Format

Send to

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

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

Author information

1
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, United States.

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

PMID:
23044428
PMCID:
PMC3556195
DOI:
10.1016/j.ijcard.2012.09.077
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center