3D bio-printing human pluripotent stem cell-derived skeletal muscle constructs for disease modelling and drug discovery

人体最大的组织是骨骼肌，负责自主运动和呼吸。肌营养不良是一组使人衰弱的遗传性疾病，其特征在于进行性骨骼肌萎缩，随后是脂肪和结缔组织的积累。目前的护理标准不能提供有效的治疗，只能延迟失去手臂，心脏和呼吸问题。杜氏肌营养不良症（DMD）是最常见的，目前无法治愈的神经肌肉疾病。已经为DMD开发了许多动物模型，包括最常用的mdx/dmd小鼠和较大的动物，如狗和猪。然而，mdx/dmd小鼠不能完全概括人类的病理生理学。不幸的是，许多可以改善mdx/dmd小鼠表型的药物在临床试验中未能显示出有效性。为了取代/减少在肌营养不良症研究中使用小鼠模型，我们建议开发人类特异性的、生理学相关的体外模型，这些模型可以用于阐明疾病机制和测试用于开发新疗法的候选药物。然而，人类原代成肌细胞在培养中大量扩增后失去了肌原性，并且缺乏用于比较的同基因对照细胞。使用CRISPR-Cas9基因组编辑，我们精确地校正了DMD突变，以获得两个CRISPR校正的iPSC系作为同基因对照。在无转基因的肌源性分化方案之后，iPSC系的等基因对分化成肌源性祖细胞，类似于人原代肌肉前体细胞。人iPSC衍生的肌源性祖细胞的终末分化形成多核、横纹肌纤维。全长肌营养不良蛋白表达在CRISPR校正的肌肉细胞中完全恢复。由于标准的3D培养不能反映体内高度对齐的肌纤维结构的复杂性，我们建议采用新的生物工程技术来弥合这一差距。我们将使用3D生物打印来制造人iPSC衍生的骨骼肌构建体，然后对3D生物打印的人骨骼肌构建体的生物物理和生物学特性进行表征。我们将通过评估生物打印的人类3D模型的等基因对来建立一系列疾病相关的功能测定，例如收缩力的产生，对化学和电刺激的反应，氧化应激以及钙处理。最后，作为概念验证，我们将在我们的生理相关的3D体外模型中测试候选药物，以研究其改善病理生理表型的功效。简而言之，人类iPSC衍生的肌源性祖细胞与3D生物打印技术相结合，可以为阐明疾病机制和促进药物发现提供急需的人类特异性平台。重要的是，我们的实验范式广泛适用于任何肌肉疾病。这个多学科项目将对开发新的人类临床前模型和取代/减少在肌营养不良症研究中使用小鼠模型产生重大影响。

The largest tissue of human body is skeletal muscle, which is responsible for voluntary movements and breathing. Muscular dystrophies are a group of debilitating inherited diseases, characterised by progressive skeletal muscle wasting, followed by accumulation of fat and connective tissue. Current standards of care do not provide effective treatment, and can only delay loss of ambulation, cardiac and respiratory problems. Some muscular dystrophies cause premature death.Duchenne muscular dystrophy (DMD) is the most common and currently incurable neuromuscular disorder. Numerous animal models have been developed for DMD, including the most commonly used mdx/dmd mice and larger animals, such as dogs and pigs. However, the mdx/dmd mice do not fully recapitulate human pathophysiology. Unfortunately, many drugs that could ameliorate phenotypes in mdx/dmd mice fail to show efficacy in clinical trials. To replace/reduce the use of mouse models in muscular dystrophy research, we propose to develop human-specific, physiology-relevant in vitro models that can be exploited for elucidating disease mechanisms and testing drug candidates for developing novel therapies. However, human primary myoblasts lose myogenicity after extensive expansion in culture and lack isogenic control cells for comparison.To overcome these challenges, we generated two iPSC lines from two patients with distinct DMD mutations. Using CRISPR-Cas9 genome editing, we precisely corrected the DMD mutations to obtain two CRISPR-corrected iPSC lines as isogenic controls. Following a transgene-free myogenic differentiation protocol, the isogenic pairs of iPSC lines were differentiated to myogenic progenitors, resembling human primary muscle precursor cells. Terminal differentiation of human iPSCs-derived myogenic progenitors formed multinucleated, striated myofibers. Full-length dystrophin expression was completely restored in the CRISPR-corrected muscle cells. As standard 3D culture does not reflect the complexity of highly aligned myofiber architecture in vivo, we propose to employ novel bioengineering technologies to bridge this gap. We will use 3D bio-printing to fabricate human iPSC-derived skeletal muscle constructs, followed by characterisation of the biophysical and biological properties of the 3D bio-printed human skeletal muscle constructs. We will establish a range of disease-relevant functional assays by assessing isogenic pairs of bio-printed human 3D models, such as contractile force generation, response to chemical and electric stimulus, oxidative stress, as well as calcium handling. Finally, as proof of concept, we will test candidate drugs in our physiology-relevant 3D in vitro models to investigating their efficacy in ameliorating pathophysiological phenotypes.In brief, human iPSC-derived myogenic progenitors in combination with 3D bio-printing technologies can provide human-specific platforms critically needed for elucidating disease mechanisms and facilitating drug discovery. Importantly, our experimental paradigms are broadly applicable to any muscle disease. This multidisciplinary project will have a significant impact on developing novel human pre-clinical models and replacing/reducing the use of mouse models in muscular dystrophy research.