Morpho-functional comparison of differentiation protocols to create iPSC-derived cardiomyocytes

Area of Science:

• Cardiovascular Research
• Stem Cell Biology

Background:

• Induced pluripotent stem cells (iPSC) can be differentiated into cardiomyocytes (iPSC-CMs).
• Patient-specific iPSC-CMs are valuable for disease modeling, diagnostics, and personalized medicine.
• Existing differentiation protocols lack comprehensive performance data.

Purpose of the Study:

• To evaluate two Wnt/β-catenin pathway modulation protocols for iPSC-CM differentiation.
• To characterize iPSC-CMs derived from control and SCN5A mutation-carrying lines.
• To compare the morpho-functional and electrophysiological properties of iPSC-CMs generated by the two protocols.

Main Methods:

• Differentiation of four iPSC lines (two control, two SCN5A mutation) into iPSC-CMs using two serum-free protocols.
• Molecular characterization via immunostaining and qPCR for cardiac markers.
• Assessment of cell morphology, contractility, and survival post-dissociation.
• Electrophysiological evaluation of action potentials (AP) and calcium transients (CT) using patch-clamping and optical imaging.

Main Results:

• Both protocols produced a high percentage of contracting iPSC-CMs with acceptable post-dissociation survival (>50%).
• Both protocols yielded iPSC-CMs with typical AP and CT characteristics.
• One protocol (sequential CHIR99021 and Wnt-C59) resulted in iPSC-CMs more amenable to patch-clamping and calcium imaging, with gene expression closer to human left ventricle samples.

Conclusions:

• Both Wnt/β-catenin modulation protocols are effective for iPSC-CM generation.
• The sequential CHIR99021/Wnt-C59 protocol offers advantages for electrophysiological studies and exhibits a more physiologically relevant gene expression profile.