Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes

Abstract

The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.

Figure 1

Stages in EB production and differentiation. (a) Schematic of the three stages in human ES cell differentiation. Initially, the ES colonies are grown on top of the MEF feeder layer (left). To induce differentiation, cells are transferred to suspension, where they aggregate to form EBs (middle). After 10 days in suspension, EBs are plated on gelatin-coated culture dishes, where they are observed for the appearance of spontaneous contractions (right). (b) Photomicrographs depicting the just-mentioned three stages: ES colony (left), EBs in suspension (middle), and a contracting area in the outgrowth on an EB (right, arrow).

Figure 2

Cumulative percentage of EBs containing spontaneously contracting areas as a function of the number of days after plating of the EB.

Figure 3

Ultrastructural analysis of ES-derived cardiomyocytes. (a) Transmission electron micrograph of sectioned beating EB 10 days after plating. Relatively unorganized myofibrillar bundles can be seen in some myocytes. (b) A cell 27 days after plating, displaying a more mature sarcomeric organization. (c) A different cell from the same EB as in b, demonstrating more organized sarcomeres and Z-bands (arrow). (d) High-power electron micrograph showing the presence of a gap junction (arrow) from a cell 16 days after plating. (e) High-power electron micrograph showing the presence of desmosomes (arrow) from a cell 16 days after plating.

Figure 4

Immunostaining of ES cell–derived cardiomyocytes. (a) Immunostaining of dispersed cells from a beating EB (day 16 after plating) with anti-cardiac α/β-myosin heavy chain mAb’s. Several cells stained positively. ×40. (b) Higher magnification of a cardiomyocyte in a more developed stage (day 16 after plating). Note the appearance of early striation pattern (arrow). ×63. (c) Positive staining with anti–sarcomeric α-actinin mAb’s (day 17 after plating). ×63. (d) Positive staining with cTnI mAb’s (day 30 after plating). ×63. (e) Positive staining with anti-desmin mAb’s (day 18 after plating). ×63. (f) Positive staining with anti-ANP antibodies (day 16 after plating). ×63.

Figure 5

Expression of specific markers in the contracting EBs. Negative images of ethidium-stained gels are shown. RNA samples from undifferentiated ES cells and contracting EBs (C-EBs) were analyzed by RT-PCR for the expression of cardiac-specific markers: cTnI, cTnT, GATA4, human ANP, MLC-2A, MLC-2V, Nkx2.5, and α-myosin heavy chain. Octamer-binding protein 4 (Oct-4) is an ES-cell marker. GAPDH served as internal standard. “–RT” indicates no cDNA.

Figure 6

Functional analysis of contracting area within the EBs. (a) Calcium transients of human ES cell–derived cardiomyocytes as determined by fura-2 fluorescence. A typical calcium transient is shown on the left, and continuous recording is shown on the right. T_p, time to peak; T_t, total transient time; T_1/2, time to half-peak relaxation. (b) Typical extracellular electrophysiological recordings from different areas of the EB. Note the presence of a sharp and slow component. R, ratio.