Billion-Scale production of functional hiPSC-derived cardiomyocytes in bioreactors through oxygen control and Wnt Activation

Producing therapeutic quantities of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for myocardial infarction treatment remains a significant challenge. We demonstrate that precise control of dissolved oxygen (DO) at low levels combined with Wnt pathway activation, through continuous perfusion of CHIR99021, enables the production of billions of functional hiPSC-CM in stirred-tank bioreactors (STB). Specifically, culturing hiPSC-CM aggregates under 10%O2 led to an upregulation of pathways involved in cell proliferation and survival, resulting in a significantly higher hiPSC-CM expansion factor when compared to hiPSC-CM under atmospheric normoxia and static culture systems. Scaling up the process to a 2L STB, while maintaining constant power input per volume, generated 4 billion highly pure hiPSC-CM, with more than 90% positive-cells for cardiac markers. hiPSC-CM aggregates also showed spontaneous beating, expression of cardiac markers and were able to further mature in culture, revealing improved gene expression, sarcomere alignment and cardiac action potential. Overall design: Bulk RNA-seq of cardiomyocytes produced in different conditions both in bioreactor (normoxia, hypoxia) and in T-flasks, as well as two weeks of maturation after bioreactor production under hypoxia