title: Development of a novel two-dimensional directed differentiation system for generation of cardiomyocytes from human pluripotent stem cells.
abstract: 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.
  
  
  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.
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, United States.
authors.forename: Sung-Hwan
authors.initials: SH
authors.lastname: Moon
authors.forename: Kiwon
authors.initials: K
authors.lastname: Ban
authors.forename: Changhoon
authors.initials: C
authors.lastname: Kim
authors.forename: Sang-Sung
authors.initials: SS
authors.lastname: Kim
authors.forename: Jaemin
authors.initials: J
authors.lastname: Byun
authors.forename: Ming-Ke
authors.initials: MK
authors.lastname: Song
authors.forename: In-Hyun
authors.initials: IH
authors.lastname: Park
authors.forename: Shan Ping
authors.initials: SP
authors.lastname: Yu
authors.forename: Young-Sup
authors.initials: YS
authors.lastname: Yoon
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology* / Q000637:transplantation*; D008297:Male; D009203:Myocardial Infarction / Q000473:pathology / Q000601:surgery; D032383:Myocytes, Cardiac / Q000502:physiology* / Q000637:transplantation*; D039904:Pluripotent Stem Cells / Q000502:physiology / Q000637:transplantation; D061251:Primary Cell Culture / Q000379:methods* / Q000639:trends; D011923:Rats, Nude