Engineering a Human Skeletal Muscle Tissue Model of LGMD2B

肢带型肌营养不良症2B(LGMD2B)是一种迟发性进行性肌营养不良症，由以下原因引起 脱铁蛋白基因的突变。脱铁蛋白是一种膜相关蛋白，在骨骼和骨骼中高度表达。 心肌纤维，它在那里协调膜修复，以应对各种损伤。目前，有以下几种 没有正在进行的临床试验或疗法来减缓疾病进展或治愈LGMD2B。虽然对活体很有用 机制研究表明，作为LGDM2B模型的缺铁蛋白缺陷(BLAJ)小鼠表现为轻度疾病表型 与人类相比，这限制了小鼠在翻译研究中的应用。建立一种高保真的体外实验模型 人类LGMD2B肌肉将补充小鼠的研究，并允许针对特定患者的疾病建模和 药物发现。因此，该项目的首要目标是设计一种新型的3D人体骨骼肌组织 复制LGMD2B主要结构、功能和代谢特征的模型(“myobundle”)。 具体地说，我们将利用来自三个健康的和三个LGMD2B捐赠者的人类IPSC系来设计LGMD2B 在肌肉收缩功能、钙稳态和脂质方面表现出可重复性缺陷的肌束 处理，同时显示出与在BLAJ小鼠和LGMD2B患者中的研究一致的药物反应。重要的是 LGMD2B肌肉的一个明显特征是异位脂肪的形成可能是由成脂作用引起的 肌肉间质细胞(MICs)的分化。因此，我们将开发一种新的组织工程模型 LGMD2B肌肉内脂肪组织(IMAT)在共培养条件下的蓄积 LGMD2B人肌肉活检和IPSC来源的肌祖细胞。在这个新奇的共同文化体系中，我们 将鉴定LGMD2B肌肉分泌的促脂肪因子，并研究它们诱导异位脂肪的能力 队形。由于免疫细胞渗透和偏向巨噬细胞极化是另一个定义特征 LGMD2B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 异种细胞相互作用和炎症环境在LGMD2B损伤反应和脂肪堆积中的作用。 最后，我们的初步研究表明，LGMD2B肌肉中的胆固醇代谢受到损害， 造成这种疾病，我们将在药理学、生化和组织学方面进一步研究 LGMD2B肌束和BLAJ小鼠。总体而言，我们预计人类新的组织工程化模型 在这个项目中开发的LGMD2B肌肉最终将使新的机械学和药理学研究成为可能 导致了LGMD2B的第一次临床试验。

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.