Figure 1.
Experimental approach depiction and vectors utilized to derive iPS cells or cardiac progenitor cells.
A. Mouse embryonic fibroblasts were epigenetically reprogrammed into induced pluripotent stem cells following their transduction with an inducible expression polycystronic lentiviral vector allowing the controlled expression of transcription factors Pou5f1, Sox2, Klf4, and Myc. Subsequently the derived iPS cells were stably transfected by electroporation with two DNA vectors allowing the genetic selection of cardiac progenitor cells (Nkx2-5 enhancer) and the detection of cardiomyocytes (Myh6 promoter). iPS cell-derived cardiac progenitors mainly differentiate into cardiomyocytes or smooth muscle and are used to assemble biosynthetic cardiac tissue constructs within hydrogel-based matrix and a polydimethylsiloxane mold. B. Schematic depiction of the lentiviral vectors used to derive iPS cells as well as the DNA vectors used to select the cardiac progenitors and identify cardiomyocytes. C. Brightfield images depicting epigenetic reprogramming of mouse embryonic fibroblasts into iPS cells. Clusters of fibroblasts with an increased proliferation rate and altered morphology ultimately becoming iPS cell colonies are indicated by white arrows.
Figure 2.
A–B. Undifferentiated iPS cells express and co-localize transcription factors Pou5f1 and Nanog in their nuclei. Expression of the surface antigen Fut4 (SSEA-1) is also detected in Pou5f1-expressing iPS cells. Importantly the cells organize in tight compact colonies similarly to colonies of undifferentiated embryonic stem cells. C. Relative gene expression analysis for Pou5f1, Sox2, Nanog, Rex1, Klf4, and Myc following three serial expansion passages of the derived iPS cells in the absence of doxycycline. All gene expression levels are normalized against mouse embryonic fibroblasts using the ΔΔCt method. Error bars represent standard deviation. *, ** and *** indicate p<0.05, 0.01, 0.0001, respectively, and computed using one-tailed Student’s t-test for comparison of the gene expression levels measured for iPS cells with that measured for primary MEFs. #indicates significant variance amongst all three groups as determined by one-way ANOVA analysis.
Figure 3.
Gene expression analysis performed on differentiating mouse iPS cells.
Temporal gene expression analysis was performed over the length of 14 days (7 timepoints) on cultures of differentiating mouse iPS cells: Pou5f1 (undifferentiated iPS cells). T, and Mesp1 (precardiac mesoderm). Nkx2-5, Gata4, Tbx5, Mef2c, and Myocd (early cardiac transcription factors). Nppa, Myl2, Myl7, Myh6, Myh7, and Tnnt2 (mature cardiomyocyte markers), Atp2a2, Atp2a3, Cacna1c, Casq2, Hc4, Kcnj2, Kcnj3, Kcnk1, Kcnd3, Pln, Ryr2, and Slc8a1 (cardiomyocyte electrophysiology genes). All gene expression levels were normalized against day 0 undifferentiated iPS (except for Pou5f1) using the ΔΔCt method. Error bars represent standard deviation. Significant differences in gene expression were determined using one-way ANOVA. Independent variable: differentiation day, and dependent variable: percentage gene expression. In all cases, the p-value was found to be very small (<<0.01; not shown), indicating highly significant changes in gene expression. Holm-Bonferroni multiple comparison t-tests were then performed to determine whether an individual data point on a particular differentiation day was significantly different from that of the previous. Holm-corrected p-values <0.05 were deemed significantly different and denoted by an *.
Figure 4.
Cardiac progenitor characterization and multipotential differentiation capacity.
A. The percentage of total cells expressing cell-surface antigens cKit, Flk1, and Sca-1 or the combinations of cKit/Flk1, and cKit/Sca-1 were determined by fluorescence-activated cell sorting before (gray) and after (black) puromycin addition for enriching Nkx2-5(+) cardiac progenitor cells. No RFP(+) cells were detected before addition of puromycin. The percentage of the five cell subpopulations also expressing RFP following puromycin addition is noted above the black columns. Error bars represent standard deviation. *denotes significant change (p<0.05) as determined by t-test statistical analysis. Isotype control antibodies were used as negative control in order to set the gates for the three cell surface antibodies. B. When cultured in suspension the derived iPS cells readily aggregated to form three-dimensional embryoid bodies which temporally differentiated into various cell types including spontaneously contracting cardiomyocytes detected as early as differentiation day 7. C. Following three days of puromycin antibiotic selection aggregates of spontaneously contracting RFP(+) cardiac progenitors were allowed to attach on gelatin-coated polystyrene. The cells in these aggregates stained positive for cardiac-specific actinin. D. Enzymatically dissociated and puromycin selected iPS-derived cardiac progenitors formed large-scale monolayers of spontaneously contracting cells. E. RFP-expressing (Myh6 promoter) and spontaneously contracting cardiomyocytes stained positive for the cardiac-specific marker Tnnt2. F. Acta2(+)/Tagln(+) smooth muscle cells were detected interspersed within the cultures of Act2(+) cardiomyocytes. G–H. Rare colonies of endothelial cells with varying size were detected within the cell monolayers as determined by immunostaining for Vwf.
Figure 5.
Immunofluorescence characterization of cardiomyocytes differentiated from the cardiac progenitor cells.
A–D. The cardiomyocytes formed well-defined cross-striated sarcomeric structures as determined by the expression and spatial organization of Actn2, Tnnt2, and Myh6 and also expressed the ventricular specific protein Myl2. E–F. The cells also formed robust intercellular electrical and mechanical connections as determined by the spatial organization of Cdh2 and Gja1. G. RFP(+) cardiomyocytes stained positive for sodium/potassium ATPase (Atp2a2).
Figure 6.
Intracellular microelectrode characterization of iPS cell-derived cardiomyocytes.
The electrophysiology of RFP-expressing cardiomyocytes matured temporally in culture. A. Representative cardiomyocyte action potential traces at the intermediate differentiation stage (IDS, Day 7+7) or late differentiation stage (LDS, Day 7+14). Between IDS and LDS in culture, B. Action potential amplitude was not significantly different (102.4±1.9 mV versus 100.9±2.2 mV). C. Maximum action potential upstroke velocity increased from 149.3±3.5V/s to 179.5±9.3V/s. D. Action potential duration at 80% repolarization decreased from 77.2±3.2 ms to 60.9±1.7 ms. E. Maximum diastolic potential was not significantly different (−74.1±1.1 mV versus −75.9±1.1 mV).
Figure 7.
Engineered tissue patches and optical mapping of action potential propagation.
Microfabricated tissue molds cast in PDMS were used to direct the alignment of iPS cell-derived cardiac progenitor cells in three-dimensional biosynthetic tissue constructs. A. PDMS tissue mold (sputtered with chrome for greater contrast). B–C. Zygometer surface profiles of the PDMS mold showing 1600 µm tall hexagonal features. Individual hexagons are 800 µm in length and 200 µm in width. D. An engineered tissue patch removed from its mold, bordered by a nylon mesh frame to facilitate handling. Elliptical void spaces are a result of tissue compaction away from the hexagonal features. E–F. Live brightfield and RFP images of the engineered tissue construct within its mold, showing tissue compaction forming elliptical pores around the hexagonal features and live cardiomyocytes within the tissue patch. G. Isochrone map showing activation times across the tissue patch during propagation (left to right) of an action potential. Blue pixels represent sites of early activation, and red pixels represent sites of late activation. H. False-color images representing a calcium transient traversing a tissue patch from left to right as a result of action potential firing. Red pixels represent a high intracellular calcium concentration, and blue pixels represent a low concentration.
Figure 8.
Confocal microscopy of three-dimensional engineered biosynthetic tissue constructs containing iPS cell-derived differentiated cardiac progenitor cells.
Representative images of the immunostained cells within the tissue constructs. This series of images was taken from an area located between the large and small pores within the construct. A. Cells within the biosynthetic tissue constructs significantly compacted the original hydrogel matrix, allowing for the formation of high-density intertwined string-like fibers. The cardiomyocyte cytoskeleton was elongated and aligned along the long-axis of the biosynthetic tissue constructs. B–C. The cytoskeletal sarcomeric arrangement was highly organized and placed perpendicular to the long axis of the biosynthetic tissues. D–F. The cardiomyocytes formed robust electromechanical connections (gap and adherens junctions) as determined by the level of expression and spatial organization of Gja1 and Cdh2 over long distances. These connections were often detected perpendicular to the long-axis of the tissue constructs.