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A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations
Ninuo Xia1, Fang Fang1, Pengbo Zhang2
1Department 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.
Abstract:
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.
