Objective: To generate and characterize TH-GFP and -mCherry reporter iPSC lines.

Background: Human induced pluripotent stem cells (iPSCs)-based dopaminergic neuronal (iDA) cultures emerged as a valuable resource for disease modeling and development of therapies for Parkinson’s disease (PD). In the last decade, several protocols for generating iDA cultures have been published. However, although efficient, these protocols result in mixed neuronal cultures containing only up to 30% of tyrosine hydroxylase (TH)-positive neurons.

Methods: Commercially available iPSCs from a healthy control were transfected with a vector coexpressing gRNA targeting a sequence of the TH gene right before the stop codon and Cas9 mRNA. In addition, a plasmid containing a Puromycin resistance cassette and GFP or mCherry DNA sequence flanked by 1kB-long DNA sequences of the TH gene upstream and downstream of the stop codon was used as a donor vector. Upon transfection, iPSCs were plated on the matrigel-coated plates and treated with Puromycin for seven days. Single colonies were picked and correct knocking in was confirmed by sequencing. The “original” and TH-mCherry/-GFP reporter iPSCs were differentiated into dopaminergic neurons according to a previously published protocol by Kriks et al., 2011.

Results: 95% of Puromycin-resistant colonies showed correct, in-frame integration of the reporters. Among them, 20% had integrations in both TH alleles. Expression analysis showed comparable levels of the floorplate marker FOXA2, of the dopaminergic midbrain progenitor marker LMX1A, as well as of markers for mature dopaminergic neurons, i.e. PITX3 and NR4A2 between the “original” and the reporter iPSCs. Biallelic knock-in of the reporters did not alter TH mRNA expression during differentiation. In differentiated iDA cultures, only GFP- or mCherry-positive cells were positively immunostained using an antibody against TH. Finally, we were able to select out mCherry-positive cells at day 17 using fluorescence-activated cell sorting and to plate them onto Poly-L-ornithine/laminin/fibronectin-coated plates for further differentiation.

Conclusions: We developed an efficient, high-throughput method for generating TH reporter iPSCs lines. Differentiated reporter lines can be sorted to obtain pure dopaminergic neuronal cultures and thereby diminish the commonly observed heterogeneity-induced variability of iPSC-derived dopaminergic cultures.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/crispr-cas9-based-fluorescent-tyrosine-hydroxylase-reporter-lines/