Hu Ruiqi, Boshans Linda L, Zhu Bohan, Cai Peiwen, Tao Yiran, Youssef Mark, Girrbach Gizem Inak, Song Yingnan, Wang Xuran, Tsankov Alexander, Buxbaum Joseph D, Ma Sai, Yang Nan
Nash Family Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
Alper Center for Neurodevelopment and Regeneration, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
bioRxiv. 2024 Dec 4:2024.12.03.626438. doi: 10.1101/2024.12.03.626438.
GABAergic interneurons play a critical role in maintaining neural circuit function, and their dysfunction is implicated in various neurodevelopmental and psychiatric disorders. Traditional approaches for differentiating human pluripotent stem cells (PSCs) into neuronal cells often face challenges such as incomplete neural differentiation, prolonged culture periods, and variability across PSC lines. To address these limitations, we developed a new strategy that integrates overexpression of transcription factors ASCL1 and DLX2 with dual-SMAD and WNT inhibition, efficiently driving the differentiation of human PSCs into diverse, region-specific GABAergic neuronal types. Using single-cell sequencing, we characterized the cellular heterogeneity of GABAergic induced neurons (iNs) generated with the patterning factors (patterned iNs) and those derived solely with transcription factors (PSC-derived iNs), uncovering the regulatory mechanisms that govern their fate specification. Patterned iNs exhibited gene expression features corresponding to multiple brain regions, particularly ganglionic eminence (GE) and neocortex, while GABAergic PSC-derived iNs predominantly resembled hypothalamic and thalamic neurons. Both iN types were enriched for genes relevant to neurodevelopmental and psychiatric disorders, with patterned iNs more specifically linked to neural lineage genes, highlighting their utility for disease modeling. We further applied this protocol to investigate the impact of an ADNP syndrome-associated mutation (p.Tyr719* variant) on GABAergic neuron differentiation, revealing that this mutation disrupts GABAergic fate specification and synaptic transmission. Overall, this study expands the toolkit for disease modeling by demonstrating the complementary advantages of GABAergic PSC-derived iNs and patterned iNs in representing distinct GABAergic neuron subtypes, brain regions, and disease contexts. These approaches offer a powerful platform for elucidating the molecular mechanisms underlying various neurodevelopmental and psychiatric disorders.
γ-氨基丁酸能中间神经元在维持神经回路功能中起关键作用，其功能障碍与多种神经发育和精神疾病有关。将人类多能干细胞（PSC）分化为神经元细胞的传统方法通常面临诸如神经分化不完全、培养周期延长以及PSC系之间的变异性等挑战。为了解决这些局限性，我们开发了一种新策略，该策略将转录因子ASCL1和DLX2的过表达与双SMAD和WNT抑制相结合，有效地将人类PSC分化为多种区域特异性的γ-氨基丁酸能神经元类型。使用单细胞测序，我们对由模式化因子产生的γ-氨基丁酸能诱导神经元（iN）（模式化iN）和仅由转录因子产生的γ-氨基丁酸能神经元（PSC衍生的iN）的细胞异质性进行了表征，揭示了控制其命运决定的调控机制。模式化iN表现出与多个脑区相对应的基因表达特征，特别是神经节隆起（GE）和新皮层，而γ-氨基丁酸能PSC衍生的iN主要类似于下丘脑和丘脑神经元。两种iN类型都富集了与神经发育和精神疾病相关的基因，模式化iN更具体地与神经谱系基因相关联，突出了它们在疾病建模中的实用性。我们进一步应用该方案来研究ADNP综合征相关突变（p.Tyr719*变体）对γ-氨基丁酸能神经元分化的影响，揭示该突变破坏了γ-氨基丁酸能命运决定和突触传递。总体而言，本研究通过展示γ-氨基丁酸能PSC衍生的iN和模式化iN在代表不同的γ-氨基丁酸能神经元亚型、脑区和疾病背景方面的互补优势，扩展了疾病建模的工具包。这些方法为阐明各种神经发育和精神疾病的分子机制提供了一个强大的平台。
