title: A systemic evaluation of cardiac differentiation from mRNA reprogrammed human induced pluripotent stem cells.
abstract: Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by "foot-print free" reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Ashish
authors: A
authors: Mehta
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Vinod
authors: V
authors: Verma
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Manasi
authors: M
authors: Nandihalli
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Chrishan J A
authors: CJ
authors: Ramachandra
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Glen L
authors: GL
authors: Sequiera
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Yuliansa
authors: Y
authors: Sudibyo
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore.
authors: Yingying
authors: Y
authors: Chung
authors: Experimental and Therapeutics Centre, A'STAR, Singapore, Singapore.
authors: William
authors: W
authors: Sun
authors: National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore; Cardiovascular and Metabolic Disorders Program, DUKE-NUS, Singapore, Singapore.
authors: Winston
authors: W
authors: Shim
mesh_terms: D002217:Carbachol / Q000494:pharmacology; D002316:Cardiotonic Agents / Q000494:pharmacology; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000187:drug effects / Q000235:genetics; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D018679:Cholinergic Agonists / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D008875:Middle Aged; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens / Q000235:genetics / Q000378:metabolism
chemical_list: D002316:Cardiotonic Agents; D018679:Cholinergic Agonists; D007473:Ion Channels; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D002217:Carbachol; D007545:Isoproterenol