Bianchi Fabio, Malboubi Majid, Li Yichen, George Julian H, Jerusalem Antoine, Szele Francis, Thompson Mark S, Ye Hua
Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, OX3 7DQ, UK.
Department Physiology, Anatomy and Genetics, University of Oxford, OX1 3QX, UK.
Stem Cell Res. 2018 Oct;32:126-134. doi: 10.1016/j.scr.2018.09.006. Epub 2018 Sep 26.
Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have some limitations of physiological accuracy. Motor neurons (MN) derived from human induced pluripotent stem cells (iPSCs) enable the generation of cell models with features relevant to human physiology. To facilitate this, it is desirable that MN protocols both rapidly and efficiently differentiate human iPSCs into electrophysiologically active MNs. In this study, we present a simple, rapid protocol for differentiation of human iPSCs into functional spinal (lower) MNs, involving only adherent culture and use of small molecules for directed differentiation, with the ultimate aim of rapid production of electrophysiologically functional cells for short-term neural injury experiments. We show successful differentiation in two unrelated iPSC lines, by quantifying neural-specific marker expression, and by evaluating cell functionality at different maturation stages by calcium imaging and patch clamping. Differentiated neurons were shown to be electrophysiologically altered by uniaxial mechanical deformation. Spontaneous network activity decreased with applied stretch, indicating aberrant network connectivity. These results demonstrate the feasibility of this rapid, simple protocol for differentiating iPSC-derived MNs, suitable for in vitro neural injury studies focussing on electrophysiological alterations caused by mechanical deformation or trauma.
原代啮齿动物神经元和永生化细胞系一直被大量用于外周和中枢神经元创伤性损伤的体外研究，但在生理准确性方面存在一些局限性。源自人类诱导多能干细胞（iPSC）的运动神经元（MN）能够生成具有与人类生理学相关特征的细胞模型。为便于实现这一点，理想的情况是MN方案能够快速且高效地将人类iPSC分化为具有电生理活性的MN。在本研究中，我们提出了一种简单、快速的方案，可将人类iPSC分化为功能性脊髓（低级）MN，该方案仅涉及贴壁培养并使用小分子进行定向分化，最终目的是快速产生用于短期神经损伤实验的具有电生理功能的细胞。我们通过量化神经特异性标志物的表达，并通过钙成像和膜片钳技术评估不同成熟阶段的细胞功能，证明了在两个不相关的iPSC系中成功实现了分化。结果表明，分化的神经元在单轴机械变形作用下电生理特性发生改变。随着施加拉伸，自发网络活动减少，表明网络连接异常。这些结果证明了这种快速、简单的方案用于分化iPSC衍生的MN的可行性，适用于聚焦于由机械变形或创伤引起的电生理改变的体外神经损伤研究。
