Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
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具有生理功能神经肌肉接头的人体运动单位生物工程模型
DOI：
10.1101/2020.05.04.076778
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Rimington R
中科院分区：
文献类型：
--
作者：
Rimington R
对人类神经肌肉接头(NMJ)的研究主要利用了实验动物、模型生物或单层细胞培养，这些培养未能代表突触的生理复杂性。因此，关于人类NMJ发育的数据仍然很少，也缺乏能够调查运动单位的系统。这项工作解决了这一需求，提供了对人体运动单位的3D模型进行生物工程的方法。IPSC来源的运动神经元前体细胞球状培养后，OLIG2、ISLET1和SMI32运动神经元 ~ 4 0 0、 ~ 15 0和 ~ 的转录分别比单层等效物高2 0 0倍。贴壁球体的轴突投射在单层超过1000μm，加入I型胶原浓度依赖的3D细胞外基质后，SMI32和Vacht mRNAs的转录进一步增强。生物工程骨骼肌产生功能性强直和抽动特征，显示乙酰胆碱受体(AChR)聚集增加以及麝香和LRP4 mRNAs的转录，表明突触后膜的组织得到增强。然后确定了对3D肌肉组织进行功能性神经支配所需的运动神经元球体或运动池的数量，产生的功能性人类NMJ证明突触前和突触后的膜和运动神经轴突共定位。3D运动单位的自发放电显著增加，证实是由运动神经通过拮抗抑制AChR而驱动的。功能分析显示，收缩的峰值时间和半松弛时间缩短，表明神经运动单位兴奋收缩偶联的生理学增强。我们的发现提供了最大化IPSC运动神经元和初级人类骨骼肌成熟的方法，利用细胞类型特定的细胞外基质和发育时间表来生物工程人类运动单位，用于神经肌肉连接生理学的研究。
Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.
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DOI：
10.1039/c7tb00721c
发表时间：
2017-06-07
期刊：
Journal of materials chemistry. B
影响因子：
--
作者：
Wu S;Xu R;Duan B;Jiang P
通讯作者：
Jiang P
影响因子：
13.6
作者：
Osaki T;Uzel SGM;Kamm RD
通讯作者：
Kamm RD
影响因子：
14
作者：
Santhanam N;Kumanchik L;Guo X;Sommerhage F;Cai Y;Jackson M;Martin C;Saad G;McAleer CW;Wang Y;Lavado A;Long CJ;Hickman JJ
通讯作者：
Hickman JJ
影响因子：
25
作者：
White MA;Kim E;Duffy A;Adalbert R;Phillips BU;Peters OM;Stephenson J;Yang S;Massenzio F;Lin Z;Andrews S;Segonds-Pichon A;Metterville J;Saksida LM;Mead R;Ribchester RR;Barhomi Y;Serre T;Coleman MP;Fallon JR;Bussey TJ;Brown RH Jr;Sreedharan J
通讯作者：
Sreedharan J
影响因子：
7.2
作者：
Barros, Claudia S.;Franco, Santos J.;Mueller, Ulrich
通讯作者：
Mueller, Ulrich