Efficient multi-lineage cardiovascular differentiation of human pluripotent stem cells in animal serum-free conditions

Human induced pluripotent stem cell (hiPSC) technologies offer human-relevant cardiac models for biomedical applications. However, workflows for differentiation of cardiac stromal cells and fabrication of engineered heart tissue (EHT) commonly rely on animal serum, contrary to growing policy demands to reduce use of these products. Applying marker analysis via COL-I, DDR2 and GATA4 for cardiac fibroblasts or CD31, CD34 and CD144 for endothelial cells, we tailored Panexin, a defined serum substitute, to support high efficiency differentiation of cardiac stromal lineages to 85% purity without additional purification steps. We evaluated fabrication of EHTs using hiPSC-cardiomyocytes only (monoculture) or further combined with cardiac fibroblasts and endothelial cells (triculture; 70%:15%:15%, respectively). Panexin poorly supported fabrication and contractility of EHTs, a finding unaltered by modulating spontaneous cardiac myofibroblast activation via TGFβ inhibition. In contrast, human serum enabled fabrication of mono- and tri-culture EHTs, wherein constructs made without TGFβ signalling inhibition delivered the strongest contractile forces and exceeded comparator tissues engineered using animal serum. Our data show that iterative evaluation of serum substitutes, human serum, cell combinations and signalling pathway modulators can mitigate use of animal serum for functional EHT generation, aligning with the UK government's roadmap for alternative methods.