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，它负责自主运动和呼吸。肌肉萎缩症是一组使人衰弱的遗传性疾病，其特点是骨骼肌逐渐萎缩，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，而且只能延迟行动不便、心脏和呼吸问题。一些肌肉萎缩症会导致过早死亡。杜氏肌营养不良症（DMD）是目前最常见且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小鼠和大型动物，如狗和猪。然而，mdx/dmd小鼠不能完全概括人类病理生理。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疗效。为了在肌萎缩症研究中取代或减少小鼠模型的使用，我们建议开发人类特异性的、与生理相关的体外模型，这些模型可以用于阐明疾病机制和测试候选药物，以开发新的治疗方法。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，并且缺乏等基因对照细胞进行比较。为了克服这些挑战，我们从两个具有不同DMD突变的患者中产生了两个iPSC系。使用CRISPR-Cas9基因组编辑，我们精确地校正了DMD突变，获得了两个crispr校正的iPSC系作为等基因对照。遵循无转基因的肌源性分化方案，等基因对iPSC细胞系分化为肌源性祖细胞，类似于人类原代肌前体细胞。人类ipscs衍生的肌源性祖细胞最终分化形成多核横纹肌纤维。在经过crispr校正的肌肉细胞中，全长肌营养不良蛋白的表达完全恢复。由于标准的3D培养不能反映体内高度排列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补这一差距。我们将使用3D生物打印来制造人类ipsc衍生的骨骼肌结构，然后表征3D生物打印的人类骨骼肌结构的生物物理和生物学特性。我们将通过评估生物打印人体3D模型的等基因对，建立一系列与疾病相关的功能分析，如收缩力产生、对化学和电刺激的反应、氧化应激以及钙处理。最后，作为概念证明，我们将在我们的生理相关3D体外模型中测试候选药物，以研究它们在改善病理生理表型方面的功效。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，我们的实验模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/减少肌肉萎缩症研究中小鼠模型的使用产生重大影响。

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.