解析移植的人纹状体GABA能神经细胞在脑损伤模型脑内修复环路的结构和功能的数据

缺氧缺血性脑病（HIE）是一种在分娩过程中因缺氧引起的脑损伤，HIE会导致包括纹状体-苍白球/纹状体-黑质在内的多个脑区域神经元丢失和神经环路破坏，对于中/重度HIE临床上无有效治疗措施。我们以小鼠HIE为模型，研究通过人多能干细胞分化得到的神经细胞治疗HIE的可行性和机制。我们通过单侧新生鼠大脑中动脉结扎建立了HIE小鼠模型。我们把hPSCs分化为纹状体神经前体细胞并原位移植到HIE模型小鼠的纹状体，我们将检测移植的纹状体神经前体细胞可以存活并逐渐分化为DARPP32+的纹状体GABA能神经元；研究移植的人纹状体神经元长出轴突，特异性修复纹状体-侧苍白球和纹状体-黑质环路；我们将结合光遗传学技术激活移植细胞的胞体和轴突，并在宿主多巴胺能神经元内记录到抑制性突触后电流（IPSCs），研究移植细胞是否可以和近端或远端宿主细胞形成功能性突触联系；我们将通过APEX2技术结合免疫电镜，研究移植细胞和宿主细胞是否形成突触结构；我们将通过记录移植细胞的自发兴奋性/抑制性突触后电流（sEPSCs/sIPSCs），研究移植神经元功能性输入的建立；我们将结合狂犬病毒依赖的上游宿主神经元示踪，研究移植细胞接受来自宿主多脑区的投射；我们将进一步研究移植hPSCs来源的纹状体GABA能神经元可以显著改善HIE模型动物的运动功能和学习记忆相关行为学障碍。我们的研究将为缺氧缺血性脑病的治疗提供新的思路和治疗策略。

在这项研究中，通过将基因标记的人类胚胎干细胞源纹状核神经前体细胞注入缺氧缺血性脑病受损小鼠的同侧纹状核，我们发现移植的细胞逐渐形态学和电生理学上成熟为GABA脊索投射神经元，并显著挽救了缺氧缺血性脑病受损脑区的损失。有趣的是，通过使用免疫组织化学染色结合增强抗坏血酸过氧化物酶基础的免疫电子显微镜和狂犬病病毒介导的跨突触示踪，我们展示了移植物开始向内源目标区域（外侧球状体、内侧球状体、黑质）延伸轴突投射，与宿主纹状核、球状体和黑质神经元形成突触，早在移植后2个月就开始接收广泛而稳定的突触输入。
重要的是，我们进一步证明，在缺氧缺血性脑病受损的大脑中，移植的纹状核脊索投射神经元而不是脊髓GABA神经元在3-6个月的移植后恢复了运动缺陷，这些缺陷可以通过抑制移植物功能来逆转。
这些发现表明，在围产期缺氧缺血性脑病受损的未成熟大脑中，通过纹状核脊索投射神经元可以解剖和功能上重建包括多个回路的基底神经节神经回路，这为围产期缺氧缺血性脑病的细胞替代治疗策略提供了可能性。

Hypoxic-ischemic encephalopathy (HIE) refers to brain injury caused by oxygen deprivation during labor, which leads to neuronal loss and neural circuit disruption in multiple brain regions including the striatum-pallidum and striatum-nigra. Currently, there are no clinically effective therapeutic options for moderate or severe HIE. In this study, we utilized a mouse model of HIE to investigate the feasibility and underlying mechanisms of neural cell transplantation derived from human pluripotent stem cells (hPSCs) for treating HIE.

We established the HIE mouse model via unilateral middle cerebral artery ligation in neonatal mice. We differentiated hPSCs into striatal neural progenitor cells (NPCs) and performed orthotopic transplantation into the striatum of HIE model mice. We first verified that the transplanted striatal NPCs could survive and gradually differentiate into DARPP32-positive striatal GABAergic neurons. Next, we examined whether the transplanted human striatal neurons could extend axons and specifically repair the striatum-pallidum and striatum-nigra circuits. We combined optogenetic techniques to activate the soma and axons of the transplanted cells, and recorded inhibitory postsynaptic currents (IPSCs) in host dopaminergic neurons to investigate whether the transplanted cells form functional synaptic connections with proximal or distal host cells. We used APEX2 technology combined with immunoelectron microscopy to examine whether synaptic structures are formed between transplanted and host cells. We recorded spontaneous excitatory/inhibitory postsynaptic currents (sEPSCs/sIPSCs) of the transplanted neurons to investigate the establishment of functional inputs to the transplanted neurons. We combined rabies virus-dependent retrograde trans-synaptic tracing to study whether the transplanted cells receive projections from multiple brain regions of the host. Finally, we demonstrated that transplanted hPSC-derived striatal GABAergic neurons could significantly improve motor function and learning and memory-related behavioral deficits in HIE model animals. Our study will provide novel insights and therapeutic strategies for the treatment of hypoxic-ischemic encephalopathy.

In this study, we injected genetically labeled human embryonic stem cell-derived striatal neural progenitor cells into the ipsilateral striatum of mice with HIE-induced damage. We found that the transplanted cells gradually matured into GABAergic projection neurons both morphologically and electrophysiologically, and significantly rescued the neuronal loss in the brain regions damaged by HIE. Intriguingly, using immunohistochemical staining combined with enhanced ascorbate peroxidase-based immunoelectron microscopy and rabies virus-mediated trans-synaptic tracing, we demonstrated that the grafts began to extend axonal projections to endogenous target regions (lateral globus pallidus, medial globus pallidus, and substantia nigra), forming synapses with host striatal, pallidal, and nigral neurons, and began to receive widespread and stable synaptic inputs as early as 2 months after transplantation.

Importantly, we further confirmed that in the HIE-damaged brain, transplanted striatal GABAergic projection neurons, rather than spinal cord GABAergic neurons, reversed motor deficits observed 3 to 6 months after transplantation, and these deficits could be restored by inhibiting graft function.

These findings indicate that in the immature brain damaged by perinatal hypoxic-ischemic encephalopathy, striatal GABAergic projection neurons can anatomically and functionally reconstruct basal ganglia neural circuits including multiple loops, which provides the possibility for cell replacement therapy strategies for perinatal hypoxic-ischemic encephalopathy.

搜集汇总

背景与挑战

背景概述

该数据集聚焦于缺氧缺血性脑病（HIE）小鼠模型，通过移植人源多能干细胞衍生的纹状体GABA能神经元，研究其在脑内修复受损神经回路的结构和功能。数据集结合了光遗传学、APEX2技术和电生理记录等方法，深入分析了移植神经元的存活、轴突生长、突触连接形成以及对运动技能和记忆缺陷的改善作用，为HIE的治疗提供了潜在见解。