Figure 1. Generation of Human iPSC Line, SCTi003-A-3, ABCA4 Knockout

ABCA4 gene structure map is shown on the top. CRISPR-Cas9-mediated gene editing was employed to introduce frameshift mutations in Exon 3 of ABCA4, resulting in loss-of-function for both alleles. The sequences of the parental SCTi003-A line (Catalog #200-0511) and the SCTi003-A-3 line were validated by Sanger sequencing and shown in the inset. A 1 bp insertion was introduced into one allele and 1 bp deletion was introduced into the other, resulting in early stop codons and a complete loss-of-function in these truncated ABCA4 alleles.

Figure 2. SCTi003-A-3 Human iPSCsDemonstrate High-Quality Morphology in Routine Culture

Cryopreserved cells from line SCTi003-A-3 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-3 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 - 450 G-bands per haploid genome. (B) Fluorescent in situ hybridization in a representative p34 iPSC using probes for 20p11 (green) and 20q11.21 (red). 96.5% 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-3 Human iPSCs Express Undifferentiated Cell Markers

Cells from line SCTi003-A-3 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-3 Human iPSCs Demonstrate a High Trilineage Differentiation Capacity

Cells from SCTi003-A-3 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-3 iPSC line Can be Differentiated to Mature RPE with Comparable Functionality to the Isogenic Control SCTi003-A

SCTi003-A-3 and SCTi003-A (Isogenic Control; Catalog #200-0511) were differentiated into RPE cells using STEMdiff™-ACF RPE Differentiation Kit (Catalog #100-1367) and subsequently subcultured on cell culture inserts or tissue culture plates in STEMdiff™-XF RPE Maturation Medium (Catalog #100-1365) for 5 weeks. Maturity and functionality of the RPE derived from SCTi003-A-3 were assessed and compared to the isogenic control SCTi003-A to ensure ABCA4 knockout does not impact correct RPE development. (A) Brightfield microscopy images of mature RPE. (B) The percentage of RPE cells expressing PMEL17, CRALBP, EZRIN, and RPE65 was assessed by flow cytometry analysis. Data are reported as mean (n = 2). (C) Mature RPE generated a strong barrier with high TER. Data shown as mean + SEM; n = 3. (D,E) Apical and basal conditioned medium were collected from mature RPE, and a sandwich ELISA was performed to quantify VEGF and PEDF secretion. Mature RPE secreted more basal VEGF and apical PEDF demonstrating that RPE display correct apicobasal polarity. Data shown as mean + SEM; n = 3. (F) Mature RPE efficiently internalized bovine POS. Data shown as mean + SEM; n = 3. iPSC = induced pluripotent stem cell; PEDF = pigment epithelial derived growth factor; POS = photoreceptor outer segments; RPE = retinal pigment epithelium; TER = transepithelial resistance; VEGF = vascular endothelial growth factor.

Figure 7. Immunostaining Confirms Absence of ABCA4 Protein in STi003-A-3 Derived RPE Cells

Mature RPE differentiated from the healthy control human iPSC line, SCTi003-A (Isogenic Control; Catalog #200-0511), display robust ABCA4 protein expression. In contrast, RPE cells derived from the SCTi003-A-3 (ABCA4 knockout) iPSC line show complete absence of ABCA4, validating the knockout and confirming the null phenotype. iPSC = induced pluripotent stem cell; RPE = retinal pigment epithelium.

Figure 8. SCTi003-A-3 Derived RPE Cells Show Impaired POS Clearance and Lipid Accumulation Compared to Isogenic Control, recapitulating Stargardt’s Disease Phenotype

iPSCs from SCTi003-A-3 and SCTi003-A (Isogenic Control; Catalog #200-0511) were differentiated into RPE cells using STEMdiff™-ACF RPE Differentiation Kit (Catalog #100-1367) and subsequently matured in STEMdiff™-XF RPE Maturation Medium (Catalog #100-1365) for 5 weeks. Mature RPE were fed bovine POS prior to being enzymatically dissociated for flow cytometry analysis or fixed with paraformaldehyde for immunostaining. (A) 24 hours after POS feeding, healthy control human iPSC line, SCTi003-A (Isogenic Control) contain less undigested POS compared to SCTi003-A-3 (ABCA4 knockout), highlighting ABCA4 knockout results in impaired POS digestion (B,C) POS feeding for 7 days induced elevated intracellular lipids in ABCA4 knockout RPE, as confirmed by increased BODIPY staining. iPSC = induced pluripotent stem cell; POS = photoreceptor outer segments; RPE = retinal pigment epithelium.