Transparent neural interface for in vivo interrogation of human organoids

摘要 多能干细胞技术的最新进展使神经细胞系的产生和 人胚胎干细胞和人诱导多能干细胞来源的脑有机体 (HiPSCs)来源于外周组织。这些有机化合物是自组装的3D细胞结构， 类似于人类大脑发育的早期阶段为人类提供了前所未有的机会 人类神经网络水平功能障碍在发育性脑疾病中的研究。然而， 在培养的有机物中缺乏自然的脑微环境可以影响表型和 重新编程的神经元的成熟。为了减轻这一限制，我们最近移植了人类 脑器官进入小鼠脑内，并用2-羟色胺证实了它们的分化和血管形成。 通过玻璃制成的颅骨“窗户”进行光子成像。在拟议的研究中，我们将取代这些 由我们团队成员开发的具有光学透明石墨烯电极微网的窗口， 以实现多模式纵向监测和询问移植物和 围绕着宿主神经元回路。 体内有机物移植和石墨烯电极阵列是现有的技术，为 拟议项目的科学前提--如果没有这些部分，我们也将建造一座桥 远远的。我们目前的目标是将这些技术结合起来，创造一个协同和变革性的结果。 关键的是，这种结合将允许检查细胞类型的特定尖峰(用双光子成像检测) 指由器官或宿主皮质(检测为局部)引起的大规模网络事件 场电位，LFPS，通过石墨烯电极栅格)。我们从以前的工作中知道有机化合物可以 足够的层流组织和突触连接来产生LFP，增加了科学前提。 我们将设计可植入的石墨烯装置，这种装置将附着在皮质表面和 能够在小鼠身上进行稳定的纵向记录、成像和光刺激的有机类化合物 移植人皮质类器官(目标1)。然后，我们将提供一个原则证明 论证了这种多通道技术在研究人类进化过程中的独特优势 类有机物在体内成熟过程中的活性。为此，我们将重点参与具体的 LFP事件中的细胞类型来自有机体或宿主(目标2)。所有的实验都将是 在清醒的小鼠身上进行，没有麻醉的混乱。 由于人脑器官在小鼠脑内的移植仍处于初级阶段，该项目将提供 这是对这一新的生物模型系统进行全面功能评估所急需的工具。进一步 在此过程中(不在当前范围内)，此模型系统将用于调查以下方面 人类大脑发育和发育障碍。

ABSTRACT Recent advances in pluripotent stem cell technology have enabled generation of neuronal cell lines and cerebral organoids from human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs) derived from peripheral tissues. These organoids are self-assembled, 3D cellular structures that resemble early developmental stages of the human brain opening unprecedented opportunities for investigation of human neuronal network-level dysfunction underlying developmental brain disease. However, the lack of the natural brain microenvironment in cultured organoids can influence the phenotype and maturation of the reprogrammed neurons. To mitigate this limitation, we recently transplanted human cerebral organoids into the mouse brain and demonstrated their differentiation and vascularization using 2- photon imaging through cranial “windows” made of glass. In the proposed study, we will replace these windows with optically transparent graphene electrode microgrids, developed by members of our team, to enable multimodal longitudinal monitoring and interrogation of neuronal activity in the graft and the surrounding host neuronal circuits. The in vivo organoid transplantation and graphene electrode arrays are existing technologies providing Scientific Premise to the proposed project – without these parts in place, we would be building a bridge too far. Our current goal is to combine these technologies creating a synergistic and transformative result. Critically, this combination will allow examination of cell-type specific spiking (detected with 2-photon imaging) referenced to large-scale network events arising either from the organoid or the host cortex (detected as Local Field Potentials, LFPs, by a graphene electrode grid). We know from our prior work that organoids achieve sufficient laminar organization and synaptic connectivity to generate LFPs, adding to the Scientific Premise. We will engineer implantable graphene devices that would adhere to the cortical surface and the organoid to enable stable, longitudinal recordings, imaging, and photostimulation in mice transplanted with human cortical organoids (Aim 1). Then, we will provide a proof-of-principle demonstration of the unique advantage of this multimodal technology for studying the evolution of organoid activity during its maturation in vivo. To this end, we will focus on participation of specific cell types in LFP events originating from either the organoid or the host (Aim 2). All experiments will be performed in awake mice without confounds of anesthesia. Since transplantation of human cerebral organoids in the mouse brain is still in infancy, this project will deliver a much needed tool for comprehensive functional assessment of this novel biological model system. Further along the road (outside the current scope), this model system will find its use for investigating aspects of human brain development and developmental disorders.