iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling.
iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling.
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DOI：
10.7554/elife.70341
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发表时间：
2022-01-25
期刊：
影响因子：
7.7
通讯作者：
Emerson CP Jr
中科院分区：
文献类型：
--
作者：
Guo D;Daman K;Chen JJ;Shi MJ;Yan J;Matijasevic Z;Rickard AM;Bennett MH;Kiselyov A;Zhou H;Bang AG;Wagner KR;Maehr R;King OD;Hayward LJ;Emerson CP Jr
利用高效、可靠的无转基因诱导和干细胞选择方案，从人诱导的多能干细胞(IPSCs)中分离出骨骼肌成肌细胞(IMyoblells)。免疫荧光、流式细胞仪、定量聚合酶链式反应、数字RNA表达谱和scRNA-Seq研究表明，肌成肌细胞是PAX3+/MYOD1+骨骼肌细胞系，具有胎儿样的转录组特征，不同于成人肌肉活检成肌细胞(BMyoblast)和iPSC诱导的肌祖细胞。肌成肌细胞可以稳定繁殖12代或30代群体倍增，同时保留其对肌管分化和储备细胞再生的双重承诺。肌成肌细胞还可以有效地异种移植到受辐射和损伤的小鼠肌肉中，在那里它们经历分化和胎儿-成人MYH亚型转换，展示了它们对成年肌肉成熟的可塑性调节，以响应宿主肌肉中的信号。异种肌肉移植保留了PAX3+的肌祖细胞，并能在继发性损伤后再生人类肌肉。作为疾病的模型，来自面肩肩周肌营养不良患者的肌成肌细胞揭示了一种先前未知的表观遗传调控机制，该机制控制着病理性DUX4基因的发育表达。来自肢体-Girdle肌营养不良症R7和R9以及Walker Warburg综合征患者的肌成肌细胞对他们的分子疾病病理进行了建模，并对小分子和基因编辑疗法产生了反应。这些发现为肌成肌细胞在体外和体内研究人类肌肉发生和疾病发病机制以及肌肉干细胞疗法的发展奠定了基础。肌营养不良是一组以进行性肌肉无力为特征的遗传性疾病。它们会导致残疾甚至死亡，目前还没有针对这些疾病的治疗方法。基因组测序的进展已经发现了许多导致肌肉营养不良的突变，为修复错误基因或重建受损肌肉的新疗法打开了大门。然而，测试这些想法需要更好的方法来在实验室重建人类肌肉营养不良症。对肌营养不良症进行建模的一种策略是，诱使个体的皮肤或其他细胞成为“诱导多能干细胞”；然后这些细胞可以成熟，形成体内几乎任何成人细胞，包括肌肉。然而，这种方法通常不会产生成肌细胞，成肌细胞是在发育过程中专门成熟为肌肉的“前体”细胞。这限制了对致病突变如何影响肌肉形成的早期研究。作为回应，郭等人。开发了一种肌肉成熟的两步方案，然后进行干细胞生长选择，从健康志愿者和肌营养不良患者的诱导多能干细胞中分离和培养“诱导成肌细胞”。这些诱导的成肌细胞既可以制造更多的自己，也可以成为肌肉，这使得郭等人。来模拟三种不同类型的肌营养不良症。这些成肌细胞在移植到成年小鼠肌肉内时也表现为干细胞：一些形成了人类肌肉组织，而另一些仍然是前体细胞，然后这些细胞可以对肌肉损伤做出反应并开始修复。诱导成肌细胞是由Guo等人开发的。将使科学家能够调查不同突变对肌肉组织的影响，并更好地测试治疗方法。它们也可以作为再生医学疗法的一部分，用来恢复患者的肌肉细胞。
Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics. Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming ‘induced pluripotent stem cells’; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the ‘precursor’ cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow ‘induced myoblasts’ from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.