Figure 1. Generation of Human iPSC Line, SCTi003-A-5, SNCA A53T

Synuclein Alpha (SNCA) gene structure is shown at the top. CRISPR-Cas9-mediated gene editing was employed to introduce the A53T mutation into both alleles of the SNCA gene in the parental Healthy Control Human iPSC Line, Female, SCTi003-A (Catalog #200-0511). Targeted nucleotide substitutions were introduced at codon 53 to convert alanine (GCA) to threonine (ACT). The genomic region surrounding codon 53 in the parental SCTi003-A and edited SCTi003-A-5 lines was validated by Sanger sequencing. A portion of the corresponding sequences is shown in the inset, confirming homozygous introduction of the SNCA A53T mutation. iPSC = induced pluripotent stem cell.

Figure 2. Cells from Human iPSC Line, SCTi003-A-5, SNCA A53T Exhibit High-Quality Morphology in Routine Culture

Cryopreserved cells from the SCTi003-A-5 line were thawed and maintained in mTeSR™ Plus (Catalog #100-1130) on Corning® Matrigel® Matrix. Cells were cultured at 37°C and subsequently analyzed on Day 7 by brightfield microscopy. (A) The resulting iPSC colonies have densely packed cells and show multi-layering when ready to be passaged. (B,C) Cells retain prominent nucleoli and high nuclear-to-cytoplasmic ratios.

Figure 3. Human iPSC Line, SCTi003-A-5, SNCA A53T Maintains a Normal Karyotype

(A) G-T-L banding for thawed cells from SCTi003-A-5 at p34 (n = 20) shows a normal karyotype with no evidence of clonal abnormalities at a band resolution of 550 G-bands per haploid genome. (B) Fluorescent in situ hybridization in a representative p20 iPSC using probes for 20p11.21 (green) and 20q11.21 (red). 95% of cells examined displayed two sets of two probe signals, indicating no aneusomy of chromosome 20 (n = 200).

Figure 4. Cells from Human iPSC Line, SCTi003-A-5, SNCA A53T Express Undifferentiated Cell Markers

Cells from the SCTi003-A-5 line were characterized using flow cytometry for undifferentiated cell markers OCT3/4 and TRA-1-60. (A) Percentage marker expression was quantified after 3 passages following thawing from the Master Cell Bank; two technical replicates were analyzed. Representative flow cytometry plots are displayed for (B) OCT3/4 and (C) TRA-1-60.

Figure 5. Cells from Human iPSC Line, SCTi003-A-5, SNCA A53T Demonstrate a High Trilineage Differentiation Capacity

Cells from the SCTi003-A-5 line were split into 3 groups, differentiated using STEMdiff™ Trilineage Differentiation Kit (Catalog #05230), and then subjected to flow cytometry analysis. Two markers for each embryonic germ layer were assessed, and bars present mean marker expression for each group of cells (n = 2 biological replicates). PAX6 and Nestin confirm differentiation to the ectoderm lineage, NCAM and Brachyury (T) to the mesoderm lineage, and CXCR4 and SOX17 to the endoderm lineage.

Figure 6. Cells from Human iPSC Line, SCTi003-A-5, SNCA A53T Can Efficiently Differentiate into Neural Progenitor Cells and Dopaminergic Neurons

(A) NPCs were generated from SCTi003-A-5 iPSCs using STEMdiff™ SMADi Neural Induction Kit (Catalog #08581) following the monolayer protocol described in the product information sheet. The resulting NPCs were fixed for immunocytochemistry. The NPCs express neural progenitor markers PAX6 and SOX1. (B) NPCs derived from SCTi003-A-5 iPSCs were further differentiated using STEMdiff™ Midbrain Neuron Differentiation Kit (Catalog #100-0038) and matured in STEMdiff™ Midbrain Neuron Maturation Kit (Catalog #100-0041) to midbrain neurons that express tubulin beta 3 class III, a general neuron marker, and TH, a dopaminergic neuron marker. NPCs = neural progenitor cells; TH = tyrosine hydroxylase