Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture

Abstract

Introduction: Myocardial infarction is accompanied by a significant loss of cardiomyocytes (CMs). Functional CMs, differentiated from human embryonic stem cells (hESCs), offer a potentially unlimited cell source for cardiac disease therapies and regenerative cardiovascular medicine. However, conventional production methods on monolayer culture surfaces cannot adequately supply the large numbers of cells required for such treatments. To this end, an integrated microcarrier (MC) bioprocessing system for hESC propagation and subsequent CM differentiation was developed. Methods: Production of hESC-derived CMs was initially established in monolayer cultures. This control condition was compared against hESC expansion on laminin-coated MC with cationic surface charge, in a stirred serum-free defined culture. Following expansion, the hESC/MC aggregates were placed in a CM differentiation medium, using Wnt signalling modulators in four different culture conditions. This process eliminated the need for manual colony cutting. The final optimized protocol was tested in stirred spinner flasks, combining expansion and differentiation on the same MC, with only media changes during the culture process. Results: In the propagation phase, a 15-fold expansion of viable pluripotent HES-3 was achieved, with homogeneous sized aggregates of 316 ± 11 μm. Of the four differentiation conditions, stirred spinner flask cultures (MC-Sp) provided the best controlled aggregate sizes and yielded 1.9 × 106 CM/ml, as compared to 0.5 × 106 CM/ml using the monolayer cultures method: a four-fold increase in CM/ml. Similar results (1.3 × 106 CM/ml) were obtained with an alternative hESC H7 line. The hESC/MC-derived CM expressed cardiac-specific transcription factors, structural, ion channel genes, and exhibited cross-striations of sarcomeric proteins, thus confirming their cardiac ontogeny. Moreover, E-4031 (0.3 μM) prolonged the QT-interval duration by 40% and verapamil (3 μM) reduced it by 45%, illustrating the suitability of these CM for pharmacological assays. Conclusions: We have demonstrated a robust and scalable microcarrier system for generating hESC-derived CM. This platform is enabled by defined microcarrier matrices and it integrates cell propagation and differentiation within a continuous process, in serum-free culture media. It can generate significant numbers of CM, which are potentially suitable for future clinical therapies.