ioGlutamatergic Neurons HTT 50CAG/WT - Human iPSC derived cells (ab303448)

Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of pan-neuronal proteins (MAP2 and TUBB3) in ioGlutamatergic Neurons HTT^50CAG/WT compared to the isogenic control. 100X magnification.

Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of glutamatergic neuron-specific transporter (VGLUT2) in ioGlutamatergic Neurons HTT^50CAG/WT compared to the isogenic control. 100X magnification.

ioGlutamatergic Neurons HTT^50CAG/WT mature rapidly and form structural neuronal networks over 11 days, when compared to the isogenic control. Day 1 to 11 post thawing; 100X magnification.

(A) Successful on-target integration into one HTT allele confirmed by gel electrophoresis. Genotyping primers flanking the endogenous HTT CAG repeat expansion region produce a band at approximately 320bp, by PCR, in both isogenic control (ioGlutamatergic Neurons) and disease model (ioGlutamatergic Neurons HTT^50CAG/WT). PCR fragments at 395bp detect on-target gene editing and introduction of a 50 CAG repeat expansion in ioGlutamatergic Neurons HTT^50CAG/WT only. (B) Amplicon PCR of the plasmid donor reveals no random integration in genomic DNA from targeted colonies via gel electrophoresis. Off-target random insertion of the donor template (used to introduce the 50 CAG repeat expansion at the WT HTT locus) is detected by PCR amplification of the donor vector backbone. This is not detected in the samples from ioGlutamatergic Neurons HTT^50CAG/WT.

Gene expression analysis demonstrates that ioGlutamatergic Neurons HTT^50CAG/WT (50CAG/WT) and the isogenic control (WT) at Day 11 lack the expression of pluripotency makers (NANOG and OCT4) whilst robustly expressing pan-neuronal (TUBB3 and SYP) and glutamatergic specific (VGLUT1 and VGLUT2) markers, as well as the glutamate receptor GRIA4. Gene expression levels assessed by RT-qPCR (data expressed relative to the parental hiPSC control (iPSC Control), normalised to HMBS). Data represents Day 11 post-revival samples; n=2 biological replicates.

RT-qPCR analysis demonstrates similar expression level of the Huntingtin gene in both wild type ioGlutamatergic Neurons (WT) and ioGlutamatergic Neurons HTT^50CAG/WT (50CAG) at day 11 post-revival (n=2 replicates). cDNA samples of the parental iPSC line
(iPSC control) were included as a reference.

NGS-Amplicon Sequencing was employed to confirm the number of CAG repeats in both the ioGlutamatergic Neurons HTT^50CAG/WT (orange) and isogenic control (black). The number of CAG repeat reads peak at 24 for both the isogenic control and ioGlutamatergic Neurons HTT^50CAG/WT. The 50 CAG repeat expansion was detected only in the ioGlutamatergic Neurons HTT^50CAG/WT (orange) confirming a successful introduction of a heterozygous 50 CAG repeat expansion.