produce abundant, patient-specific, and disease-specific human myogenic cells, which are highly desirable for therapeutic applications and disease modeling. As compared to deriving myogenic cells from hiPSCs, direct reprogramming is substantially quicker, and reprogrammed cells avoid the risk of teratoma formation and retain aging- and disease-associated epigenetic signatures, which is particularly important for modeling aging-related muscle diseases. Despite these advantages, applications of directly reprogrammed myogenic cells are hampered by low reprogramming efficiency and their immature nature. It is challenging to improve reprogramming through rational design, as molecular mechanisms underlying the process remain largely unknown; therefore, high-throughput screening (HTS) is an important strategy to expedite discovery of more efficient direct reprogramming technologies. A major hurdle to effective HTS for direct reprogramming technologies is the difficulty to establish a simple, low-cost phenotypic readout that truly represents an integrative biological endpoint defining the target lineage. We recently discovered that myogenic cells cultured on surfaces patterned with parallel nanogrooves/ridges and functionalized with Matrigel form myotubes aligning nearly perpendicular to the nanogrooves, and this phenotype is unique and universal for all non-diseased myogenic cells, regardless of their origin and species. Quantitative analysis of myotube orientations reveals a single peak near 90°; furthermore, when normal myogenic cells are mixed with diseased cells that do not exhibit this phenotype, myotube orientation angle decreases with the percentage of the normal myogenic cells. We hypothesize that when cultured on nanogrooved, Matrigel-functionalized surface, myotubes derived from reprogrammed cells will exhibit increased orientation angles relative to the nanogrooves with increasing reprogramming efficiency, and this highly reproducible and quantifiable phenotype will provide a simple, low- cost, and physiologically relevant readout for effective HTS to discover efficient myogenic reprogramming technologies. We plan to test our hypothesis by (1) developing a high-throughput screening platform using myotube orientation relative to nanogrooves as a physiologically relevant readout and use this platform to discover novel small compounds capable of enhancing myogenic reprogramming efficiency, (2) characterizing the myotubes at molecular, structural, and functional levels, and (3) dissecting transcriptional and epigenetic mechanisms underlying the positive effects of the novel compounds. The proposed study will result in new technologies to generate directly reprogrammed human myogenic cells exhibiting more similarities to true myogenic cells. The established HTS platform can be used to discover other types of enhancers for myogenic reprogramming (transcription factors, microRNAs) and the enriched pathways and motifs identified in the cells reprogrammed with the novel compounds will indicate novel targets to further improve reprogramming efficiency.

项目摘要 非肌肉体细胞的肌原性直接重编程已成为 产生丰富的，特异性和疾病特异性的人类肌生成细胞，这是非常需要的 治疗应用和疾病建模。与从HIPSC衍生肌细胞相比，直接 重新编程要快得多，重编程的细胞避免了形成畸胎瘤的风险并保留 衰老和疾病相关的表观遗传学特征，这对于与衰老相关的建模特别重要 肌肉疾病。尽管有这些优势，但直接重编程的肌原性细胞的应用是 由于低重编程效率及其不成熟的性质而阻碍了。改进是一个挑战 通过合理设计重新编程，因为该过程的分子机制在很大程度上保持不变 未知;因此，高通量筛查（HTS）是加快更多发现的重要策略 有效的直接重编程技术。有效HTS直接重新编程的主要障碍 技术很难建立一个真正代表一个集成的简单，低成本的表型读数 定义目标谱系的生物端点。我们最近发现肌原细胞在表面上培养 用平行的纳米室/脊形成图案，并用矩阵形成肌管形成肌管几乎对齐 垂直于纳米植物，这种表型是独特而通用的 细胞，无论其起源和物种如何。肌管方向的定量分析揭示了一个峰 接近90°；此外，当正常的肌生成细胞与不存在的患病细胞混合时 表型，肌管方向角度随正常肌原细胞的百分比而降低。我们 假设，当在纳米改性的，矩阵官能化的表面培养时，源自 重编程的细胞将暴露于增加的方向角相对于纳米室，随着增加 重编程效率，这种高度可重复和可量化的表型将提供简单，低 - 成本和物理相关的读数，以发现有效的HT发现有效的肌源重编程 技术。我们计划通过（1）使用（1）使用使用高通量筛选平台来检验我们的假设 Myotube方向相对于纳米植物作为物理相关的读数，并使用此平台进行 发现能够提高肌原性重编程效率的新型小型化合物，（2）表征 分子，结构和功能水平的肌管，以及（3）剖析转录和表观遗传学 新化合物的积极作用的基础机制。拟议的研究将导致新的 直接生成直接重编程的人类肌生成细胞的技术表现出与真实的相似之处 肌原细胞。已建立的HTS平台可用于发现其他类型的肌源性增强剂 重编程（转录因子，microRNA）以及在细胞中鉴定的富集途径和基序 用新颖的化合物重编程将指示新的靶标，以进一步提高重编程效率。