Patient-derived induced pluripotent stem cells to study non-canonical splicing variants associated with Hypertrophic Cardiomyopathy

Fig. 1. Schematic representation of MYBPC3 showing Sanger sequencing of the regions where the c.1224-52G > A and c.1898-23A > G variants are located.

Fig. 2. Characterisation of the 3_H24 and 4_H17 iPSC lines. (A) Schematic of the Sendai virus reprogramming protocol of PBMCs to iPSCs. (B) On the left, bright field photomicrograph of iPSC colonies. Scale bar, 200 μm. On the right, representative immunofluorescence images for OCT4 and SOX2 staining. Scale bar, 20 μm. (C) Flow cytometry analysis of the indicated pluripotent stem cell markers. (D) G-banding karyotyping.

Fig. 3. Directed differentiation of iPSCs to cardiomyocytes. (A) Timeline and cell culture conditions used for differentiation. (B) qRT-PCR analysis of MYBPC3 and TNNT2 cardiac markers in iPSC-CMs derived from a healthy individual at day 0 (d0) and day 13 (d13) of differentiation. Graphs depict mRNA expression levels (2–ΔCt) relative to U6 snRNA. Dots represent data from 3 independent differentiation experiments. The mean ± SEM is depicted. (C) Immunofluorescence of iPSC-CMs derived from a healthy individual at day 13 of differentiation. Sarcomeric proteins c-MyBP-C and cTnT are stained in green. Nuclei stained with DAPI (in blue).

Fig. 4. Impact of the MYBPC3 intronic variants on RNA and protein levels.(A, B) On the left, semi-quantitative RT-PCR analysis using the indicated primers (black arrows) in iPSC-CMs derived from a healthy individual (WT) and HCM patients with the c.1224-52G > A (A) and c.1898-23A > G (B) variants. Cells were either untreated (−CHX) or treated (+CHX) with cycloheximide for 6 h. Molecular size markers are indicated. On the right, Sanger sequencing of amplified RT-PCR products. (C) qRT-PCR analysis of MYBPC3 mRNA relative to TNNT2 in the indicated iPSC-CMs at day 13 of differentiation. Dots represent data from 3 independent differentiation experiments. The mean ± SEM is depicted. * p < 0,05; n.s. not significant. (D) Immunofluorescence of mutant iPSC-CMs at day 13 of differentiation. Sarcomeric proteins c-MyBP-C and cTnT are stained in green. Nuclei stained with DAPI (in blue). (E) Western blot analysis of the indicated iPSC-CMs at day 13 of differentiation. Molecular mass markers are indicated on the left. Quantification of the c-MyBP-C band intensity relative to cTnT is shown, in percentage, below. Data from 3 independent differentiation experiments.

Supplementary Fig. S1. Characterisation of the 6_H35 and 4_H22 iPSC lines. (A) On the left, bright field photomicrograph of iPSC colonies. Scale bar, 200 μm. On the right, representative immunofluorescence images for OCT4 and SOX2 staining. Scale bar, 20 μm. (B) Flow cytometry analysis of the indicated pluripotent stem cell markers. (C) G-banding karyotyping. (D) Sanger sequencing of the c.1898-23A>G and c.1224-52G>A iPSC clones, 6_H35 and 4_H22, respectively.

Supplementary Fig. S2. Trilineage differentiation potential of 3_H24, 4_H17, 6_H35, and 4_H22 iPSC lines. (A, C) Representative immunofluorescence images for trilineage differentiation. Endoderm (SOX17 and CD184), Mesoderm (αSMA) and Ectoderm (PAX6 and NESTIN). Scale bar, 20 μm. (B, D) qRT-PCR analysis of the expression of Brachyury, upon mesoderm differentiation of iPSC clones for 5 days, compared to non-differentiated iPSCs. Graphs depict mRNA expression levels relative to U6 snRNA.

Supplementary Fig. S3. Splicing and NMD analysis in iPSC-CMs carrying the MYBPC3 c.1898-23A>G variant. (A) qRT-PCR analysis of MYBPC3 intron 19 retention in WT and mutant iPSC-CMs at day 13 of differentiation. The graph depicts relative expression levels normalized to total MYBPC3 mRNA (2–ΔCt). Dots represent data from 3 independent differentiation experiments. The mean ± SEM is depicted. ** p<0,01. (B) Schematic representation of the construct used to evaluate NMD activity. (C) qRT-PCR analysis of firefly luciferase mRNA levels in WT and mutant iPSC-CMs transfected at day 13 of differentiation. One day after transfection, cells were untreated (-CHX) or treated (+CHX) with cycloheximide for 6 hours. The graph depicts relative expression levels normalized to the U6 snRNA (2–ΔCt). Dots represent data from 2 independent transfection experiments. The mean ± SEM is depicted. * p<0,05.