Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;767:419-31.
doi: 10.1007/978-1-61779-201-4_31.

Methods for the Derivation and Use of Cardiomyocytes From Human Pluripotent Stem Cells

Affiliations
Free PMC article

Methods for the Derivation and Use of Cardiomyocytes From Human Pluripotent Stem Cells

Wei-Zhong Zhu et al. Methods Mol Biol. .
Free PMC article

Abstract

The availability of human cardiomyocytes derived from embryonic stem cells (ESCs) has generated -considerable excitement, as these cells are an excellent model system for studying myocardial development and may have eventual application in cell-based cardiac repair. Cardiomyocytes derived from the related induced pluripotent stem cells (iPSCs) have similar properties, but also offer the prospects of patient-specific disease modeling and cell therapies. Unfortunately, the methods by which cardiomyocytes have been historically generated from pluripotent stem cells are unreliable and typically result in preparations of low cardiac purity (typically <1% cardiomyocytes). We detail here the methods for a recently reported directed cardiac differentiation protocol, which involves the serial application of two growth factors known to be involved in early embryonic heart development, activin A, and bone morphogenetic protein-4 (BMP-4). This protocol reliably yields preparations of 30-60% cardiomyocytes, which can then be further enriched to >90% cardiomyocytes using straightforward physical methods.

Figures

Figure 1
Figure 1. Directed cardiac differentiation protocol
A: Timeline for the protocol used to generate cardiomyocytes from human pluripotent stem cells. In brief, after growth to confluence in the undifferentiated state in MEF-CM plus bFGF, cultures are switched to differentiating conditions in RPMI-B27 medium and are serially pulsed with two growth factors, activin A (day 0) and BMP-4 (day 1). After day 5 post-induction with activin A, the differentiating cultures are grown in RPMI-B27 medium in the absence of exogenous cultures. Spontaneous beating activity commences on ~day 9-11 post-induction, and cultures may be harvested after day 14. B: Photomicrographs illustrating morphological changes in cultures during the protocol. Prior to treatment with activin A (day 0), the cultures should have formed a compact, 100% confluent monolayer that is composed of cells with a high nuclear-to-cytoplasmic ratio but greater irregularity in cell shape than do hESCs in a usual undifferentiated colony. By day 5, cells in the monolayer are phase-bright, more loosely attached, and have a rounded morphology. Some cell death is to be expected at this stage, but the monolayer should still occupy >90% of the well surface area. By day 14, the cultures are far more heterogeneous and include widespread areas with spontaneous beating activity. After dispersion and re-plating at low density (day 25), the resultant differentiated cultures are comprised of individual and small clusters of contractile cardiomyocytes with either a spindled- or triangular morphology. Scale bar = 50 μm.

Similar articles

See all similar articles

Cited by 38 articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback