Ascl1, Nurr1 and Lmx1 convert mouse and human fibroblasts into functional dopaminergic neurons without passing through an intermediate precursor state. Mus musculus

Lineage-specific transcription factors, which drive cellular identity during embryogenesis, have been shown to convert cell fate when express ectopically in heterologous cells. Herein, we screened the key molecular factors governing the dopaminergic neuronal specification during brain development for their ability to generate similar neurons directly from mouse and human fibroblasts. Remarkably, we found a minimal set of three factors Mash1, Nurr1 and Lmx1a/b able to elicit such cellular reprogramming. Molecular and transcriptome studies showed reprogrammed DA neurons to faithfully recapitulate gene expression of their brain homolog cells while lacking expression of other catecholaminergic neuronal types. Induced neurons showed spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain DA neurons. The three factors were able to elicit DA neuronal conversion in human fibroblasts from prenatal or adult fibroblasts of healthy donors and a Parkinson’s disease patient. Generation of DA induced neurons from somatic cells might have significant implications in studies of neural development, disease in vitro modeling and cell replacement therapies. Overall design: We infected mouse embryonic fibroblasts (MEFs) isolated from TH-GFP knock-in mice embryos at E14.5, with lentiviruses expressing the three dopaminergic transcription factors Ascl1, Lmx1a and Nurr1. TH-GFP MEFs infected (Ind) by a pool of the three previously mentioned dopaminergic lentiviruses were shifted in a neuronal medium for 12 days and sorted for GFP-positive cells. Thus we extracted mRNA from Ind-GFP-positive cells and compared them to not infetced (NI) cells by means of RNA-microarray analysis.

谱系特异性转录因子（lineage-specific transcription factors）可在胚胎发生过程中调控细胞身份，已有研究表明，此类因子在异源细胞中异位表达时可转换细胞命运。在此研究中，我们筛选了脑发育过程中调控多巴胺能神经元（dopaminergic neuron）特化的关键分子因子，以考察其能否直接从小鼠和人类成纤维细胞（fibroblast）中生成同类神经元。值得注意的是，我们发现仅需Mash1、Nurr1与Lmx1a/b这三种因子即可触发细胞重编程（cellular reprogramming）。

分子与转录组学研究显示，重编程得到的多巴胺能（DA）神经元可忠实再现其脑内同源细胞的基因表达谱，且不表达其他儿茶酚胺能神经元（catecholaminergic neuron）的相关基因。诱导产生的神经元展现出自发电活动，其以规则的spike形式呈现，与脑内多巴胺能神经元的起搏活动（pacemaker activity）特征相符。

这三种因子可在健康供体的产前或成体成纤维细胞，以及帕金森病患者的成纤维细胞中触发多巴胺能神经元转化。从体细胞（somatic cell）生成多巴胺能诱导神经元，有望在神经发育研究、体外疾病建模（in vitro disease modeling）以及细胞替代疗法（cell replacement therapies）领域产生重要意义。

整体实验设计：我们将从E14.5期TH-GFP敲入小鼠（TH-GFP knock-in mouse）胚胎中分离得到的小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs），用携带三种多巴胺能转录因子Ascl1、Lmx1a与Nurr1的慢病毒（lentivirus）进行感染。用上述三种多巴胺能慢病毒混合液感染的TH-GFP MEFs（诱导组，Ind）在神经元培养基中培养12天后，分选GFP阳性细胞。我们由此从诱导组GFP阳性细胞中提取mRNA，并通过RNA微阵列分析（RNA-microarray analysis）与未感染（NI）组细胞进行对比。