Functional cardiomyocytes derived from human induced pluripotent stem cells

Abstract

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

Figure 1

Development of contracting EBs from human iPS and ES cells. (A) Percentage of contracting EBs formed from four different iPS clones (IMR90 C1, n=11; IMR90 C4, n=23; Foreskin C1, n=13; Foreskin C2, n=8) after 30 days of differentiation. (B) Time course of forming contracting EBs from iPS and ES cell lines (IMR90 C4, n=23; Foreskin C1, n=13; H1, n=3; H9, n=3). (C) Comparison of the contraction rate of iPS and ES cell contracting EBs over time (IMR90 C4, n=72-139; Foreskin C1, n=97-157; H1, n=14-38; H9 n=106-118). Error bars represent SEM. Data were compared using one-way ANOVA and Tukey post tests with * indicating H9 is significantly different from Foreskin C1, P < 0.05.

Figure 2

Cardiac and Pluripotency Gene Expression in Cardiomyocytes Derived from iPS and ES Cells. (A) RT-PCR analyses of pluripotency genes, OCT4 and NANOG, and cardiac genes in undifferentiated iPS and ES cells, day 60 EBs, and adult left ventricular myocardium (LV). (B and C) Quantitative RT-PCR analyses of total OCT4 (B) and NANOG (C) expression in undifferentiated iPS and ES cells compared to differentiated contracting areas from day 60 EBs. Error bars represent SEM (n=3), *** indicate P < 0.001 comparing gene expression in undifferentiated cells and d60 EBs using t-test.

Figure 3

Transgene expression of OCT4 and NANOG in undifferentiated and differentiated iPS cells. Quantitative RT-PCR analyses of total and endogenous OCT4 and NANOG expression in undifferentiated iPS and ES cells (A and B), and in day 60 EB contracting areas (C and D). Error bars represent SEM (n=3), and *** indicate P < 0.001 comparing total and endogenous gene expression using t-test. (E) Double immunolabeling for Oct4 and cTnT in undifferentiated Foreskin C1 iPS cells (left panel) and Foreskin C1 iPS cell-derived cardiomyocytes from day 60 EBs (right panel). (F) Double immunolabeling for Nanog and cTnT in undifferentiated IMR90 C4 iPS cells (left panel) and IMR90 C4 iPS cell-derived cardiomyocytes from day 60 EBs (right panel).

Figure 4

Proliferation of cardiomyocytes differentiated from iPS and ES cells. (A) Co-labeling for sarcomeric myosin with the MF20 antibody (red) and BrdU (green) in cardiomyocytes isolated from contracting areas of early EBs (10-20 days) from H9 and IMR90 C4 following a 17-hour pulse of BrdU to identify dividing cells. Nuclei were stained with Hoechst (blue). Scale bars are 50 μm. (B) Average percentage of BrdU positive nuclei in cardiomyocytes (MF20 positive) differentiated from H9 ES cells, IMR90 C4 and Foreskin C1 iPS cells from early and late (day 60) EBs. Error bars represent SEM (n=3), ** for P < 0.01 and *** indicate P < 0.001 in t-test comparisons of early and late EBs.

Figure 5

Sarcomeric organization in cardiomyocytes derived from iPS and ES cells. Cardiomyocytes isolated from contracting areas of day 60 EBs from IMR90 C4, Foreskin C1, H1 and H9 were co-labeled for α-actinin (green) and MLC2a (red). Nuclei were stained with Hoechst (blue). Overlap of α-actinin and MLC2a labeling demonstrated an alternating pattern of sarcomeric labeling consistent with MLC2a present in the A band of the sarcomere between the Z-lines demonstrated by the α-actinin labeling. Scale bars are 20 μm.

Figure 6

Immunolabeling of cTnT and MLC2v in iPS and ES cell-derived cardiomyocytes. Single cardiomyocytes isolated from contracting areas of day 60 EBs from IMR90 C4, Foreskin C1, H1 and H9 were immunolabeled for cTnT, a cardiac-specific myofilament protein, and MLC2v, a ventricular-specific protein. Nuclei were stained with Hoechst (blue). Scale bars are 20 μm.

Figure 7

Electrophysiological characterization of iPS cell-derived cardiomyocytes. (A) Representative recordings from 3 iPS cell-derived EB outgrowths demonstrating that each of the 3 major action potentials types were observed. Right, action potentials shown at an expanded timescale taken from the region indicated (•) on the left. Dotted lines indicate 0 mV. (B) Cardiomyocytes within a given EB outgrowth display similar electrophysiological properties: Action potential durations at 90% repolarization (APD90s) are shown for consecutively studied iPS (top) or ES (bottom) cell-derived EBs from which 3 or more cells were examined.

Figure 8

Action potentials of iPS cell-derived cardiomyocytes exhibit rate adaptation. (A) Electrical field stimulation of a ventricular-like cardiomyocyte derived from the Foreskin C1 line at three frequencies as indicated. Dashed line represents 0 mV. (B) Overlay of single action potentials from the cell in (A) obtained at 1, 2, and 3 Hz stimulation rates. Action potentials were normalized to correct for a slight differences in amplitude (for this cell the average amplitudes were 79.2, 83.1, and 84.7 mV at 1, 2, and 3 Hz, respectively). Electrical artifacts corresponding to the stimulus were manually removed for normalization. (C) Average (± SEM) fractional changes in APD90 and APD50 during 2 and 3 Hz stimulation. Durations were normalized to the respective values at 1 Hz stimulation. The number of cells is given in parentheses below each bar. Data were compared using a one-way ANOVA and Tukey post tests to the durations at 1 Hz with ** P < 0.01 and *** P < 0.005.

Figure 9

Effect of Isoproterenol on spontaneous electrical activity of iPS cell-derived cardiomyocytes. (A) Time course of responses for an H9- (top) and a Foreskin C1- derived (bottom) cardiomyocyte before, during, and after perfusion with Tyrode's solution containing 1 μmol/L ISO as indicated by measurement of the instantaneous frequencies. Bar at the top of each graph shows the duration of ISO application. (B) Action potentials recorded from the cells in (A) before and during perfusion of ISO. (C) Bar graphs of the average rate and APD90 data for H9, IMR90 C4, and Foreskin C1 derived cardiomyocytes before (light gray bars) and during (black bars) application of ISO. Number of cells examined for each cell type is indicated in parentheses below each graph. * P < 0.05, ** P < 0.01 in paired t-test comparisons.