Functional integration and plasticity of human ESC-derived neurons

描述(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结合的能力。完全整合要求神经元既可以发送和接收突触信息，也可以改变其突触强度，以应对突触连接神经元细胞行为的变化。由于细胞跟踪和刺激技术有限，以往的报道仅显示移植神经元可以通过突触刺激从宿主神经元接收信息。因此，没有直接证据证明它们向宿主细胞发送信息或经历突触可塑性的能力。为了验证这些假设，我们建议使用光激活的通道视紫红质-2(ChR2)离子通道，该通道与人胚胎干细胞(HESC)来源的神经元中的mCherry荧光团相连。在将hESC衍生的前脑模式神经元移植到小鼠海马区后，我们将使用光刺激来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(以及随后的动作电位产生)将引起宿主神经元强大的突触激活。然后，各种光刺激方案的应用将测试人类神经元是否可以在宿主细胞中触发短期和长期形式的突触可塑性。同时，我们将在ChR2表达和非表达的hESC来源的神经元的混合培养上进行类似的实验。在这里，光刺激方案的应用将测试hESC来源的神经元是否可以经历突触后的变化，这是突触效力持续变化所必需的。总而言之，这些数据试图提供证据，证明人类神经元有能力在宿主组织的神经网络中发挥全功能单位的作用。 公共卫生相关性：成功的神经细胞替换被认为依赖于移植细胞与宿主组织通过形成突触连接的整合。直到最近，技术限制一直无法确定干细胞来源的神经元是否可以向宿主细胞发送信息，或者是否可以经历真正的电路整合所必需的突触效率的变化。这项拟议的研究将使用人类胚胎细胞来源的神经元中新鉴定的光激活通道视紫红质-2来调节移植神经元的兴奋性，以检验这些假说。

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. PUBLIC HEALTH RELEVANCE: Successful neuronal cell replacement is thought to rely on the integration of transplanted cells with host tissue via the formation of synaptic connections. Until recently, technical limitations have prevented the determination of whether stem cell-derived neurons can send information to host cells or undergo changes in synaptic efficacy that are necessary for true circuit integration. The proposed research will use the newly characterized light activated Channelrhodopsin-2 in human embryonic cell-derived neurons to regulate the excitability of transplanted neurons to test these hypotheses.