Cardiac Differentiation of Human iPS Cells in Embryoid Bodies
As an initial test of the ability of human iPS cells to undergo cardiac differentiation, we generated embryoid bodies (EBs) from four previously described iPS cell clones (IMR90 C1, IMR90 C4, Foreskin C1, Foreskin C2).3
A fraction of the EBs generated from each of the tested iPS cell lines formed contracting outgrowths of cells suggesting cardiac differentiation. The efficiency of forming contracting EBs after 30 days in culture varied significantly among iPS cell clones from less than 1% to 10% of the EBs having contracting regions (). Based on these results, we chose two iPS clones of different origins (IMR90 C4 and Foreskin C1) which readily underwent cardiogenesis for comparison to two well-characterized human ES cell lines, H1 and H9.
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 (more ...)
We first compared the in vitro
differentiation of the iPS and ES cell lines by determining the time courses for formation of spontaneously contracting EBs and their associated rates of contraction. Contractions were first observed 8-9 days after EB formation for all of the iPS and hES cell lines (supplemental movies 1
). As shown in , contracting EBs developed over a similar time course during 60 days of observation, although the overall efficiency varied from line to line. H9 ES cells showed the greatest efficiency, reaching a maximum of 22%, in contrast to the least efficient, Foreskin C1, which reached 4.2%. The human iPS cell line IMR90 C4 and ES cell line H1 displayed nearly identical efficiencies (~10%) for the formation of contracting outgrowths. Comparison of the rates of spontaneous contractions showed an increase in rate over the period of observation with some differences observed between the lines (). H9 EBs exhibited the highest contraction rates while Foreskin C1 was slowest at most time points. Again, IMR90 C4 and H1 EBs performed comparably. In summary, there was no observable difference in the time course for the development of contraction for iPS- and ES- derived EBs, although the efficiency of forming contracting EBs varied for both iPS and ES cell lines.
Cardiac and Pluripotency Gene Expression in Cardiomyocytes Derived from iPS Cells
To provide a more detailed comparison of the cardiomyocytes derived from iPS cells and ES cells, we examined the gene expression patterns present in microdissected contracting regions from EBs using RT-PCR. Pluripotency and cardiac muscle gene expression was analyzed in day 60 EBs when cardiogenesis reached a stable plateau. The gene expression patterns of stem cell-derived cardiomyocytes were compared to those of undifferentiated iPS and ES cells as well as adult human left ventricular myocardium.
We examined a variety of cardiac genes: the transcription factor Nkx2.5 (NKX2-5); several myofilament protein genes including cardiac troponin T (TNNT2), α-myosin heavy chain (MYH6), α-actinin (ACTN2), myosin light chain 2 atrial isoform (MYL7) and myosin light chain 2 ventricular isoform (MYL2); atrial natriuretic factor (HPPA); and phospholamban (PLN). The cardiac genes showed no detectable expression in undifferentiated iPS or ES cells with the exception of low levels of transcript detected for TNNT2 and MYL7. In contrast, by day 60, there was robust expression of the full range of cardiac genes in cardiomyocytes derived from iPS and ES cell lines, comparable to that observed in adult human ventricular myocardium (). Overall, the cardiac gene expression pattern was quite similar in iPS and ES cell-derived cardiomyocytes, with a strong increase in expression following cardiac differentiation.
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 (more ...)
For pluripotency genes, we focused on the most extensively studied genes, OCT4 and NANOG. Expression of OCT4 and NANOG was high in undifferentiated iPS and ES cells, and the expression greatly decreased with differentiation, with the exception of the Foreskin C1 iPS line. In the Foreskin C1 line, expression of OCT4 and NANOG persisted to some extent (). To provide a more quantitative assessment of OCT4 and NANOG expression during differentiation, we performed quantitative RT-PCR. OCT4 gene expression was significantly downregulated in EBs from all lines compared to undifferentiated iPS or ES cells (, P < 0.001), and the degree of downregulation was similar for H1 (114-fold), H9 (105-fold) and IMR90 C4 (105-fold), but less for Foreskin C1 (14-fold). There was also significant downregulation of NANOG expression in all lines during differentiation (, P < 0.001), although the decrease in NANOG in differentiated cardiomyocytes was relatively less than that of OCT4 (H1: 34-fold; H9: 45-fold; IMR90 C4: 4-fold; Foreskin C1: 4-fold). These results confirmed a reduction in pluripotency gene expression for both iPS and ES cells during cardiogenesis in EBs, but the reduction may be variably blunted in the iPS cell lines.
To investigate if persistent expression of lentiviral transgenes contributes to the blunted downregulation of OCT4 and NANOG during cardiogenesis of iPS cells, we measured the total OCT4 or NANOG gene expression using primers located in the coding region and compared it to the expression of endogenous OCT4 or NANOG using primers located in 3′ UTR. The difference between total and endogenous gene expression is due to expression of the transgene. In undifferentiated ES cells, the total and endogenous OCT4 and NANOG expression was not different as predicted given the lack of transgenes (). The iPS cells exhibited significantly greater total than endogenous OCT4 expression indicating residual transgene expression most prominently in the case of the Foreskin iPS cell line (). For NANOG, the range of total expression varied in the undifferentiated cells, but there was no significant difference between total and endogenous expression of NANOG in either of the iPS cell lines ().
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 (more ...)
To determine whether transgene expression accounted for the difference in iPS and ES cell expression of pluripotency genes following differentiation, we compared total and endogenous expression of OCT4 and NANOG in contracting outgrowths isolated from each of the four cell lines. Total expression of OCT4 was comparable in differentiated H1, H9 and IMR90 C4 EBs, but the level of OCT4 transcript in Foreskin C1 EBs was significantly greater (P < 0.001) which could largely be accounted for by persistent expression of the transgene (). In the case of NANOG, the relative total expression in EBs also varied from line to line (), and both IMR90 C4 and Foreskin C1 EBs exhibited a relatively higher total expression of NANOG than observed for EBs from H1 and H9 (P < 0.001), an effect which, again, was largely attributable to transgene expression. Together, these results demonstrate that there is some persistent expression of the OCT4 and NANOG transgenes in the two iPS cell lines studied following differentiation, and this is especially evident for the OCT4 transgene in the Foreskin C1 iPS cell line. Nevertheless, a strong downregulation in total OCT4 and NANOG gene expression occurs during cardiogenesis of iPS cells. Although we did not evaluate LIN28 and SOX2 transgene expression, it is possible that some level of expression for these transgenes persists as well.
Given the findings of persistent OCT4
transgene expression in differentiated iPS cell-derived cardiomyocytes, we examined whether Oct4 and Nanog protein expression could be detected using standard immunolabeling approaches. Immunolabeling for Oct4 and Nanog was examined in undifferentiated iPS cells and differentiated cells from day 60 EBs which were co-labeled for cardiac troponin T (cTnT) to detect cardiomyocytes. In experiments using Foreskin C1 cells (), we detected nuclear localized Oct4 immunolabeling in the undifferentiated iPS cells but not in the surrounding mouse fibroblast feeder layer cells. There was no detectable cTnT immunolabeling in the undifferentiated cells. In contrast, cells isolated from contracting outgrowths from Foreskin C1 EBs subjected to the identical immunolabeling protocol revealed that the majority of cells were cTnT positive and showed DAPI labeled nuclei without detectable Oct4 labeling. Nor was Oct4 labeling detected in any surrounding cTnT negative cells. Similar results were observed for Nanog and cTnT immunolabeling in the IMR90 C4 line (). Nanog was detected in the undifferentiated iPS cells but not detectable in IMR90 iPS cell-derived cardiomyocytes. These results show that Oct4 and Nanog protein expression is strongly downregulated during differentiation of Foreskin C1 and IMR90 C4 iPS cells despite some persistent mRNA expression. This apparent discrepancy between mRNA and protein expression could be due to a variety of regulatory effects such as microRNA regulation, which may be particularly important for pluripotency genes.18
Proliferation of Cardiomyocytes Derived from iPS and ES Cells
Because the proliferative activity of cells may be impacted by overexpression of genes associated with pluripotency, we next compared cellular proliferation in iPS cell- and ES cell-derived cardiomyocytes. In normal embryonic cardiac development, the proliferative activity of cardiomyocytes declines steeply at later stages, and a similar phenomena has also been observed during in vitro
differentiation of human ES cell-derived cardiomyocytes.11, 19, 20
In this experiment, cardiomyocytes were isolated from early (day 10-20) and late (day 60) contracting EBs. Immunofluorescence analysis showed that a fraction of the MF20 positive cells contained BrdU positive nuclei indicating proliferating cardiomyocytes for both iPS and ES cell-derived populations (). In early EBs, there was no difference in the percentage of BrdU positive cardiomyocyte (MF20 positive) nuclei for H9 and IMR90 C4 (~15%), but Foreskin C1 showed a significantly lower percentage of dividing cardiomyocytes (~11%, P
< 0.05) compared to the other two cell lines (). Cells from late EBs revealed significantly less proliferation compared to early EBs in all the ES and iPS cells tested (P
< 0.01). H9 late EBs exhibited greater proliferation of cardiomyocytes (4%) compared to late EBs from IMR90 C4 and Foreskin C1 (~1%, P
< 0.05). These results demonstrate that iPS cell-derived cardiomyocytes like ES-cell derived cardiomyocytes show a marked reduction in proliferation during 60 days in culture, and the proliferative activity of the iPS cardiomyocytes from the cell lines studied tended to be slightly less than for cardiomyocytes formed from H9 ES cells.
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 (more ...)
iPS Cell-derived Cardiomyocytes Exhibit Sarcomeric Organization
To evaluate the expression of myofilament proteins and the sarcomeric organization in iPS cell-derived cardiomyocytes, we performed immunolabeling with antibodies for specific myofilament proteins using enzymatically isolated cells from day 60 EBs. Cells were co-labeled with the antibodies for α-actinin, which is present at the Z-line of the sarcomere, and myosin light chain 2 atrial isoform (MLC2a) which is typically present at the A-band of the sarcomere. Immunofluorescence analysis revealed a clear striated pattern for α-actinin labeling in cardiomyocytes from IMR90 C4 and Foreskin C1 comparable to that observed for H1 and H9 cardiomyocytes (). Striated MLC2a labeling was also observed in cardiomyocytes from all cell lines. Overlap of α-actinin and MLC2a labeling demonstrated an alternating pattern in the sarcomeres in agreement with the known localization of MLC2a to the A-band of the sarcomere, which lies between the Z-lines highlighted by the α-actinin labeling.
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 (more ...)
We also performed immunolabeling for cTnT, which is a highly cardiac-specific myofilament protein. We observed comparable sarcomeric labeling of iPS and hES cell cardiomyocytes (). Likewise, immunolabeling with an antibody to the ventricular specific protein, myosin light chain 2 ventricular isoform (MLC2v), detected presumed ventricular cardiomyocytes derived from iPS cells and ES cells (). In summary, immunolabeling of multiple myofilament proteins indicates that a well-organized sarcomeric structure can similarly develop in iPS and ES cell-derived cardiomyocytes.
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, (more ...)
Action Potentials Reveal Multiple Types of Cardiomyocytes Derived from iPS Cells
To provide an initial assessment of the functional competence of iPS cell-derived cardiomyocytes, we performed sharp microelectrode recordings from spontaneously contracting EB outgrowths at 56 – 70 days post-EB formation. A total of 54 and 47 stable recordings from 23 and 20 IMR90 C4 and Foreskin C1 derived EBs, respectively, were obtained. Three major types of action potential were observed: ventricular, atrial, and nodal. Cells with ventricular-like action potentials (, bottom) were the most frequently encountered in both IMR90 C4 and Foreskin C1 derived EBs, and typically displayed a more negative maximum diastolic potential (MDP), a rapid action potential upstroke, and a distinct plateau phase. Atrial-like cells were distinguished from ventricular-like cells by the absence of a distinct plateau during repolarization and typically also exhibited spontaneous activity that was higher in frequency than that observed for ventricular cells (, middle). Finally, nodal-like cells were distinguished by MDPs that were less negative than those of ventricular- and atrial- like cells, smaller amplitude action potentials, a slower action potential upstroke, and a pronounced phase 4 depolarization preceding the action potential upstroke (, top).
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 (more ...)
Comparison of recordings from cardiomyocytes within the same EB revealed that a given action potential phenotype was predominant in each EB, as has previously been shown for human ES cell-derived EBs.12
plots action potential durations measured at 90% repolarization from the action potential peak (APD90) for EBs from which three or more recordings were obtained. APD90 values obtained from myocytes within the same EB clustered together much more closely than would be predicted if EBs contained myocytes of each class. The relative proportions of EBs exhibiting the nodal, atrial, and ventricular phenotypes are shown in and compared to the proportions observed for EB outgrowths derived from the H1 and H9 cell lines. Although sample sizes are limited due to the difficulty inherent in recording, iPS and ES cells appear comparably efficient in generating cardiac myocytes of each major class.
Comparison of cardiomyocyte types in H9, H1, IMR90 C4, and Foreskin C1 derived cardiomyocytes.
presents a comparison of the properties of iPS and ES cell-derived action potentials for ventricular-like cells, which were the predominant class of cardiomyocytes encountered in EBs formed from each line. Specifically, we compared spontaneous beating rate (in beats per minute, BPM), action potential durations (APD), the maximum rates of rise during the action potential upstroke (dV/dtmax), action potential amplitudes (APA), and maximum diastolic potentials (MDP). As observed for H9 and H1 cardiomyocytes, the action potentials of IMR90 C4 and Foreskin C1 cardiomyocytes exhibited properties that were clearly more comparable to those of human embryonic, than neonatal or adult, cardiac muscle. Measurements from IMR90 C4- and Foreskin C1-derived cells fell within range of the measurements obtained for cardiomyocytes from H9 and H1 ES cell lines and, although there were modest differences in means, these were no larger than the differences between H9 and H1 cardiomyocytes.
Comparison of action potential properties of ventricular-like cardiomyocytes derived from H9, H1, IMR90 C4, and Foreskin C1 cell lines.
Cardiomyocytes typically respond to increases in heart rate with a compensatory decrease in action potential duration, and this property is likewise present in ES cell-derived cardiomyocytes.12
Therefore, we tested whether iPS cell-derived cardiomyocytes exhibit this typical rate adaptation in response to changing rates of electrical stimulation. As shown in , field stimulation of microdissected Foreskin C1-derived contracting outgrowths at 1, 2, and 3 Hz resulted in incremental shortening of the observed action potential durations. Increasing the stimulus frequency from 1 to 2 and 3 Hz decreased the average APD90 and APD50 by approximately 20 and 30 percent (), respectively, with little or no change in other action potential features.
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 (more ...)
β-adrenergic Regulation of iPS Cell-derived Cardiomyocytes
The above experiments suggest that iPS and ES cell-derived cardiomyocytes exhibit similar functional potential in regard to their electrical activity. We next sought to determine whether β-adrenergic signaling, a canonical cardiomyocyte signaling pathway, is operational in iPS cell-derived myocytes by examining the responsiveness to isoproterenol (ISO), a nonselective β-adrenergic receptor agonist. In spontaneously active myocytes such as those derived from embryonic or neonatal hearts as well as nodal cells in adult hearts, β-adrenergic receptor stimulation, via protein kinase A-mediated regulation of several different ion channels, results in a positive chronotropic effect which is accompanied by a shortening of APD.21
panels A and B show individual responses to isoproterenol for ventricular-like myocytes derived from H9 (top
) and Foreskin C1 (bottom
) lines. Both cells displayed initial contraction frequencies slightly above 0.6 Hz during perfusion of control Tyrode's solution and responded to perfusion of ISO (1 μmol/L) with a 2-fold or greater increase in rate. As shown in , the increases in rate were also accompanied by decreases in action potential duration as observed in native cardiomyocytes. Summary data are presented in ; perfusion with ISO resulted in statistically significant increases in rate and decreases in action potential duration for IMR90 C4- and Foreskin C1-, as well as H9-, derived cardiomyocytes. These results suggest that β-adrenergic receptors and their associated intracellular signaling partners are present and functional in cardiomyocytes derived from iPS cells.
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 (more ...)