Rakovic Aleksandar, Voß Dorothea, Vulinovic Franca, Meier Britta, Hellberg Ann-Katrin, Nau Carla, Klein Christine, Leipold Enrico
Institute of Neurogenetics, University of Lübeck, Lübeck, Germany.
Department of Anesthesiology and Intensive Care, Center of Brain, Behavior and Metabolism (CBBM), University of Lübeck, Lübeck, Germany.
Front Cell Neurosci. 2022 Mar 23;16:817198. doi: 10.3389/fncel.2022.817198. eCollection 2022.
Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH) dopaminergic neurons (DNs) is a powerful method for creating patient-specific models to elucidate mechanisms underlying Parkinson's disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a "floor plate protocol" generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH neurons compared to the TH cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH and TH neuronal cell populations and that the "floor plate protocol" is particularly efficient in generating electrophysiologically mature TH DNs, which are the most vulnerable neuronal subtype in PD.
基于诱导多能干细胞（iPSC）生成酪氨酸羟化酶阳性（TH）多巴胺能神经元（DNs）是一种强大的方法，可用于创建患者特异性模型，以在细胞和分子水平阐明帕金森病（PD）的潜在机制并进行药物筛选。然而，目前可用的分化方案会导致细胞群体高度异质性，通常产生的与PD相关的TH DNs比例令人失望（<50%）。为便于对该细胞群体进行靶向分析并表征其电生理特性，我们采用了CRISPR/Cas9技术，生成了一种基于mCherry的人类TH报告基因iPSC系。随后，报告基因iPSCs通过“底板方案”（直接从iPSCs生成DNs）或涉及iPSC衍生神经元前体的另一种方法（NPC衍生的DNs）进行多巴胺能分化。为确定向成熟神经元转化效率最高的策略，在通过免疫化学和单细胞电生理学触发神经元分化后，对两种培养物进行了为期8周的检查。我们证实mCherry表达与内源性TH的表达相关，并且基因编辑既不影响iPSC和NPC衍生的DNs的分化过程，也不影响内源性TH表达。尽管两种培养物产生的TH细胞比例相同（≈30%），但全细胞膜片钳实验表明，与分化2周后的NPC衍生的DNs相比，iPSC衍生的DNs产生的由电压门控钠通道和钾通道介导的电流更大，表现出更高程度的突触活性，并且更频繁地发放成熟的自发动作电位序列。此外，与TH细胞相比，在TH神经元中更频繁地检测到自发动作电位发放，这提供了直接证据，表明这两个神经元亚群表现出不同的内在电生理特性。总之，数据揭示了iPSC衍生的TH和TH神经元细胞群体在电生理特性上的显著差异，并且“底板方案”在生成电生理成熟的TH DNs方面特别有效，而TH DNs是PD中最易受损的神经元亚型。
