Evaluation of Cardiomyocyte Proliferation after Transplantation of Engineered Heart Tissue Patches

Abstract

Transplantation of engineered heart tissue (EHT) derived from human induced pluripotent stem cells (hiPSC) represents a promising regenerative strategy. Recent studies provided first evidence that hiPSC-derived cardiomyocytes re-enter the cell cycle after transplantation. The aim of this project was to analyze cardiomyocyte proliferation after transplantation in detail. Large mesh-structured EHT patches (15 × 106 cells; 15 mm x 27 mm x 1.5 mm) were generated from hiPSC. Cardiac cryo-injury was induced in guinea pigs and EHT patches were transplanted seven days after injury. Hearts were harvested at varying time points after transplantation and prepared for histological analysis. Immunofluorescence staining for either Ki67 (proliferation marker) + PCM1 (cardiomyocyte nuclei marker) or Ki67 + human Ku80 (human cell marker) was performed to unequivocally analyze cell cycle activity in human cardiomyocytes. The percentage of double-positivity was examined at baseline as well as two and four weeks after transplantation. To unambiguously clarify whether the observed cell cycle activity was true cellular division or not, immunofluorescence staining of Aurora B kinase (marker for midbody positioning) and PCM1 was performed. At baseline, approximately 9 % of human cardiomyocytes in the EHTs expressed the cell cycle marker Ki67 (10 % co-expression of PCM1/Ki67 and 8 % of human Ku80/Ki67). EHT patch transplantation resulted in large human grafts. Hearts that were harvested two weeks after transplantation showed an even higher cell cycle activity of the human cardiomyocytes (21 % co-expression of PCM1/Ki67 or 14 % human Ku80/Ki67 respectively). Four weeks after transplantation, the latest time point analyzed so far, about 12% of human cardiomyocytes were still in the cell cycle (13% PCM1/Ki67 and 10% human Ku80/Ki67 double positive cells). Preliminary data depicted a symmetrical localization of Aurora B kinase in several cases, indicating true cellular division. The current study describes the optimization of EHT geometry. EHT patches structurally and physiologically resembled adult human ventricular myocardium and EHT patch transplantation resulted in large human myocardial grafts that led to complete coverage of the scar area.