Functional integration and plasticity of human ESC-derived neurons

描述（由申请人提供）：神经细胞替代疗法的成功取决于移植细胞与宿主组织突触整合的能力。完全整合要求神经元既可以发送和接收突触信息，也可以根据突触连接神经元的细胞行为变化来调整其突触强度。由于细胞跟踪和刺激技术的限制，以前的报道表明只有移植的神经元可以通过突触刺激从宿主神经元接收信息。因此，没有直接证据表明它们有能力向宿主细胞发送信息或进行突触可塑性。为了验证这些假设，我们建议在人类胚胎干细胞（hESC）来源的神经元中使用与mCherry荧光团相连的光激活通道视紫红质-2 （ChR2）离子通道。将hesc来源的前脑模式神经元移植到小鼠海马后，我们将使用光刺激选择性地激活人类神经元，同时记录小鼠细胞。我们假设光刺激（和随后的动作电位产生）将引起宿主神经元的强大突触激活。各种光刺激方案的应用将测试人类神经元是否可以触发宿主细胞中短期和长期形式的突触可塑性。同时，我们将在表达chr2和不表达hesc来源的神经元的混合培养中进行类似的实验。在这里，光刺激方案的应用将测试hesc来源的神经元是否能够经历突触后变化，这是突触功效持久变化所必需的。总之，这些数据试图提供证据，证明人类神经元在宿主组织的神经网络中作为一个全功能单元的能力。

DESCRIPTION (provided by applicant): The success of neuronal cell replacement therapy depends on the ability of transplanted cells to synaptically integrate with host tissue. Complete integration requires that neurons can both send and receive synaptic information, as well as to modify their synaptic strength in response to changes in the cellular behavior of synaptically connected neurons. Due to limited cell tracking and stimulation techniques, previous reports have shown only that transplanted neurons can receive information from host neurons via synaptic stimulation. Thus, no direct evidence exists for their ability to send information to host cells or undergo synaptic plasticity. To test these hypotheses, we propose to use the light-activated Channelrhodopsin-2 (ChR2) ion channel linked to the mCherry fluorophore in human embryonic stem cell (hESC)-derived neurons. Following transplantation of hESC-derived forebrain-patterned neurons to the mouse hippocampus, we will use light stimulation to selectively activate human neurons while recording mouse cells. We hypothesize that light stimulation (and subsequent action potential generation) will give rise to robust synaptic activation of host neurons. Application of various light stimulus protocols will then test whether human neurons can trigger short-term and long-term forms of synaptic plasticity in host cells. In parallel, we will perform similar experiments on mixed cultures of ChR2-expressing and non-expressing hESC-derived neurons. Here, application of light stimulation protocols will test whether hESC-derived neurons can undergo post-synaptic changes required for enduring changes in synaptic efficacy. Together, these data seek to provide evidence of the ability of human neurons to act as a fully functional unit within a neural network in host tissues.