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.

项目概要 非肌肉体细胞的肌原性直接重编程已成为一种重要的策略 产生丰富的、患者特异性和疾病特异性的人类肌原细胞，这是非常需要的 与从 hiPSC 衍生的生肌细胞相比，直接的治疗应用和疾病建模。 重编程速度大大加快，并且重编程的细胞避免了畸胎瘤形成和保留的风险 与衰老和疾病相关的表观遗传特征，这对于建模衰老相关的模型尤其重要 尽管有这些优点，直接重编程的肌原细胞的应用仍然有限。 重编程效率低且不成熟的性质阻碍了改进。 通过理性设计重新编程，因为该过程背后的分子机制在很大程度上仍然存在 未知；因此，高通量筛选（HTS）是加速发现更多物质的重要策略。 高效的直接重编程技术是有效进行直接重编程的主要障碍。 技术的难点在于建立一个简单、低成本的表型读数，真正代表综合性 我们最近发现在表面上培养的生肌细胞定义了目标谱系。 用平行的纳米凹槽/脊图案化，并用基质胶功能化，形成几乎对齐的肌管 垂直于纳米凹槽，这种表型对于所有非疾病肌源性来说是独特且普遍的 细胞，无论其来源和种类如何，肌管方向的定量分析都显示出单个峰。 接近 90°；此外，当正常生肌细胞与不表现出这种情况的患病细胞混合时 表型中，肌管定向角随着正常肌源细胞的百分比而减小。 培养发现，当在纳米凹槽、基质胶功能化表面上培养时，肌管源自 随着纳米凹槽的增加，重编程的细胞将表现出相对于纳米凹槽增加的方向角。 重编程效率，这种高度可重复和可量化的表型将提供一个简单的、低 有效 HTS 的成本和生理相关读数，以发现有效的肌源性重编程 我们计划通过（1）开发一个高通量筛选平台来检验我们的假设。 肌管相对于纳米凹槽的方向作为生理相关的读数，并使用该平台 发现能够增强生肌重编程效率的新型小化合物，(2) 表征 在分子、结构和功能水平上分析肌管，以及（3）剖析转录和表观遗传 拟议的研究将产生新化合物的积极作用的机制。 直接产生重新编程的人类肌原细胞的技术与真实的肌原细胞表现出更多的相似性 已建立的HTS平台可用于发现其他类型的肌原性增强剂。 重编程（转录因子、microRNA）以及细胞中鉴定的丰富途径和基序 用新化合物进行重编程将表明新的靶标，以进一步提高重编程效率。