Busskamp Volker, Lewis Nathan E, Guye Patrick, Ng Alex H M, Shipman Seth L, Byrne Susan M, Sanjana Neville E, Murn Jernej, Li Yinqing, Li Shangzhong, Stadler Michael, Weiss Ron, Church George M
Department of Genetics, Harvard Medical School, Boston, MA, USA Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA.
Department of Genetics, Harvard Medical School, Boston, MA, USA Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA Department of Biology, Brigham Young University, Provo, UT, USA Department of Pediatrics, University of California, San Diego, CA, USA.
Mol Syst Biol. 2014 Nov 17;10(11):760. doi: 10.15252/msb.20145508.
Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types.
细胞重编程和干细胞分化技术的进展，使得现在能够对人类神经元分化进行体外研究。然而，阐明其潜在调控程序仍然具有挑战性，因为分化方案繁琐，且神经元产量往往较低。在此，我们在人诱导多能干细胞中过表达两种神经生成素转录因子，4天内获得了具有双极形态的神经元，纯度超过90%。高纯度使得能够在神经发生过程中进行mRNA和微小RNA表达谱分析，从而揭示了参与从干细胞快速转变为神经元的遗传程序。所得细胞表现出分化神经元的转录、形态和功能特征，与产前人类脑样本的转录相似性最高。我们的分析揭示了一个关键转录因子和微小RNA网络，该网络通过祖细胞状态促进多能性丧失和快速神经发生。关键转录因子的扰动影响了所得神经元的同质性和表型特性，这表明从系统层面看待分化的分子生物学可能会指导后续对人类干细胞的操作，以快速获得多种神经元类型。
