Generation of human induced pluripotent stem cells (hiPSCs) and subsequent differentiation to cardiomyocytes (hiPSC-CMs) has become trivial, in part due to the tools developed within our group. This hiPSC-CM model is essential to many projects funded by the AHA, including our recent Northwestern SFRN grant for which we are applying for this supplement. There, we proposed to evaluate patient-specific electrophysiological phenotypes in 60 hiPSC lines. This creates a need for large-scale differentiation of many hiPSC lines to produce high-quality, highly replicated and replicable data. This project has raised our awareness that the existing process to make hiPSC-CMs is slow and cumbersome, requiring constant differentiation of hiPSC lines, resulting in a major bottleneck for large cardiac genome/phenotype projects. We have previously worked extensively on methodologies for negligible-cost hiPSC culture (B8) and cardiac differentiation, including the first chemically defined protocol (CDM3). Most recently we have developed the first efficient protein-free differentiation media (PFDM) protocol. This simplification of the differentiation process, enhancement of robustness, and near elimination of media cost allows previously unachievable scale of hiPSC-CM production. Furthermore, for the last two years, we have worked on adapting our differentiation protocol to glass spinner flask suspension which allows much larger volumes of cells (0.1 L) within a single vessel. We have optimized a spheroid aggregation protocol utilizing B8 and basic cardiac differentiation achieving yields of >1e6 cardiomyocytes per mL. The next step up from spinner flasks is to use stirred tank perfusion bioreactors. These have been described numerous times for pluripotent hESC and hiPSC culture, with the most advanced work describing that, with carefully controlled perfusion, densities of 35e6 per mL can be achieved. Human cardiac differentiation in similar devices has produced yields of ~1e6 at 90% purity at a 0.1 L scale, yet these systems are expensive and the media costs prohibitive. Here we propose to tackle the issue of cumbersome and onerous hiPSC cardiac differentiation by adapting our protein-free differentiation to fully automated bioreactors. We intend for this system to be essentially hands-off for the entire 16-day process. Importantly we intend to develop this system using cost-effective modular parallel perfusion bioreactors that can easily be adopted by other laboratories.
|Effective start/end date||7/1/21 → 6/30/23|
- American Heart Association (874276)
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