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Huntington ’ s disease (HD) is an inherited neurodegenerative disorder caused by an abnormal length of CAG repeats in the gene HTT , leading to an elongated poly-glutamine (poly-Q) sequence in huntingtin (HTT). We used non-integrative Sendai virus to reprogram fibroblasts from a patient with juvenile onset HD to induced pluripotent stem cells (iPSCs). Reprogrammed iPSCs expressed pluripotency-associated markers, exhibited a normal karyotype, and following directed differentiation generated cell types belonging to the three germ layers. PCR analysis and sequencing confirmed the HD patient-derived iPSC line had one normal HTT allele and one with elongated CAG repeats, equivalent to ≥ 180Q.


Resource utility
Huntington's disease (HD) is a rare neurodegenerative disorder with suspected broader pathophysiology including cardiovascular dysfunction. Patient-derived iPSCs, in particular those with a severe HTT poly-CAG genotype, and their neuronal or cardiac derivatives, could provide advances in the mechanistic understanding of the disease pathogenesis and the discovery of potential treatment strategies (see Table 1).

Resource details
As part of a long term study into HD, we began reprogramming samples from multiple HD patients into iPSCs (Miller et al., 2022). In order to broaden this study and identify the pathophysiology arising from extremely elongated forms of the protein Huntingtin (HTT) forming extended glutamine stretches (poly-Q), we obtained human dermal fibroblasts from an anonymous male juvenile patient suffering early onset HD. Cells were reprogrammed to iPSCs using Sendai virus, with individual colonies isolated and outgrown under feeder-free pluripotent culture conditions. Following several passages one of the clones exhibiting stable and uniform iPSC morphology was selected for expansion and further characterisation (Fig. 1A). Single nucleotide polymorphism (SNP) microarray as late as passage 31 (P31) revealed a normal karyotype with no chromosome alterations (Fig. 1B), with several small variations marginally above cut-off of 3x10 5 bases (O'Shea et al., 2020) (Fig. S1A). Analysis by reverse-transcription PCR (RT-PCR) at P28 indicated an absence of viral transgene expression (Fig. S1B). Cells were also negative for mycoplasma at P31 (Fig. S1C), as well as other human pathogenic viruses (data available upon request). STR profiling also confirmed that the donor fibroblasts and P31 reprogrammed iPSCs had matching identity (archived data). Following fixing and immunostaining, expression of pluripotency-associated transcription factors and cell surface markers was confirmed by immunofluorescence microscopy (P31) and flow cytometry (P29) analysis ( Fig. 1C-D). Directed differentiation of BIHi035-A iPSCs produced cell types from all three germ layers, with robust expression of definitive endoderm, cardiomyocyte (mesoderm), and neuronal (ectoderm) markers detected (Fig. 1E).
To confirm the pathogenic poly-CAG genotype of HTT, PCR primers flanking exon 1 were used to amplify the region from fibroblast and iPSC genomic DNA (Fig. 1F). Sanger sequencing of excised bands confirmed the poly-CAG motif in both alleles (Fig. 1G, black arrows indicate 5 ′ end of reverse complement). Interestingly, the pathogenic larger band in derivative iPSCs had increased in length compared to the parental fibroblasts, from ~180 to ~195 poly-Q. This is in line with previously observed instability of very long HTT poly-CAG sequences in iPSC culture over time (Goold et al., 2019).

Immunostaining and microscopy
Cells were fixed with 4 % paraformaldehyde (Science Services) for 20 min at room temperature (RT), washed with PBS -, and incubated with blocking solution containing 10 % normal goat serum (Abcam) and 0.1 % Triton X-100 (Sigma-Aldrich) in PBSwith 0.05 % Tween 20 (Sigma-Aldrich) for 1 h at RT. Primary antibodies were incubated overnight at 4 • C and secondary antibodies for 1 h at RT (see Table 2). Nuclei were counterstained with 1:10,000 Hoechst (Thermo). All microscopy images were acquired on a DMi8 microscope fitted with a K5 camera, with images processed using LASX software (all from Leica).

RT-PCR
Total RNA was isolated using RNeasy Mini Kit (Qiagen). Mycoplasma testing was performed using the Venor®GeM qOneStep Kit according to manufacturer's instructions. Thermocycling and real-time analysis were performed using a QuantStudio 6 (Thermo). Sendai clearance was tested by RT-PCR following conversion of 1 µg RNA using iScript cDNA Synthesis Kit (BioRad). PCR was performed using polymerase KAPA2G Hotstart (KAPABiosystems) and primers listed in Table 2, in a Sim-pliAmp thermocycler (Thermo): initial denaturation 95 • C for 3 min,

Flow cytometry analysis
Cells were harvested using TrypLE, stained for viability using Vio-Bility Blue (Miltenyi), fixed and permeabilised using FoxP3 staining buffer kit (Miltenyi), and stained with conjugated antibodies (Table 2). Expression was analysed using a MACSQuant VYB flow cytometer (Miltenyi) with gating and plots generated using FlowJo 10.

Directed differentiation
Differentiations were performed on Geltrex at 37 • C in 5 % CO 2 and 20 % O 2 (normoxia). For ectodermal differentiation, iPSCs were enzymatically dissociated and replated at 2 × 10 5 cells/well of a 12-well plate in ectoderm-specific medium from the StemMACS™ Trilineage differentiation kit (Miltenyi), followed by daily medium changes. For mesodermal differentiation, cells were specified to the cardiac lineage using an adapted version of an established differentiation protocol (Lian et al., 2012). Metabolic selection using sodium lactate (Sigma) was applied during days 10-12. For endodermal differentiation, iPSCs were dissociated and replated at 2 × 10 6 cells/well of a 6-well plate in pluripotency medium. Next day, medium was replaced with Medium 1 of StemMACS™ Definitive Endoderm differentiation kit (Miltenyi), and on subsequent days with Medium 2, according to manufacturer's instructions.

HTT genotyping
Genomic DNA was isolated with FlexiGene DNA Kit (QIAGEN). Exon 1 of the HTT locus was amplified by PCR using PrimeSTAR GXL polymerase and primers listed in Table 2. A SimpliAmp thermocycler was used: initial denaturation 98 • C for 2 min, 36 cycles of [denaturation 98 • C for 15 s, annealing 60 • C for 15 s, extension 68 • C for 1 min 30 s], final extension 68 • C for 2 min. For Sanger sequencing, PCR products were excised from a low-melt agarose gel (Carl Roth), purified and submitted to LGC Genomics. Chromatograms were analysed using SnapGene.

Karyotyping and STR
SNP karyotyping was assessed using Infinium OmniExpressExome-8 Kit and the iScan system from Illumina. Copy number variations (CNVs) and SNP visualizations were determined using KaryoStudio v1.3 (Illumina). For STR, ten microsatellite loci were amplified via PCR and labelled using the GenePrint® 10 system (Promega). Analysis was performed with ABI 3730xl DNA analyser (Thermo Fisher).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.