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Functional Coculture of Sympathetic Neurons and Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.
American Journal of Physiology-Heart and Circulatory Physiology ( IF 4.8 ) Pub Date : 2020-08-21 , DOI: 10.1152/ajpheart.00546.2020
Annika Winbo 1, 2, 3 , Suganeya Ramanan 1, 2 , Emily Eugster 4 , Stefan Jovinge 4, 5 , Jonathan R Skinner 2, 3 , Johanna M Montgomery 1, 2
Affiliation  

Sympathetic neurons (SNs) capable of modulating the heart rate of murine cardiomyocytes (CMs) can be differentiated from human stem cells. The electrophysiological properties of human stem cell derived SNs remain largely uncharacterised, and human neurocardiac cocultures remain to be established. Here we have adapted previously published differentiation and coculture protocols to develop feeder-free SNs using human induced pluripotent stem cells (hiPSCs). hiPSC-SNs were characterized in mono- and coculture with hiPSC-CMs, using antibody labelling, enzyme-linked immunosorbent assay, and whole-cell patch clamp electrophysiology techniques. hiPSC-SNs stained positive for peripherin, tyrosine hydroxylase and nicotinic acetylcholine receptors, the latter two colocalising in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average -61±0.7 mV), secreted noradrenaline upon activation, and generated synaptic- and action currents and inward and outward voltage-dependent currents. All hiPSC-SNs fired action potentials in response to current injection, local application of potassium, or spontaneously, followed by short-medium afterhyperpolarisations. hiPSC-SNs could successfully be maintained in coculture with hiPSC-CMs, and this induced further development of hiPSC-SN action potential kinetics. To test functional coupling between the neurons and cardiomyocytes, hiPSC-CM beating response to nicotine-induced-noradrenaline-release was assessed. In neurocardiac coculture, nicotine exposure significantly increased hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monoculture, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterisation of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multi-cellular model to study neurocardiac modulation under physiological and pathological conditions.

中文翻译:

从人诱导的多能干细胞衍生的交感神经元和心肌细胞的功能共培养。

能够调节鼠心肌细胞(CMs)心率的交感神经元(SN)可以与人干细胞区分开。人类干细胞来源的SN的电生理特性仍未完全表征,人类神经心脏共培养物仍有待建立。在这里,我们已经采用人为诱导的多能干细胞(hiPSC)调整了以前发表的分化和共培养方案,以开发无饲养层的SN。使用抗体标记,酶联免疫吸附测定和全细胞膜片钳电生理技术,在与hiPSC-CM的单培养和共培养中鉴定hiPSC-SN。hiPSC-SNs的外周血蛋白,酪氨酸羟化酶和烟碱乙酰胆碱受体染色呈阳性,后两者在体细胞和突触静脉曲张中共定位。hiPSC-SNs在体外功能成熟,表现出健康的静息膜电位(平均-61±0.7 mV),在激活时分泌去甲肾上腺素,并产生突触和动作电流以及内向和外向电压依赖性电流。所有的hiPSC-SNs都响应当前的注射,局部应用钾离子或自发激发动作电位,随后进行中速超极化。hiPSC-SNs可以成功地与hiPSC-CMs共培养,并诱导了hiPSC-SN动作电位动力学的进一步发展。为了测试神经元和心肌细胞之间的功能偶联,评估了hiPSC-CM对尼古丁诱导的去甲肾上腺素释放的跳动反应。在神经心共培养中,烟碱暴露可显着提高hiPSC-CM自发搏动率,但在hiPSC-CM单培养中则不然,支持烟碱神经元的hiPSC-SN刺激直接影响hiPSC-CM功能。我们的数据表明,能够在体外调节hiPSC-CM跳动率的电生理功能hiPSC-SNs的开发和表征。这些人类共培养物提供了一种新颖的多细胞模型,用于研究生理和病理条件下的神经心脏调节。
更新日期:2020-08-22
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