A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Ninuo Xia, Fang Fang, Pengbo Zhang, Jun Cui, Chhavy Tep-Cullison, Tim Hamerley, Hyun Joo Lee, Theo Palmer, Brian Bothner, Jin Hyung Lee, Renee Reijo Pera
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.

Graphical abstract

Teaser

Xia et al. create a knockin human tyrosine hydroxylase (TH) reporter line to isolate TH-positive dopaminergic neurons from heterogeneous population and examine their transcriptional landscape.


http://ift.tt/2lVpv4p