Abstract
Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, existing approaches to cardiomyocyte production from human iPS cells are inefficient, limiting the application of iPS cells in basic and translational cardiac research. Furthermore, strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging, we showed that these induced cardiomyocytes expressed typical sarcomeric markers, exhibited normal rhythmic Ca(2+) transients, and responded to both β-adrenergic and electric stimulation. Furthermore, human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus, modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct, rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics.
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📋 Methods
Human ES and iPS Cells Culture and differentiation
The human ES and iPS cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and were maintained in undifferentiated ES culture medium for 5–6 days. Cells were detached with collagenase type IV (Invitrogen) to make embryoid bodies (EBs) of about 100–200 cells. Upon aggregation, ES cells begin to differentiate in a manner mimicking embryonic development. EBs were cultured in suspension in differentiation medium for 4 days. EBs were attached to plates coated with 0.1% gelatin on day 4 at 1:1 ratio, and either small molecules or DMSO were added on day 5. The medium was changed every other day. After 10 days, the medium’s FBS content (20%) was reduced to 2.5%. EBs were examined daily for beating starting on day 12. Detailed information of human ES and iPS cell culture can be found in Supplemental Methods .
Quantitative RT-PCR See Supplemental
Methods for details.
Immunostaining and Confocal Microscopy See Supplemental
Methods for details.
Fluorescence-activated cell sorting
(FACS) analysis See Supplemental Methods for details.
Western Blots See Supplemental
Methods for details.
Calcium Transient Measurements
For calcium imaging, beating EBs were manually dissected and digested with Collagenase A and Collagenase B (1:1, Roche, 6.67 mg/mL) and plated onto gelatin-coated cover slips. After 2–3 days in culture the beating cardiomyocyte clusters were loaded with 25 μM cell permeant calcium indicator dye fluo-4 AM (Invitrogen) together with 0.1% Pluronic F-127 (Invitrogen) in Tyrode solution. Before imaging, the cells were allowed to de-esterify the fluo-4 AM for 30 minutes in Tyrode solution at 37°C. Fluorescence was measured by manually defining each region of interest and quantified in relation to baseline fluorescence (F/F 0 ). The samples were electrically stimulated by field pacing (10 V, 10 ms bipolar pulses at 0.2–4 Hz). The samples were stimulated with the β-adrenoceptor agonist isoproterenol (Sigma) at 500 nM. See Supplemental Methods for details.
Show full methods section
Human ES and iPS Cells Culture and differentiation
The human ES and iPS cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and were maintained in undifferentiated ES culture medium for 5–6 days. Cells were detached with collagenase type IV (Invitrogen) to make embryoid bodies (EBs) of about 100–200 cells. Upon aggregation, ES cells begin to differentiate in a manner mimicking embryonic development. EBs were cultured in suspension in differentiation medium for 4 days. EBs were attached to plates coated with 0.1% gelatin on day 4 at 1:1 ratio, and either small molecules or DMSO were added on day 5. The medium was changed every other day. After 10 days, the medium’s FBS content (20%) was reduced to 2.5%. EBs were examined daily for beating starting on day 12. Detailed information of human ES and iPS cell culture can be found in Supplemental Methods .
Quantitative RT-PCR See Supplemental
Methods for details.
Immunostaining and Confocal Microscopy See Supplemental
Methods for details.
Fluorescence-activated cell sorting
(FACS) analysis See Supplemental Methods for details.
Western Blots See Supplemental
Methods for details.
Calcium Transient Measurements
For calcium imaging, beating EBs were manually dissected and digested with Collagenase A and Collagenase B (1:1, Roche, 6.67 mg/mL) and plated onto gelatin-coated cover slips. After 2–3 days in culture the beating cardiomyocyte clusters were loaded with 25 μM cell permeant calcium indicator dye fluo-4 AM (Invitrogen) together with 0.1% Pluronic F-127 (Invitrogen) in Tyrode solution. Before imaging, the cells were allowed to de-esterify the fluo-4 AM for 30 minutes in Tyrode solution at 37°C. Fluorescence was measured by manually defining each region of interest and quantified in relation to baseline fluorescence (F/F 0 ). The samples were electrically stimulated by field pacing (10 V, 10 ms bipolar pulses at 0.2–4 Hz). The samples were stimulated with the β-adrenoceptor agonist isoproterenol (Sigma) at 500 nM. See Supplemental Methods for details.
Action Potential Recordings
Beating clusters were derived as described above and were stained with 7.5μM di-4-ANEPPS (Invitrogen), washed and imaged during perfusion with Tyrode’s solution at 1 to 2 ml/min at 37°C. To test drugs, 25 μM procainamide (Sigma, P9391) or 375 nM verapamil (Sigma, V4629) were added to the perfusion solution. These concentrations were chosen to simulate the tissue concentration resulting from clinical administration of these drugs. Action potentials were induced by field stimulation and recordings were captured before and during drug perfusion and after a washout by Tyrode’s solution. Action potential duration (APD) was calculated from the time of maximal rise in membrane potential to the time at 90% of repolarization for each beating cluster from three consecutive beats. See Supplemental Methods for details.
Statistical Analysis
Data were presented as mean ± standard error of the mean (SEM) from three independent experiments. Sigma Plot was used to administer a one-tailed Student’s t-test to action potential measurements in experiments using arrhythmogenic drugs because it was known that those drugs have particular lengthening or shortening effects on APD. All other experiments were analyzed with two-tailed Student’s t-tests. A probability value
📊 Figures
Figure 1
Effect of BMP-4 on cardiac differentiation in human ES cells
A, Schematic diagram of protocol used for cardiac differentiation of human ES and iPS cells. B, Undifferentiated human ES cells (hESCs), human iPS cells (hiPSCs) and EBs derived from hESCs and hiPSCs....
Figure 2
Effects of Wnt inhibitors on the expression of u03b2-catenin protein and cardiac mesodermal/progenitor marker genes
A, EBs made from H7 human ES cells were cultured in the presence of BMP-4 (25 ng/ml) for 4 days in suspension, followed by a 2-day treatment with IWR-1 (10 u03bcM) or DMSO. EBs were then harvested for...
Figure 3
Effects of Wnt inhibitors on cardiac differentiation of human ES and iPS cells
EBs were cultured in the presence or absence of BMP-4 (25 ng/ml) in suspension culture for 4 days, followed by a treatment of DMSO (vehicle control) or Wnt small molecule inhibitors for 2 additional d...
Figure 4
Immunolabeling of cTnT and u03b1-actinin in hES-CMs and hiPS-CMs
Beating EBs around day 20 of H7 human ES cells (A) or fetal lung fibroblasts IMR90 C1 iPS cells (B) were manually dissected and enzymatically dissociated into small cardiomyocyte clusters, which were ...
Figure 5
Calcium transients in hES-CMs and hiPS-CMs
The beating cardiomyocyte clusters derived from beating EBs were loaded with the cell permeant calcium indicator dye fluo-4 AM. Fluorescence was measured by manually defining a region of interest cent...
Figure 6
Effect of channel modulating drugs on the action potential durations of hES-CMs and hiPS-CMs
Beating cardiomyocyte clusters derived from H7 human ES and fetal lung fibroblasts IMR90 C1 iPS cells were stained with the voltage sensitive dye di-4-ANEPPS and electrically paced at 0.5 Hz. Insets i...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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