Department of Bioengineering, Behavioral Sciences, Stanford University, Stanford, USA.
Stem Cells. 2011 Jan;29(1):78-88. doi: 10.1002/stem.558.
Membrane depolarization has been shown to play an important role in the neural differentiation of stem cells and in the survival and function of mature neurons. Here, we introduce a microbial opsin into ESCs and develop optogenetic technology for stem cell engineering applications, with an automated system for noninvasive modulation of ESC differentiation employing fast optogenetic control of ion flux. Mouse ESCs were stably transduced with channelrhodopsin-2 (ChR2)-yellow fluorescent protein and purified by fluorescence activated cell sorting (FACS). Illumination of resulting ChR2-ESCs with pulses of blue light triggered inward currents. These labeled ESCs retained the capability to differentiate into functional mature neurons, assessed by the presence of voltage-gated sodium currents, action potentials, fast excitatory synaptic transmission, and expression of mature neuronal proteins and neuronal morphology. We designed and tested an apparatus for optically stimulating ChR2-ESCs during chronic neuronal differentiation, with high-speed optical switching on a custom robotic stage with environmental chamber for automated stimulation and imaging over days, with tracking for increased expression of neural and neuronal markers. These data point to potential uses of ChR2 technology for chronic and temporally precise noninvasive optical control of ESCs both in vitro and in vivo, ranging from noninvasive control of stem cell differentiation to causal assessment of the specific contribution of transplanted cells to tissue and network function.
膜去极化已被证明在干细胞的神经分化以及成熟神经元的存活和功能中发挥重要作用。在这里，我们将一种微生物视蛋白引入胚胎干细胞中，并开发出用于干细胞工程应用的光遗传学技术，该技术采用快速光遗传学控制离子流，实现了对 ESC 分化的非侵入性调节的自动化系统。通过荧光激活细胞分选（FACS）对通道视紫红质-2（ChR2）-黄色荧光蛋白进行稳定转导的鼠胚胎干细胞。用蓝色光脉冲照射产生的 ChR2-ESC 会引发内向电流。这些标记的 ESC 保留了分化为功能性成熟神经元的能力，通过存在电压门控钠电流、动作电位、快速兴奋性突触传递以及成熟神经元蛋白和神经元形态的表达来评估。我们设计并测试了一种在慢性神经元分化过程中用光刺激 ChR2-ESC 的设备，该设备使用带有环境室的定制机器人台进行高速光学切换，用于在数天内进行自动刺激和成像，并进行跟踪以增加神经和神经元标记物的表达。这些数据表明 ChR2 技术在体外和体内具有用于慢性和时间精确的非侵入性光控 ESC 的潜在用途，范围从对干细胞分化的非侵入性控制到对移植细胞对组织和网络功能的具体贡献的因果评估。
