Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture

Abstract

Summary: Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350–500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives. : Directed differentiation of human pluripotent stem cell cardiomyocytes (CMs) is required for clinical translation. Here, xeno-free suspension culture production in stirred bioreactors is shown to enable routine generation of ∼1.3 × 108 CMs/150 mL and ∼4.0 × 108 CMs/500 mL scale at >90% purity. Uninterrupted chemical WNT pathway control and the previously unappreciated role of ROR1/CD13 as predictive differentiation markers facilitate large-scale production of ventricular-like CMs. Keywords: human pluripotent stem cells, chemically defined process, cardiac differentiation, cardiomyocyte, suspension culture, stirred tank bioreactor, ROR1, WNT, process development, upscaling