Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Abstract

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.

Figure 1. Cardiac cell expansion from mouse iPSC-derived Flk1⁺ mesoderm by CSA.

A. Gross appearance of cardiomyocyte induction by CSA. Six days after the Flk1⁺ cell culture on OP9 cells (Flk-d6). cTnT staining (brown). Left panel: control. Right panel: CSA treatment. Scale bars = 400 µm. B. Quantitative evaluation of cardiomyocyte induction by fluorescent intensity of cTnT staining. Relative fluorescent intensity is indicated (n = 4, †, p<0.001 vs control). C. Representative action potential of iPSC-derived spontaneously beating cardiomyocytes. D. Sarcomeric organization in TMRM-purified cardiomyocytes at Flk-d8. Immunostaining with anti-sarcomeric α-actinin antibody (red) and DAPI (blue). Right panel shows higher magnification of boxed area. Scale bar = 25 µm. E–H. Double immunostaining of TMRM-purified cardiomyocytes at Flk-d8 for connexin43 (Cx43) (green) and cTnT (orange) (E), Cav3.2 (green) and cTnT (orange) (F), HCN4 (green) and cTnT (orange) (G), Kir2.1 (green) and cTnT (orange) (H). Nuclei are visualized with DAPI. Scale bars = 25 µm. I. FACS analysis for cardiac progenitor induction from mouse iPSCs by CSA. X axis: CXCR4. Y axis: Flk1. Percentages of FCV cardiac progenitor cells (double positive population; red boxes) in total Flk1⁺ cell progenies are indicated.

Figure 2. Induction and expansion of cardiomyocytes from human iPSCs.

Human iPSCs were co-cultured with END-2 cells to differentiate cardiomyocytes. A. Gross morphology of a beating colony from human iPSCs (captured photo from Movie S1). B. cTnT staining of a beating colony on END-2 cells. Left panel: phase contrast image. Right panel: human cTnT staining (green). Scale bar  = 50 µm. C. RT-PCR analysis for differentiation markers during cardiomyocyte differentiation of human iPSCs (from END2-d0 to d12). Oct3/4: Undifferentiated cell marker, Brachyury-T: mesendoderm marker, KDR (human Flk1): mesoderm marker, Islet1: mesoderm and cardiac progenitor marker, Nkx2.5: cardiac progenitor and cardiomyocyte marker, cTnT: cardiomyocyte marker. D. Representative gross appearance of human iPSC-derived beating colonies at END2-d12 in 12-well dishes. Left panels: control. Right panels: CSA treatment from END2-d8. Upper panels: phase contrast images. Beating colonies are shown by red arrows. Lower panels: cTnT staining (green). E–G Quantitative evaluation of beating colony appearance. E. Total colony count (control; 177±9.7/well (12-well dishes)(n = 8), CSA; 162±8.0/well (n = 9); N.S., p = 0.237), F. Beating colony count (control; 9.1±1.2/well (12-well dishes)(n = 8), CSA; 36.4±2.5/well (n = 9); †, p<0.0001), and G. Percentages of beating colonies (control; 5.4±0.9% (n = 8), CSA; 23.5±2.8% (n = 9); †, p<0.0001) in total colonies that appeared at END2-d12. H. Immunostaining of actinin (red) and DAPI (blue) in dissociated cardiomyocyte colonies. The same colony is shown in Movie S2. Right panel shows higher magnification of boxed area. Sarcomere structures are evident. Scale bar = 50 µm.

Figure 3. Functional analysis of expanded human cardiomyocytes.

A. Ca⁺⁺ transient in dissociated beating colonies. Cytoplasmic Ca⁺⁺ change was monitored with fluo-8. Left panel: a transmission image of fluo-8 loaded iPSC colony. Middle and right panels: Fluo-8 images at the end (a) and the peak (b) of the fluorescence change. Scale bar = 50 µm. Lower panel: Time course of fluo-8 intensity change. The intensity was measured at the periphery (1), the entire colony (2) and the center (3) (ROIs shown in middle panel). Ratios (F1/F0) of the intensity to the one at the beginning of recording (F0) are indicated. Note that Ca transient is well synchronized within the colony. Real time video is shown in Movie S3. B. Representative action potential recorded from a cell in a beating colony. C. Representative single whole cell patch-cramp recording of a non-self beating human iPSC-derived cardiomyocyte after electrical stimulation.

Figure 4. Pharmacological responses of human iPSC-derived cardiomyocytes.

Field potential recordings of replated beating colonies after stimulation with isoproterenol (A), propranorol (B), and E-4031 (C). Photos; array of multi-electrode and replated colonies. Data recorded at electrodes in red squares are shown. A, B. Beating frequency (beating/minite). C. QT elongation. The time period from the first negative peak (open triangle) to the first positive peaks (closed triangles) reflects QT time in electrocardiogram. n = 3, †, p<0.001.

Figure 5. Ultrastructural analysis of human iPSC-derived cardiomyocytes.

Transmission electron microscopic images of beating colonies. Myofibrils with Z-bands (white closed arrowheads in A–D.), mitochondria (black closed arrowheads in B–D.), intercalated disk-like structure with desmosome (white open arrow in D.), atrial sercetory granules (electron-dense granules surrounded by double membranes. White open arrowheads in A. and E. (magnified image of A.)), glycogen granules (electron-dense small granules. Black open arrowheads in D. and F. (magnified image of D.)), ribosomal granules (electron-lucent small granules. White arrows in B–D. and G. (magnified image of B.)). N: nucleus. Scale bar  = 2 µm, direct magnify, ×3000 (A), ×7000 (B), ×4000 (C), ×5000 (D).