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Development of a novel two-dimensional directed differentiation system for generation of cardiomyocytes from human pluripotent stem cells

 Sung-Hwan Moon  ;  Kiwon Ban  ;  Changhoon Kim  ;  Sang-Sung Kim  ;  Jaemin Byun  ;  Ming-Ke Song  ;  In-Hyun Park  ;  Shan Ping Yu  ;  Young-Sup Yoon 
 International Journal of Cardiology, Vol.168(1) : 41-52, 2013 
Journal Title
 International Journal of Cardiology 
Issue Date
Animals ; Cell Differentiation/physiology* ; Humans ; Induced Pluripotent Stem Cells/physiology* ; Induced Pluripotent Stem Cells/transplantation* ; Male ; Myocardial Infarction/pathology ; Myocardial Infarction/surgery ; Myocytes, Cardiac/physiology* ; Myocytes, Cardiac/transplantation* ; Pluripotent Stem Cells/physiology ; Pluripotent Stem Cells/transplantation ; Primary Cell Culture/methods* ; Primary Cell Culture/trends ; Rats, Nude
2D ; AP ; AP duration from the peak to 90% repolarization ; APD 90 ; CMs ; Cardiomyocytes ; Directed differentiation ; EB ; Human embryonic stem cells ; Human induced pluripotent stem cells ; action potential ; cardiomyocytes ; embryoid body ; hESCs ; hPSCs ; hiPSCs ; human embryonic stem cells ; human induced pluripotent stem cells ; human pluripotent stem cells ; two dimensional
BACKGROUND: Human pluripotent stem cells (hPSCs) hold great promise for treating ischemic heart disease. However, current protocols for differentiating hPSCs either result in low yields or require expensive cytokines. METHODS: Here we developed a novel two dimensional (2D) stepwise differentiation system that generates a high yield of cardiomyocytes (CMs) from hPSCs without using special cytokines. Initially, undifferentiated hPSCs were transferred onto Matrigel-coated plates without forming embryoid bodies (EBs) for a few days and were cultured in bFGF-depleted human embryonic stem cells (hESCs) medium. When linear cell aggregation appeared in the margins of the hPSC colonies, the medium was changed to DMEM supplemented with 10% fetal bovine serum (FBS). Thereafter when cell clusters became visible, the medium was changed to DMEM with 20% FBS. RESULTS AND CONCLUSIONS: At about two weeks of culture, contracting clusters began to appear and the number of contracting clusters continuously increased, reaching approximately 70% of all clusters. These clusters were dissociated by two-step enzyme treatment to monolayered CMs, of which ~90% showed CM phenotypes confirmed by an α-myosin heavy chain reporter system. Electrophysiologic studies demonstrated that the hPSC-derived CMs showed three major CM action potential types with 61 to 78% having a ventricular-CM phenotype. This differentiation system showed a clear spatiotemporal role of the surrounding endodermal cells for differentiation of mesodermal cell clusters into CMs. In conclusion, this system provides a novel platform to generate CMs from hPSCs at high yield without using cytokines and to study the development of hPSCs into CMs.
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Yonsei Authors
Yoon, Young Sup(윤영섭)
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