Forebrain assembloids support the development of fast-spiking human PVALB+ cortical interneurons and uncover schizophrenia-associated defects.

Disruption of parvalbumin positive (PVALB+) cortical interneurons is implicated in the pathogenesis of schizophrenia. However, how these defects emerge during development is not well understood. The protracted maturation of these cells during postnatal development has made their derivation from human pluripotent stem cells (hPSCs) extremely difficult, precluding hPSC-based disease modeling of their role in neuropsychiatric diseases. Here we present a cortical assembloid system that supports the development of PVALB+ cortical interneurons which match the molecular profiles of primary PVALB+ interneurons and display their distinctive electrophysiological features. Further, we characterized cortical interneuron development in a series of CRISPR-generated isogenic structural variants associated with schizophrenia and identified variant-specific phenotypes in cortical interneuron migration and the molecular profile of PVALB+ cortical interneurons. These findings reveal plausible mechanisms on how disruption of cortical interneuron development may impact schizophrenia risk and provide an exciting human experimental platform to facilitate the study of PVALB+ cortical interneurons. Cortical assembloids were generated by seperately patterning dorsal and ventral forebrain organoids using dual SMAD and WNT inhibition followed by Sonic activation for the ventral forebrain. Organoids were fused at day 30 to make assembloids and collected for scRNA seq at days 60, 75 or 125 via a papain dissociation.

小白蛋白阳性（PVALB+）皮层中间神经元的功能紊乱与精神分裂症的发病机制密切相关。然而，这类神经元的发育缺陷如何在发育过程中产生，目前仍未得到充分阐明。由于这类神经元在产后发育阶段的成熟过程极为漫长，使其从人类多能干细胞（human pluripotent stem cells，hPSCs）中诱导分化极具挑战性，进而阻碍了基于hPSCs的疾病模型构建，以研究这类神经元在神经精神疾病中的作用。本研究开发了一种皮层类组装体（cortical assembloid）培养体系，该体系可支持PVALB+皮层中间神经元的发育，其分子特征与原代PVALB+中间神经元高度匹配，并展现出这类神经元特有的电生理特性。进一步，本研究对一系列经CRISPR编辑获得的、与精神分裂症相关的同基因结构变异体中的皮层中间神经元发育过程进行了表征，并在皮层中间神经元迁移以及PVALB+皮层中间神经元的分子特征中，鉴定出了变异体特异性的表型。本研究结果阐明了皮层中间神经元发育紊乱影响精神分裂症患病风险的潜在机制，并提供了一个极具应用前景的人类实验平台，以推动PVALB+皮层中间神经元的相关研究。皮层类组装体的构建方式为：分别通过双重SMAD与WNT信号通路抑制，对背侧前脑细胞类器官和腹侧前脑细胞类器官进行模式化诱导，其中腹侧前脑细胞类器官还需辅以音猬因子（Sonic hedgehog，SHH）激活。两类类器官于培养第30天进行融合以构建类组装体，并分别于培养第60、75或125天通过木瓜蛋白酶解离，收集样本用于单细胞RNA测序（single-cell RNA sequencing，scRNA-seq）。