A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons

Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions <i>in vitro</i>. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) – the predominant neuronal sub-type afflicted in PD – have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based <i>in vitro</i> assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells. <b>Abbreviations:</b> AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4ʹ,6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco’s modified eagle’s medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

巨自噬（macroautophagy）/自噬（autophagy）介导的细胞质质控通路在神经发育过程中不可或缺，对成年大脑功能性神经元群体的维持亦至关重要。目前已有充分证据表明，神经元自噬通路功能衰退与人类神经退行性疾病相关，其中包括帕金森病（Parkinson disease, PD）。因此，亟需可靠且贴合生理病理状态的人类神经元模型系统，以解析疾病易感神经群体的生物学特性、阐明神经退行性疾病的潜在致病机制，并开发可用于体外（in vitro）治疗干预检测的实验方法。人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）神经模型系统可满足这一需求：其可作为可再生资源，通过分化获得区域特异性神经元亚型以用于功能检测；可扩增构建高通量筛选平台；还可经优化后用于移植或神经修复治疗。然而迄今为止，用于生成腹侧中脑多巴胺能神经元（midbrain dopaminergic neuron, mDAN）——帕金森病中主要受累的神经元亚型——的hiPSC分化方案，仍受限于较低的分化效率和/或不适用于功能及成像类体外检测的缺陷。本文描述了一种可靠的单层分化方案，可从hiPSC中快速且可重复地获得大量mDANs，且该体系适配自噬/线粒体自噬研究。我们对这些细胞的神经元分化能力与巨自噬功能进行了表征，并详述了可用于研究此类重要细胞中自噬与线粒体自噬的定性与定量检测方法。**缩写：** 抗坏血酸（ascorbic acid, AA）；自噬相关蛋白（autophagy-related, ATG）；脑源性神经营养因子（brain derived neurotrophic factor, BDNF）；羰基氰化物间氯苯腙（carbonyl cyanide m-chlorophenylhydrazone, CCCP）；双丁酰环腺苷酸（dibutyryl cAMP, dbcAMP）；多巴胺能神经元（dopaminergic neuron, DAN）；4',6-二脒基-2-苯基吲哚（4ʹ,6-diamidino-2-phenylindole, DAPI）；N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰]-苯基甘氨酸（N-[N-(3,5-difluorophenacetyl)-L-alanyl]-phenylglycine, DAPT）；盘状大蛋白MAGUK支架蛋白4（discs large MAGUK scaffold protein 4, DLG4/PSD95）；达尔伯克改良伊格尔培养基（Dulbecco’s modified eagle’s medium, DMEM）；拟胚体（embryoid body, EB）；细胞外酸化率（extracellular acidification rate, ECAR）；表皮生长因子（epidermal growth factor, EGF）；荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）；羰基氰化物对三氟甲氧基苯腙（carbonyl cyanide p-trifluoromethoxyphenylhydrazone, FCCP）；成纤维细胞生长因子（fibroblast growth factor, FGF）；甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase, GAPDH）；胶质细胞源性神经营养因子（glial cell derived neurotrophic factor, GDNF）；人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）；溶酶体相关膜蛋白2A（lysosomal associated membrane protein 2A, LAMP2A）；溶酶体Tracker Red染料（LysoTracker Red, LT-R）；微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3, MAP1LC3）；中脑多巴胺能神经元（midbrain dopaminergic neuron, mDAN）；小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）；MitoTracker Green染料（MitoTracker Green, MT-GR）；MitoTracker Red染料（MitoTracker Red, MT-R）；正常α-突触核蛋白2（normal SNCA2, NAS2）；神经前体扩增培养基（neuroprogenitor expansion media, NEM）；核受体亚家族A组成员2（nuclear receptor subfamily group A member 2, NR4A2/NURR1）；寡霉素与抗霉素A混合物（oligomycin and antimycin A, OA）；氧消耗率（oxygen consumption rate, OCR）；帕金森病（Parkinson disease, PD）；音猬因子（sonic hedgehog signaling molecule, SHH）；α-突触核蛋白（synuclein alpha, SNCA/α-synuclein）；酪氨酸羟化酶（tyrosine hydroxylase, TH）；玻连蛋白（vitronectin, VTN）。