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 来自非肌肉体细胞的肌源性直接重编程已经成为一种重要的策略 产生丰富的、患者特异性的和疾病特异性的人类肌源性细胞，这是非常理想的 治疗应用和疾病建模。与从HiPSC获得肌源性细胞相比，直接 重新编程的速度大大加快，重新编程的细胞避免了畸胎瘤的形成并保留了 衰老和疾病相关的表观遗传学特征，这对于衰老相关模型尤为重要 肌肉疾病。尽管有这些优点，直接重新编程的肌源性细胞的应用 受制于低的重新编程效率和他们不成熟的本性。要改进是很有挑战性的 通过合理的设计重新编程，因为这个过程背后的分子机制在很大程度上仍然存在 未知；因此，高通量筛查(HTS)是加速发现更多 高效的直接重新编程技术。直接重新编程的有效HTS的主要障碍 技术的困难在于建立一种简单、低成本的表型读数来真正代表一种 定义目标血统的生物终点。我们最近发现，在表面培养的肌源性细胞 具有平行纳米槽/脊的图案，并具有近乎对齐的Matrigel形肌管的功能化 垂直于纳米凹槽，这种表型在所有非病理性肌源性疾病中都是独特和普遍的。 细胞，无论它们的来源和物种。肌管取向的定量分析显示为单峰 接近90°；此外，当正常的肌源性细胞与没有表现出这一点的病变细胞混合时 表型、肌管取向角随正常肌原细胞比例的增加而减小。我们 假设当培养在纳米沟槽的Matrigel功能化表面时，肌管来源于 重新编程的细胞将表现出相对于纳米沟槽的定向角随着 重新编程效率，这种高度可重复性和可量化的表型将提供简单、低- 成本和生理相关读数，用于有效的HTS以发现有效的肌源性重编程 技术。我们计划通过(1)开发一个高通量的筛查平台来验证我们的假设 肌管相对于纳米牙根的取向作为生理相关读数，并使用该平台 发现能够提高肌源性重编程效率的新的小分子化合物，(2)表征 在分子、结构和功能水平上的肌管，以及(3)转录和表观遗传的解剖 新化合物积极作用的潜在机制。拟议的研究将产生新的 产生直接重编程的人类肌源性细胞的技术显示出与TRUE更相似的地方 肌源性细胞。所建立的HTS平台可用于发现其他类型的生肌增强剂 重新编程(转录因子、microRNAs)和细胞中确定的丰富的途径和基序 用新化合物重新编程将指示新的目标，以进一步提高重新编程效率。