Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype...

Area of Science:

• Biomedical Engineering
• Cell Biology
• Tissue Engineering

Background:

• Traditional in vitro methods struggle to replicate the complex cardiac environment due to single-cue manipulation.
• Native myocardial niches involve multiple biophysical cues crucial for cardiomyocyte development and phenotype maintenance.
• Understanding these cues is vital for advancing cardiac research and regenerative medicine.

Purpose of the Study:

• To establish a novel biofabrication workflow for studying human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a multiplexed biophysical environment.
• To investigate the response of single hiPSC-CMs to independently tunable 3D shape and substrate stiffness.
• To elucidate the role of biophysical cues in dictating hiPSC-CM phenotype and functionality in isolation.

Main Methods:

• Fabrication of silicon masters to create inverse 3D microwell patterns in hydrogels.
• Modification of polyacrylamide (PAAm) hydrogels to independently control 3D shape and stiffness while enabling adhesion motif conjugation.
• Isolation of single hiPSC-CMs within the microwell platform to study responses to biophysical cues without cell-cell interactions.

Main Results:

• Under physiologic-like conditions (3D shape, 9.83 kPa stiffness), isolated hiPSC-CMs showed increased Cx-43 density, cell membrane stiffness, calcium transient amplitude, MYL2-MYL7 co-expression, and anisotropism.
• Cells cultured in pathologic-like conditions (flat surface, 112 kPa stiffness) exhibited distinct cellular features and a differential phenotype.
• Demonstrated significant plasticity of hiPSC-CMs in response to varying biophysical cues, even in isolation.

Conclusions:

• Multiplexed biophysical cues, specifically 3D shape and substrate stiffness, profoundly influence isolated hiPSC-CM phenotype and functionality.
• The biofabrication platform enables precise control over the cellular microenvironment, revealing hiPSC-CM plasticity.
• This technology holds potential for generating fit-for-purpose hiPSC-CMs and advancing the understanding of cardiac development and other tissue engineering applications.