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

INTERNATIONAL JOURNAL OF CARDIOLOGY

卷:168

Article

Moon, Sung-Hwan¹  Ban, Kiwon¹  Kim, Changhoon¹  Kim, Sang-Sung¹  Byun, Jaemin¹  Song, Ming-Ke²  Park, In-Hyun³  Yu, Shan Ping²  Yoon, Young-Sup¹ 

[1] Emory Univ, Sch Med, Div Cardiol, Dept Med, Atlanta, GA 30322 USA

[2] Emory Univ, Sch Med, Dept Anesthesiol, Atlanta, GA 30322 USA

[3] Yale Univ, Sch Med, Dept Genet, New Haven, CT 06510 USA

关键词: Human embryonic stem cells;    Human induced pluripotent stem cells;    Cardiomyocytes;    Directed differentiation;   

DOI  :  10.1016/j.ijcard.2012.09.077

来源: Elsevier

【 摘 要 】

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 similar to 90% showed CM phenotypes confirmed by an a-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. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

【 授权许可】

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