Co-transplanted non-cardiomyocytes enhance early persistence of induced pluripotent stem cell derived cardiomyocytes after intramyocardial injection but they also proliferate in loco over time

Abstract

Purpose: Induced pluripotent stem cell derived cardiomyocytes (iPS-CM) are suitable for cardiac cell replacement therapy since they can integrate into host myocardium after transplantation and thereby improve heart function. However, highly purification is needed to avoid teratogenic risk but it leads to poor persistence of transplanted iPS-CM which we here tried to improve by co-transplantation of non-cardiomyocytes (non-CM). Methods: Male iPS-CM were derived from transgenic murine induced pluripotent stem cells and highly purified using an antibiotic resistance under a cardiac specific promoter. Intramyocardial injection of 300,000 iPS-CM with or without 300,000 male wild-type murine non-CM (embryonic fibroblasts [+MEF] or adult mesenchymal bone marrow cells [+MSC]) was performed into healthy hearts of syngeneic female wild-type mice. Immediately (0h) or after 24h, 48h or 7 days (each n≥4 per group), hearts were harvested and the number of persisting transplanted cells was determined by quantitative real-time PCR with specific primer for SRY or transgene. One additional cell aliquot was mixed with an explanted native heart ex vivo as control (=100%) for every surgery day. Results: Immediately after transplantation of iPS-CM alone (0h), 28.4±4.0% of transplanted cells were detected in recipient hearts and the number decreased to 2.6±0.9% at 6h (p<.001 vs. 0h) and 0.6±0.2% at 24h (p<.0001 vs. 0h). After co-transplantation with non-CM the number of detectable male cells (iPS-CM+non-CM) at 24h was more than 4-fold increased to 2.7±1.5% (+MEF) and 2.8±1.0% (+MSC, p=.06 vs. iPS-CM alone) and it increased over time in both groups up to 15.8±5.6% (+MEF) or 4.7±1.7% (+MSC) at 48h and 12.3±5.2% (+MEF) or 6.2±1.6% (+MSC) at 7d (p for linear trend <.01 in both groups). The number of detectable transgenic cells (iPS-CM) after co-transplantation was more than 2-fold higher than in iPS-CM alone at 24h with 1.3±0.7% (+MEF) and 1.5±0.2% (+MSC, p<.05 vs. iPS-CM alone), and it remained stable over time in +MEF (48h: 2.4±1.2%, 7d: 2.4±0.9%) and it tended to decrease in +MSC (48h: 0.3±0.1%, 7d: 0.7±0.4%, p=.1 vs. 24h). Accordingly, the fraction of iPS-CM within the persisting transplanted cells decreased over time. Conclusions: Persistence of highly purified iPS-CM at 24h after intramyocardial injection into syngeneic mouse hearts is poor (<1%) but it is increased by co-transplantation of non-CM (MEF or MSC). While this higher persistence remains stable over time only with co-transplanted MEF, proliferation of non-CM is seen in both co-transplantation groups and must be cautiously monitored.