ABSTRACT
A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/transplantation , rho-Associated Kinases/antagonists & inhibitors , Amides/pharmacology , Animals , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Cell Differentiation , Cells, Cultured , Disease Models, Animal , Down-Regulation/drug effects , Humans , Mice , Myocardial Infarction/pathology , Myocardial Infarction/therapy , Protein Subunits/metabolism , Pyridines/pharmacology , Troponin/metabolism , Verapamil/pharmacology , rho-Associated Kinases/metabolismABSTRACT
Aims: The effectiveness of cell-based treatments for regenerative myocardial therapy is limited by low rates of cell engraftment. Y-27632 inhibits Rho-associated protein kinase (ROCK), which regulates the cytoskeletal changes associated with cell adhesion, and has been used to protect cultured cells during their passaging. Here, we investigated whether preconditioning of cardiomyocytes, derived from human-induced pluripotent stem cells (hiPSC-CM), with Y-27632 improves their survival and engraftment in a murine model of acute myocardial infarction (MI). Methods and results: After MI induction, mice were subjected to intramyocardial injections of phosphate-buffered saline, hiPSC-CM cultured under standard conditions (hiPSC-CM-RI), or Y-27632-preconditioned hiPSC-CM (hiPSC-CM+RI). The resulting engraftment rate calculated 4 weeks after implantation was significantly higher and the abundance of apoptotic transplanted cells was significantly lower in hiPSC-CM+RI recipients than in hiPSC-CM-RI animals. In cultured hiPSC-CM, Y-27632-preconditioning reversibly reduced contractile activity and the expression of troponin genes, while increasing their attachment to an underlying mouse cardiomyocyte (HL1) monolayer. Y-27632 preconditioning also increased the expression of N-cadherin and integrin ß1, the two cell junction proteins. hiPSC-CM+RI were also larger in cell area with greater cytoskeletal alignment and a more rod-like shape than hiPSC-CM-RI, both after transplantation (in vivo) and in culture. The effects of Y-27632 preconditioning on contractile activity and morphology of hiPSC-CMs in culture, as well as on their engraftment rate and apoptotic death in MI mouse grafts, could be recapitulated by hiPSC-CM treatment with the L-type calcium-channel blocker verapamil. Conclusion: Preconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.
