Figure 1. Generation of Human iPSC Line, SCTi003-A-2, APP K670N/M671L (Swedish Mutation)

APP gene structure map is shown at the top. CRISPR-Cas9-mediated gene editing was employed to introduce the K670N/M671L (Swedish) double mutation into both alleles of the APP gene in the parental SCTi003-A line (Catalog #200 -0511). Targeted nucleotide substitutions were introduced at codons 670 and 671 to convert Lysine (AAG) to Asparagine (AAC) and Methionine (ATG) to Leucine (CTT/CTC ), respectively. The sequences of the parental SCTi003-A line and the edited SCTi003-A-2 line were validated by Sanger sequencing and shown in the inset. Homozygous introduction of the Swedish mutation (K670N/M671L) was confirmed.

Figure 2. SCTi003-A-2 Human iPSCs Demonstrate High-Quality Morphology in Routine Culture

Cryopreserved cells from line SCTi003-A-2 were thawed and maintained in mTeSR™ Plus (Catalog # 100-1130) on Corning® Matrigel® Matrix. (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. iPSC = induced pluripotent stem cell.

Figure 3. SCTi003-A-2 Human iPSCs Maintain a Normal Karyotype

(A) G-T-L banding for thawed cells at p34 (n = 20) shows a normal karyotype with no evidence of clonal abnormalities at a band resolution of 400 - 425 G-bands per haploid genome. (B) Fluorescent in situ hybridization in a representative p34 iPSC using probes for 20p11 (green) and 20q11.21 (red). 94% of cells examined displayed two sets of two probe signals, indicating no aneusomy of chromosome 20 (n = 200). iPSC = induced pluripotent stem cell.

Figure 4. SCTi003-A-2 iPSCs Express Undifferentiated Cell Markers

Cells from line SCTi003-A-2 were characterized using flow cytometry for undifferentiated cell markers OCT3/4 and TRA-1-60. (A) Percentage marker expression was quantified 5 passages after thawing from the Master Cell Bank from analyses of three technical replicates. Representative flow cytometry plots are displayed for (B) TRA-1-60 and (C) OCT3/4. iPSC = induced pluripotent stem cell.

Figure 5. SCTi003-A-2 Human iPSCs Demonstrate a High Trilineage Differentiation Capacity

Cells from line SCTi003-A-2 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 represent mean marker expression for each group of cells (n = 2 biological replicates). Expression of 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. iPSC = induced pluripotent stem cell.

Figure 6. SCTi003-A-2 Human iPSCs Can Efficiently Differentiate into Neural Progenitor Cells

Neural progenitor cells were generated from SCTi003-A-2 iPSCs using STEMdiff™ SMADi Neural Induction Kit (Catalog #08581) following the mono layer protocol described in the Product Information Sheet. (A) The resulting NPCs were fixed for immunocytochemistry. (B) The NPCs express neural progenitor markers PAX6 and SOX1 with low expression of SOX10. Error bars represent standard deviation (n = 3 biological replicates). NPCs = neural progenitor cells; iPSCs = induced pluripotent stem cells.

Figure 7. SCTi003-A-2 Human iPSCs Can Efficiently Differentiate into Forebrain Neurons and Astrocytes

(A) Neural progenitor cells derived from SCTi003-A-2 iPSCs were further differentiated using the STEMdiff™ Forebrain Neuron Differentiation Kit (Catalog #08600) and STEMdiff™ Forebrain Neuron Maturation Kit (Catalog #08605) to forebrain neurons th at express class III β-tubulin with low levels of GFAP. Similar differentiation of NPCs to astrocytes that express GFAP and S100B was achieved using the STEMdiff™ Astrocyte Differentiation Kit (Catalog #100-0013) and STEMdiff™ Astrocyte Serum-Free Maturation Kit (Catalog #100-1666). NPCs = neural progenitor cells; iPSCs = induced pluripotent stem cells.