Enhance myogenic transdifferentiation efficiency using engineering approaches

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肌源性细胞，这是非常需要的， 治疗应用和疾病建模。与从hiPSC衍生肌源性细胞相比，直接 重编程实质上更快，并且重编程的细胞避免了畸胎瘤形成的风险，并且保留了 衰老和疾病相关的表观遗传特征，这对于建模衰老相关的 肌肉疾病。尽管有这些优点，但直接重编程的肌原细胞的应用仍然是不可能的。 受到低重编程效率和它们的不成熟性质的阻碍。改进是具有挑战性的 通过合理的设计重新编程，因为该过程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）是一个重要的战略，以加快发现更多的 高效的直接重编程技术。直接重编程的有效HTS的主要障碍 技术的困难在于建立一个简单的，低成本的表型读数，真正代表了一个整合的 定义目标谱系的生物学终点。我们最近发现， 用平行的纳米凹槽/脊图案化并用Matrigel功能化形成几乎对齐的肌管 这种表型对于所有未患病的肌源性肌细胞是独特和普遍的。 细胞，无论其来源和物种。肌管方向的定量分析揭示了一个单峰 接近90°;此外，当正常的生肌细胞与不表现出这种情况的病变细胞混合时， 表型，肌管取向角随正常肌源性细胞的百分比而减小。我们 假设当在纳米开槽的基质胶功能化表面上培养时， 重编程的单元将表现出相对于纳米凹槽增加的取向角， 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 技术.我们计划通过（1）开发一个高通量筛选平台来测试我们的假设， 肌管相对于纳米凹槽的取向作为生理相关的读出，并使用该平台 发现能够增强肌源性重编程效率的新型小化合物，（2）表征 在分子、结构和功能水平上对肌管进行研究，以及（3）解剖转录和表观遗传 新化合物的积极作用的潜在机制。这项研究将产生新的 技术，以产生直接重编程的人类肌原细胞，表现出更多的相似性， 生肌细胞已建立的HTS平台可用于发现肌原性增强剂的其他类型。 重编程（转录因子，microRNA）和细胞中鉴定的富集途径和基序 用新化合物重编程的新靶点将指示进一步提高重编程效率。

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.