Figure 1. Generation of Human iPSC Line, SCTi003-A-4, MYH7 R403Q

Myosin heavy chain 7 (MYH7) gene structure is shown at the top. CRISPR-Cas9-mediated gene editing was employed to introduce the R403Q mutation into both alleles of the MYH7 gene in the parental SCTi003-A line (Catalog #200-0511). Targeted nucleotide substitutions were introduced at codon 403 to convert arginine (CGG) to glutamine (one allele CAG, the other CAA). The genomic region surrounding codon 403 in the parental SCTi003-A and edited SCTi003-A-4 lines was validated by Sanger sequencing. A portion of the corresponding sequences is shown in the inset, confirming homozygous introduction of the MYH7 R403Q mutation. iPSC = induced pluripotent stem cell.

Figure 2. Cells from Human iPSC Line, SCTi003-A-4, MYH7 R403Q Exhibit High-Quality Morphology in Routine Culture

Cryopreserved cells from the SCTi003-A-4 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-4, MYH7 R403Q Maintains a Normal Karyotype

(A) G-T-L banding for thawed cells from SCTi003-A-4 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 p34 iPSC using probes for 20p11.21 (green) and 20q11.21 (red). 93.8% 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-4, MYH7 R403Q Express Undifferentiated Cell Markers

Cells from the SCTi003-A-4 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. iPSC = induced pluripotent stem cell.

Figure 5. Cells from Human iPSC Line, SCTi003-A-4, MYH7 R403Q Demonstrate a High Trilineage Differentiation Capacity

Cells from the SCTi003-A-4 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-4, MYH7 R403Q Can Efficiently Differentiate into Functional Ventricular Cardiomyocytes

Ventricular cardiomyocytes were generated from SCTi003-A-4 iPSCs and the Healthy Control Human iPSC Line, Female, SCTi003-A (isogenic control; Catalog #200-0511) using STEMdiff™ Ventricular Cardiomyocyte Kit (Catalog #05010). (A) Immunocytochemistry analysis shows expression of the cardiomyocyte markers cardiac troponin T (cTnT; red) and α-actinin (green) in cardiomyocytes derived from both SCTi003-A-4 and the isogenic control. Nuclei were labeled with DAPI (blue). Differences in cell morphology and α-actinin organization are shown between SCTi003-A-4 cardiomyocytes and the isogenic control. Images were acquired at 63X magnification. (B) Flow cytometry was performed on Days 15 and 30 to assess the percentage of cTnT_ cells in the culture. (C) Quantification of cTnT_ cells showed that cardiomyocytes generated from SCTi003-A-4 iPSCs differentiated with comparable efficiency to the isogenic control, with high culture purity maintained through Day 30.

Figure 7. Cells from Human iPSC Line, SCTi003-A-4, MYH7 R403Q Exhibit Expected Morphological and Electrophysiological Phenotypes

Ventricular cardiomyocytes were generated from SCTi003-A-4 iPSCs and the Healthy Control Human iPSC Line, Female, SCTi003-A (isogenic control; Catalog #200-0511) using STEMdiff™ Ventricular Cardiomyocyte Kit (Catalog #05010). (A) Quantification of cell size by forward scatter (FSC) using flow cytometry showed a significant increase in cell area in Day 30 SCTi003-A-4 ventricular cardiomyocytes compared to the isogenic control (p < 0.05), with no difference observed on Day 15. Cell populations were categorized by size: on Day 15, small (FSC < 50) and large (FSC > 50) cells; and on Day 30, small (FSC < 65) and large (FSC > 65) cells. (B) Spontaneous field potential recordings of SCTi003-A-4-derived cardiomyocytes were collected using the Axion Maestro Pro™ MEA system on Day 15 and 30. Prior to recording, hPSC-derived ventricular cardiomyocytes were harvested and replated onto a CytoView MEA™ plate using STEMdiff™ Cardiomyocyte Dissociation Kit (Catalog #05025) and STEMdiff™ Cardiomyocyte Plating Supplement (Catalog #100-1120). (C) Quantification of spontaneous recordings showed that SCTi003-A-4 derived ventricular cardiomyocytes exhibited a detectable beat profile on Day 15; however, long-term culture resulted in no detectable beating on Day 30, despite maintaining high cTnT+ purity. MEA = multi-electrode array.