Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

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Abstract
It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES‐derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large‐scale culture of ES‐derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched‐blade‐turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high‐density suspension cultures containing 12.5 × 106 cells/mL after 9 days of differentiation. Approximately 30%–40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte‐restricted alpha myosin heavy chain (αMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES‐derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 × 109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large‐scale generation of ES‐derived cells for therapeutic and industrial applications.