Engineering a Human Skeletal Muscle Tissue Model of LGMD2B

肢带型肌营养不良症2B（LGMD 2B）是一种迟发性进行性肌营养不良症， dysferlin基因的突变Dysferlin是一种膜相关蛋白，在骨骼肌和 在心肌纤维中，它协调膜修复以应对各种损伤。目前有 没有正在进行的临床试验或治疗来减缓疾病进展或治愈LGMD 2B。虽然可用于体内 机制研究中，作为LGDM 2B模型的dysferlin缺陷（布拉伊）小鼠表现出轻度疾病表型 与人类相比，限制了小鼠用于翻译研究。建立一个高保真的体外模型， 人LGMD 2B肌肉将补充小鼠研究，并允许患者特异性疾病建模， 药物发现。因此，该项目的总体目标是设计一种新型的3D人体骨骼肌组织 模型（“肌束”），其复制LGMD 2B的主要结构、功能和代谢特征。 具体而言，我们将利用来自三个健康供体和三个LGMD 2B供体的人iPSC系来工程化LGMD 2B。 在肌肉收缩功能、钙稳态和脂质方面表现出可再现缺陷的肌束 处理，同时显示与布拉伊小鼠和LGMD 2B患者中的研究一致的药物应答。重要的是， LGMD 2B肌肉的一个定义特征是异位脂肪形成， 肌间质细胞（MIC）的分化。因此，我们将开发一种新的组织工程模型， 通过共培养从LGMD 2B肌肉中分离的MIC， LGMD 2B人肌肉活检和iPSC衍生的肌肉祖细胞。在这个新的共同文化体系中，我们 将鉴定从LGMD 2B肌肉分泌的促脂肪形成因子，并研究它们诱导异位脂肪的能力 阵由于免疫细胞浸润和偏向的巨噬细胞极化是免疫缺陷的额外定义特征， LGMD 2B肌肉，我们将设计共培养和三培养的肌肉-巨噬细胞肌束，以进一步表征 异源细胞相互作用和炎症环境在LGMD 2B损伤反应和脂肪积累中的作用。 最后，我们的初步研究表明，LGMD 2B肌肉中的胆固醇代谢受损， 有助于这种疾病，我们将进一步研究它的生物化学，生物化学和组织学， LGMD 2B肌束和布拉伊小鼠。总的来说，我们希望新的组织工程化的人类模型， 在这个项目中开发的LGMD 2B肌肉将使新的机制和药理学研究，最终 导致LGMD 2B的首次临床试验。

Limb girdle muscular dystrophy 2B (LGMD2B) is a late-onset progressive muscular dystrophy resulting from mutations in the dysferlin gene. Dysferlin is a membrane-associated protein, highly expressed in skeletal and cardiac muscle fibers where it orchestrates membrane repair in response to various injuries. Currently, there are no ongoing clinical trials or therapies to slow disease progression or cure LGMD2B. While useful for in vivo mechanistic studies, dysferlin-deficient (BLAJ) mice, a model of LGDM2B, exhibit a mild disease phenotype compared to humans, limiting mouse utility for translational studies. Developing a high-fidelity in vitro model of human LGMD2B muscle would complement mouse studies and allow patient-specific disease modeling and drug discovery. Thus, the overarching goal of this project is to engineer a novel 3D human skeletal muscle tissue model (“myobundle”) that replicates the main structural, functional, and metabolic features of LGMD2B. Specifically, we will utilize human iPSC lines from three healthy and three LGMD2B donors to engineer LGMD2B myobundles that exhibit reproducible deficits in muscle contractile function, calcium homeostasis, and lipid handling, while showing drug responses consistent with studies in BLAJ mice and LGMD2B patients. Importantly, a defining feature of LGMD2B muscle is the ectopic fat formation suggested to occur due to adipogenic differentiation of muscle interstitial cells (MICs). We will thus develop a novel tissue-engineered model of intramuscular adipose tissue (IMAT) accumulation in LGMD2B muscle by co-culturing MICs isolated from LGMD2B human muscle biopsies and iPSC-derived muscle progenitor cells. In this novel co-culture system, we will identify pro-adipogenic factors secreted from LGMD2B muscle and study their ability to induce ectopic fat formation. Since immune cell infiltration and biased macrophage polarization are additional defining features of LGMD2B muscle, we will engineer co- and tri-cultured muscle-macrophage myobundles to further characterize roles of heterocellular interactions and inflammatory milieu in injury response and fat accumulation in LGMD2B. Finally, our preliminary studies suggest that the cholesterol metabolism in LGMD2B muscle is impaired and contributes to the disease, which we will further study pharmacologically, biochemically, and histologically in LGMD2B myobundles and BLAJ mice. Overall, we expect that the novel tissue-engineered model of human LGMD2B muscle developed in this project will enable new mechanistic and pharmacological studies, eventually leading to first clinical trials for LGMD2B.