Table_1_Ventral Telencephalic Patterning Protocols for Induced Pluripotent Stem Cells.DOCX

The differentiation of human induced pluripotent stem cells (hiPSCs) into specific cell types for disease modeling and restorative therapies is a key research agenda and offers the possibility to obtain patient-specific cells of interest for a wide range of diseases. Basal forebrain cholinergic neurons (BFCNs) play a particular role in the pathophysiology of Alzheimer’s dementia and isolated dystonias. In this work, various directed differentiation protocols based on monolayer neural induction were tested for their effectiveness in promoting a ventral telencephalic phenotype and generating BFCN. Ventralizing factors [i.e., purmorphamine and Sonic hedgehog (SHH)] were applied at different time points, time intervals, and concentrations. In addition, caudal identity was prevented by the use of a small molecule XAV-939 that inhibits the Wnt-pathway. After patterning, gene expression profiles were analyzed by quantitative PCR (qPCR). Rostro-ventral patterning is most effective when initiated simultaneously with neural induction. The most promising combination of patterning factors was 0.5 μM of purmorphamine and 1 μM of XAV-939, which induces the highest expression of transcription factors specific for the medial ganglionic eminence, the source of GABAergic inter- and cholinergic neurons in the telencephalon. Upon maturation of cells, the immune phenotype, as well as electrophysiological properties were investigated showing the presence of marker proteins specific for BFCN (choline acetyltransferase, ISL1, p75, and NKX2.1) and GABAergic neurons. Moreover, a considerable fraction of measured cells displayed mature electrophysiological properties. Synaptic boutons containing the vesicular acetylcholine transporter (VACHT) could be observed in the vicinity of the cells. This work will help to generate basal forebrain interneurons from hiPSCs, providing a promising platform for modeling neurological diseases, such as Alzheimer’s disease or Dystonia.

将人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）定向分化为特定细胞类型用于疾病建模与修复治疗，是当前核心研究议题之一，且有望获取针对多种疾病的患者特异性目标细胞。基底前脑胆碱能神经元（Basal forebrain cholinergic neurons, BFCNs）在阿尔茨海默病痴呆与孤立性肌张力障碍的病理生理过程中发挥特殊作用。本研究针对多种基于单层神经诱导的定向分化方案开展有效性验证，以评估其促进腹侧端脑表型形成并生成BFCNs的能力。研究团队在不同时间点、时间间隔及浓度下施加腹侧化因子[即音猬因子（Sonic hedgehog, SHH）与普尔莫芬]，同时通过添加靶向Wnt信号通路的小分子抑制剂XAV-939以阻断尾侧细胞命运的产生。模式化诱导完成后，通过定量聚合酶链式反应（quantitative PCR, qPCR）分析细胞的基因表达谱。结果表明，若在神经诱导启动的同时开展腹侧模式化诱导，可获得最优的诱导效果。其中最具潜力的模式化因子组合为0.5 μM普尔莫芬与1 μM XAV-939，该组合可最高水平诱导内侧神经节隆起（medial ganglionic eminence）特异性转录因子的表达——该结构乃是端脑内γ-氨基丁酸能中间神经元（GABAergic interneurons）与胆碱能神经元的发育起源位点。待细胞成熟后，研究团队对其免疫表型与电生理特性进行了检测，结果显示细胞表达BFCNs特异性标志物蛋白（胆碱乙酰转移酶（choline acetyltransferase）、ISL1、p75以及NKX2.1）与γ-氨基丁酸能神经元标志物。此外，相当比例的被测细胞展现出成熟的电生理特性，且可在细胞周边区域观察到含有囊泡乙酰胆碱转运体（vesicular acetylcholine transporter, VACHT）的突触扣结。本研究将助力从hiPSCs中制备基底前脑中间神经元，为阿尔茨海默病、肌张力障碍等神经系统疾病的建模提供极具前景的研究平台。