Embedment of highly purified murine iPS-derived cardiomyocytes in biodegradable macroporous microspheres as microcarriers facilitates intramyocardial injection of huge absolute cell numbers without altering low fractional cell engraftment

摘要

Objectives: Heart failure is a major cause of morbidity and mortality in the world and cardiac cell replacement therapy is a promising strategy to restore cardiac function in heart failure. Since functional cardiomyocytes are the lacking cells in heart failure, iPS derived cardiomyocytes (iPS CM) are good candidates for transplantation. However, engraftment efficiency is very limited for purified iPS CM and the small volumes suitable for intramyocardial injection in mice limits the absolute number of transplanted cells. Therefore, we tested embedment of purified murine iPSCM in biodegradable macroporous microspheres as microcarriers for intramyocardial injection. Methods: Male murine Acta2 iPS expressing an antibiotic resistance and eGFP under the control of the Acta2 promotor we re differentiated to Acta2 iPS CM in hanging drops and highly purified with respective antibiotic treatment. Prepared single cell suspensions were either used directly for intramyocardial injections (iPS CM alone) or they were cocultured with biodegradable macroporous microspheres and the loaded microcarriers were intramyocardially injected (iPS CM in microspheres). Healthy syngeneic female mice served as recipients in an open chest surgery with 2 intramyocardial injections of 10 l each. Hearts were excised immediately after surgery (0h) or after 24h, genomic DNA was prepared and the number of persisting transplanted cells was determined using quantitative realtime PCR with y chromosome specific primers.For every surgery day, 1 aliquot was mixed with an explanted heart ex vivo and served as control and known dilutions of male in female DNA were included to derive a calibration curve