Cardiomyocyte production in mass suspension culture: Embryonic stem cells as a source for great amounts of functional cardiomyocytes

Transplantation of cardiomyocytes derived from embryonic stem (ES) cells into damaged or non-functional areas of the myocardium might be a valuable approach to treat heart failure, provided that they can be generated in sufficient quantity and with sufficient purity. Cultivation of ES cells in a standard stirred tank bioreactor makes it possible to generate large numbers of therapeutically useful cells such as cardiomyocytes. We have used this technique to expand genetically engineered ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MyHc) promotor driving the aminoglycoside phosphotransferase gene (neomycin resistance). Addition of G418 to the growth medium allowed an efficient enrichment of cardiomyocytes. We developed a draw-and-fill process in a controlled stirred tank reactor at the 2L scale to facilitate long-term cultivation under stable, reproducible conditions. Throughout the culture period cell viability and EB numbers were measured monitored by flow cytometry, immunohistochemistry and fluorescence microscopy of cardiac specific antigens. About 9e8 cardiomyocytes in 2e5 cardiac bodies were generated. The purity of the population was about 99% measured by MF20 immunohistochemistry and fluorescence microscopy. To analyze the ability of ES-cell derived cardiomyocytes to reconstitute damaged heart tissue cardiac bodies were transplanted into female ICR-mice 7 days after myocardial infarction induced by LAD ligation. Both echocardiographic follow-up and histological analysis 28d after transplantation showed an improved left ventricular function of hearts that received a transplant in comparison to sham-treated mice. We here demonstrate that it is feasible to generate functional cardiomyocytes derived from embryonic stem cells in a bioreactor at a large-scale that retain their functionally in vivo and improve cardiac functions of infarcted hearts. With only one bioreactor process it is possible to provide sufficient cardiomyocytes for a cell based therapy.

Acknowledgement: The author S. Niebruegge is supported by the Max-Buchner-Forschungsstiftung, DECHEMA. H. Ebelt was supported by the Wilhelm-Roux-Program for Research of the Martin-Luther-University.