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Improved in vitro methods for differentiating induced pluripotent stem cells to cardiomyocytes

University of British Columbia

Human adult cardiomyocytes are hard to obtain and difficult to culture. This has led to patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iCMs) becoming an attractive alternative, serving as a platform for cardiovascular disease modeling, drug toxicity screening, regenerative therapies, and the understanding of human physiology. Unfortunately, current protocols of generating iCMs remain unpredictable, yielding cellular subtypes (ventricular, atrial, nodal cells, cardiac fibroblasts and others) that vary in proportion and levels of maturity, depending primarily on the methodology employed. This led to the scope of my research, which was to understand the minimal biological and physical parameters governing cardiac differentiation from iPS cells, so as to improve current cardiac differentiation systems. My experiments into the effects of substrate stiffness on differentiation revealed that cardiac maturity could be accelerated for iPS cells differentiated on hard substrates (~1.72MPa) compared to cells differentiated on soft (~5kPa) or intermediate (~130kPa) substrates. Experiments investigating the effects of topography also showed that freshly differentiated iCMs could be directed to form uniaxial contracting cardiac tissues by seeding them into millimeter-sized linear grooved channels. Finally, proof-of-principle experiments into inductive co-cultures revealed that previously differentiated iCMs co-cultivated with iPS cells constituted a sufficient stimulatory system to induce cardiac differentiation – as determined by the exhibition of spontaneous self-contractions, expression of cardiac specific markers, and structural organization of sarcomeres. This process was achieved without the exogenous addition of pathway inhibitors and morphogens, suggesting that ‘older’ iCMs serves as an adequate stimulatory source capable of recapitulating the necessary culture environment for cardiac differentiation.

Text

2018-03-28

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/64988

Biomedical Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/