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

Int J Cardiol. 2013 Sep 20;168(1):41-52. doi: 10.1016/j.ijcard.2012.09.077. Epub 2012 Oct 6.

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.

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.

Keywords: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 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