Characterization of Dopaminergic Neurons Derived from A Tyrosine Hydroxylase Reporter Stem Cell Line

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. Tyrosine hydroxylase (TH), the first and rate-limiting enzyme for dopamine synthesis, is a well-established marker for mDA neurons. Here, we report the generation of a human embryonic stem cell (hESC) line with a TH-RFP (red fluorescent protein) that was generated via insertion of an RFP sequence downstream of the TH coding sequence. We validated that endogenous TH expression was unaffected by genetic modification; 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 tools and data described here will contribute to development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD.