Xia Ninuo, Fang Fang, Zhang Pengbo, Cui Jun, Tep-Cullison Chhavy, Hamerley Tim, Lee Hyun Joo, Palmer Theo, Bothner Brian, Lee Jin Hyung, Pera Renee Reijo
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Cell Rep. 2017 Mar 7;18(10):2533-2546. doi: 10.1016/j.celrep.2017.02.023.
Generation of midbrain dopaminergic (mDA) neurons from human pluripotent stem cells provides a platform for inquiry into basic and translational studies of Parkinson's disease (PD). However, heterogeneity in differentiation in vitro makes it difficult to identify mDA neurons in culture or in vivo following transplantation. Here, we report the generation of a human embryonic stem cell (hESC) line with a tyrosine hydroxylase (TH)-RFP (red fluorescent protein) reporter. We validated that RFP faithfully mimicked TH expression during differentiation. Use of this TH-RFP reporter cell line enabled purification of mDA-like neurons from heterogeneous cultures with subsequent characterization of neuron transcriptional and epigenetic programs (global binding profiles of H3K27ac, H3K4me1, and 5-hydroxymethylcytosine [5hmC]) at four different stages of development. We anticipate that the tools and data described here will contribute to the development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD.
从人类多能干细胞生成中脑多巴胺能（mDA）神经元为帕金森病（PD）的基础研究和转化研究提供了一个探究平台。然而，体外分化的异质性使得在培养物中或移植后在体内鉴定mDA神经元变得困难。在此，我们报告了一种带有酪氨酸羟化酶（TH）-红色荧光蛋白（RFP）报告基因的人类胚胎干细胞（hESC）系的生成。我们验证了RFP在分化过程中忠实地模拟了TH的表达。使用这种TH-RFP报告细胞系能够从异质培养物中纯化出类mDA神经元，并随后在四个不同发育阶段对神经元转录和表观遗传程序（H3K27ac、H3K4me1和5-羟甲基胞嘧啶[5hmC]的全基因组结合谱）进行表征。我们预计，本文所述的工具和数据将有助于开发用于疾病建模和/或药物筛选以及PD细胞替代疗法的mDA神经元。
