Generation of a Tyrosine Hydroxylase Reporter Line

A human iPSC line (Zanon et al., 2017) generated from human foreskin fibroblasts (ATCC, CRL-2522) was used as starting material to engineer a TH-mCherry iPSC reporter line. To facilitate gene editing of CRL-2522 cells, we used plasmid pX330S-2 (a gift from Takashi Yamamoto; Addgene plasmid # 58778¹; RRID:Addgene_58778) (Sakuma et al., 2014). Custom guide RNA (gRNA) targeting the sequence 5′- GTGCCATTGGCTAGGTGCA-3′ in exon 14 of the TH gene was cloned into the BbsI sites as annealed oligonucleotides. The donor template for homology-directed repair (HDR) was generated using Gibson cloning as described previously (Calatayud et al., 2019). In brief, for the TH donor template, 1 kb-long homology arms (HA) were amplified from genomic DNA extracted from unmodified iPSCs and verified by Sanger sequencing. HA were inserted into the pBS-SK (−) vector using KpnI and ApaI restriction sites for the 5′ HA and SpeI and XbaI restriction sites for the 3′ HA sites of pBS-SK (−). A sequence encoding the T2A peptide was fused to the 5′ end of the mCherry open reading frame. Finally, the pRex1-Neo-SV40 cassette was inserted between the XhoI and SpeI restriction sites of the plasmid.

Next, iPSCs were transfected with the HDR template and the pX330S-2 plasmid expressing the gRNA mentioned above. In total, one million iPSCs were disaggregated into single cells using Accutase (Stemcell) on the day of transfection. Disaggregated iPSCs were diluted in 100 μl of human stem cell nucleofector Kit 2 solution (Lonza Group Ltd., Switzerland) supplemented with 2 μg of pX330S-2 plasmid and 2 μg HDR template and transfected using the 2b Nucleofector device (Lonza Group Ltd., Switzerland). Transfected cells were plated onto Matrigel (BD)-coated 6-well culture plates containing mTeSR1 medium (Stemcell), supplemented with ROCK inhibitor Y-27632 (EMD Biosciences). On the following day, the medium was replaced with fresh mTeSR1 containing 50 μg/mL G418 (Sigma) and cells were maintained for 2 weeks. At that time, one half of each resistant colony was manually picked, resuspended in 1x PCR buffer containing Proteinase K and incubated for 50 min at 56°C followed by 10 min incubation at 95°C. Integration of the resistance cassette into the TH locus was verified by PCR. Positive colonies were expanded and cryopreserved.

To excise the selection cassette, positive iPSCs were transfected with 2 μg of a Cre recombinase-expressing plasmid (pCAG-Cre was a gift from Connie Cepko; Addgene plasmid # 13775²; RRID:Addgene_13775) (Matsuda and Cepko, 2007) diluted in 100 μl of human stem cell nucleofector Kit 2 solution using the 2b Nucleofector device (both Lonza Group Ltd., Switzerland). Subsequently, transfected cells were plated onto Matrigel (BD)-coated dishes in mTeSR1 (Stemcell), supplemented with ROCK inhibitor Y-27632 (EMD Biosciences Ltd.). One week post transfection, one half of each resistant colony was manually picked, resuspended in 1x PCR buffer containing Proteinase K and incubated 50 min at 56°C followed by 10 min incubation at 95°C. The colonies were analyzed for complete excision of the resistance cassette using a PCR-based strategy. Clones with successfully excised resistance cassette were expanded, the iPSC lines were comprehensively characterized including expression analyses of pluripotency markers, and cryopreserved.

To yield DN-cultures, unedited and edited iPSCs were subjected to dopaminergic differentiation following either a “floor plate differentiation method” or an alternative strategy involving iPSC-derived NPCs. The associated procedures are detailed in the Supplemental Material.