Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP(+) C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit(+)), endothelial cells (CD31(+), CD34(+)), and mesenchymal cells (CD90(+)). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used

Cellular Postconditioning: Allogeneic Cardiosphere-Derived Cells Reduce Infarct Size and Attenuate Microvascular Obstruction When Administered After Reperfusion in Pigs With Acute Myocardial Infarction

Intracoronary (IC) delivery of cardiosphere-derived cells (CDCs) has been demonstrated to be safe and effective in porcine and human chronic myocardial infarction (MI). However, IC delivery of CDCs after reperfusion in acute MI has never been assessed in a clinically-relevant large animal model. We tested CDCs as adjunctive therapy to reperfusion in a porcine model of MI

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+).Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency

Effect of intra-aortic balloon pump counterpulsation on left ventricular systolic and diastolic function

We investigated the effects of IABP counterpulsation on left ventricular (LV) contractility, relaxation and energy consumption and probed the underlying physiologic mechanisms in 12 farm pigs, using an ischemia-reperfusion model of acute heart failure. During both ischemia and reperfusion, IABP support unloaded the LV, decreased LV energy consumption (pressure volume area, stroke work) and concurrently improved LV mechanical performance (ejection fraction, stroke volume, cardiac output). During reperfusion exclusively, IABP also improved LV relaxation (tau) and contractility (Emax, PRSW). The beneficial effects of IABP support on LV relaxation and contractility correlated with IABP-induced augmentation of coronary blood flow. In conclusion, we find that during both ischemia and reperfusion, IABP support optimizes LV energetic performance (decreases energy consumption and concurrently improves mechanical performance) by LV unloading. During reperfusion exclusively, IABP support also improves LV contractility and active relaxation, possibly due to a synergistic effect of unloading and augmentation of coronary blood flow.Μελετήσαμε την επίδραση της υποβοήθησης με ΕΑΑ στη συσταλτικότητα, στην ενεργητική χάλαση και στην ενεργειακή κατανάλωση της αριστερής κοιλίας και διερευνήσαμε τους υποκείμενους παθοφυσιολογικούς μηχανισμούς σε 12 χοίρους με οξεία καρδιακή ανεπάρκεια κατά την ισχαιμία και την επαναιμάτωση. Βρήκαμε πως η υποβοήθηση με ΕΑΑ αποφορτίζει την αριστερή κοιλία, μειώνει την ενεργειακή της κατανάλωση (pressure volume area, έργο παλμού) και παράλληλα αυξάνει τη μηχανική της απόδοση (κλάσμα εξώθησης, όγκος παλμού, καρδιακή παροχή) τόσο κατά την ισχαιμία όσο και κατά την επαναιμάτωση. Κατά την επαναιμάτωση, η υποβοήθηση με ΕΑΑ επιπρόσθετα αυξάνει τη συσταλτικότητα (μέγιστη ελαστικότητα, preload-recruitable stroke work) και βελτιώνει την ενεργητική χάλαση (σταθερά χρόνου της ισοογκωτικής χάλασης) της αριστερής κοιλίας. Η αύξηση της συσταλτικότητας και η βελτίωση της χάλασης κατά την επαναιμάτωση συσχετίζονται εν μέρει με την αύξηση της στεφανιαίας ροής από την ΕΑΑ. Συμπερασματικά, η υποβοήθηση με ΕΑΑ βελτιστοποιεί την μηχανοενεργητική της αριστερής κοιλίας (μειώνει την ενεργειακή κατανάλωση και παράλληλα αυξάνει τη μηχανική της απόδοση) τόσο κατά την ισχαιμία όσο και κατά την επαναιμάτωση μέσω κοιλιακής αποφόρτισης. Κατά την επαιναμάτωση, η υποβοήθηση με ΕΑΑ επιπρόσθετα βελτιώνει τη συσταλτικότητα και την ενεργητική χάλαση της αριστερής κοιλίας, μέσω αύξησης της στεφανιαίας αιμάτωσης