Assessment of temporal functional changes and miRNA profiling of human iPSC-derived cardiomyocytes

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca²⁺ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.

Figure 1

Functional analysis of cardiomyocytes on multielectrode array (MEA) system. (A) One well of an MEA 6 well plate consisting of 64 electrodes. (B) Magnified view of MEA electrodes. (C) Phase-contrast image of hiPSC-CMs cultured on sterile MEA plate for four weeks. (D) Experimental design of the study. The continuous waveform on 64 electrodes of a single well (E) and on one electrode (F). Four wells (n = 4) were selected to acquire data (64 electrodes/well). (G) The cardiac beat detector interprets continuous waves into cardiac beat shown in the form of a cardiac beat plot, representative image. (H) Long-time culture has shown changes in activity map which shows the decrease in beats per minute (BPM), representative image. (I) Conduction plot showing propagation delay of hiPSC-CMs cultured for Wk-1, Wk-2, and Wk-4, the blue region represents the origin of the beat (start electrode) while different colors showing propagation delay time as shown in scale bar, representative image. (J) Change in beat rate beat per minute (BPM) and (K) beat period in hiPSC-CMs cultured for Wk-1, Wk-2, and Wk-4 (s = second). (L) Max. Delay (Difference in beat detection time between electrodes in a well) (ms = millisecond). (M) Conduction velocity (m/s = meter per second) and, (N) Field potential duration (FPD) in hiPSC-CMs cultured for Wk-1, Wk-2, and Wk-4. (ms = millisecond) *p < 0.001 vs Wk-1, **p < 0.05 vs Wk-1, ^§p < 0.001 vs Wk-2, ^§§p < 0.05 vs Wk-2. Data expressed as mean ± SD (n = 4).

Figure 2

The contractile waveform analysis by particle image velocimetry (PIV) showing quicker contraction in mature cardiomyocytes. (A) Phase contrast microscopic images of differentiated and beating cardiomyocytes visualized by high framerate PIV. (B) The spontaneous beat pattern (PIV-derived displacements, measured relative to a stationary reference state and averaged over a 100-pixel radius large field of view) indicates that cardiomyocyte contractility is periodic with a steady waveform. (C) Fourier power spectra of beat patterns show the dominant beat frequency as a peak (asterisks). (D) Contractility analysis overlayed onto the original phase-contrast image. Contractile behavior is indicated by warmer colors, while cooler colors indicate local expansion of the cell collective. (E) Evolution of the spontaneous beating frequency during a four-week interval, obtained from recordings of Wk-1, Wk-2, and Wk-4 hiPSC-CMs. The beat frequency is stable in the investigated time period; the differences are not statistically significant and reflect a wide distribution of beat frequencies in various cultures. (F) The length of the contractile events is decreasing with culture age (p < 0.05). (G) Average contractile profiles indicate progressively quicker contraction and a prolonged relaxation as cultures mature in vitro. Black, red and green colors indicate progressively older (Wk-1, Wk-2, and Wk-4 hiPSC-CMs) cultures, respectively.

Figure 3

Time-dependent changes in Ca²⁺ transient (single-cell analysis) during maturation. (A) Representative line scan images with corresponding spatially averaged profiles of cytosolic Ca²⁺ transients evoked by electrical field stimulation at 0.5 Hz in cells cultured for Wk-1, Wk-2, and Wk-4, respectively. (B) Average values for systolic and end decay of Ca²⁺ cyt. (C) Time to Ca²⁺ transient peaks. (D) Rate of decay of Ca²⁺ transients recorded in Wk-1 (n = 51–59), Wk-2 (n = 41–46), and Wk-4 (n = 71–76), where ‘n’ indicates number of cells. *p < 0.05 vs Wk-1, **p < 0.01 vs Wk-1, ^§p < 0.05 vs Wk-2, ^§§p < 0.001 vs Wk-2 (t-tests with Bonferroni correction).

Figure 4

Immunofluorescence microscopy showing the metabolic maturation of cardiomyocytes by switching towards fatty acids β-oxidation pathways. (A) Cells were cultured for Wk-1, Wk-2, and Wk-4 and then immunostained with anti-ACADVL and anti-HADHA. (B) An image depth comparison showing a significantly increased level of ACADVL and HADHA in prolonged cultured hiPSC-CMs as compared to short-time cultured. The image depth was measured on Olympus FLUOVIEW Ver. 4.2a Viewer. Data expressed as mean ± SD, n = 4, *p < 0.05 vs Wk-1, ^#p < 0.05 vs Wk-2.

Figure 5

Refinement of ultrastructural features of hiPSC-CMs as compared to NF adult human heart. TEM illustrates overall refinement in hiPSC-CMs structure with increased cell culture time. (A–C) hiPSC-CMs show increased myofibril organization and cardiomyocyte structure from Wk-1 (A) to Wk-2 (B) and Wk-4 (C) in culture. (A’–C’) Internal membranes (white arrow) are punctate and disorganized at Wk-1 and Wk-2 but are organized at Wk-4, where internal membrane formation and incorporation between the myofilaments is evident. (A”–C”) Sarcomere structure shows refinement over the time span with hiPSC-CMs cultured at Wk-4 showing highly prominent and well-defined M-bands, I-bands, and A-bands as well as the alignment of the Z-discs. Z = Z-disc, M = M-band. (A’”–C’”) Mitochondria (Mit) are present in cells cultured at all three-time points. (E–G) TEM identifies intercalated discs for Wk-1, Wk-2, and Wk-4 hiPSC-CMs. Length of the representative intercalated disc is traced. (E’–G’) Increased magnification shows advanced ultrastructure of intercalated discs including the development of highly electron-dense regions, likely representative of desmosomes. (D) TEM sections from the left ventricle of NF adult human heart exhibit advanced myofibril organization and cardiomyocyte structure. (D’) Internal membranes are developed and present throughout the sarcomeres. (D”) Sarcomeres show distinct separation into Z-disk, M-band, A-band, and I-band with mitochondria present with visible cristae (D’”). (H) The intercalated disc is well defined in human heart tissue. The length of the intercalated disc is traced. (H’) Human heart tissue shows advanced ultrastructural features of the intercalated disc including the presence of desmosomes. Scale bar, 500 nm.

Figure 6

RT-qPCR analysis showing the expression of pluripotency and cardiac maturity markers in hiPSC-CMs. Cells were cultured for Wk-1, Wk-2, and Wk-4 and the result show expression of (A) pluripotency markers, (B) cardiac transcription factors, (C,D) contractile genes, (E) Gap junction, (F) Ca²⁺ handling, and (G) K⁺/Na⁺ channel genes. Data expressed as mean ± SD, n = 3 for cells, n = 9 for NF adult human heart, *p < 0.05 vs Wk-1, ^#p < 0.05 vs Wk-2, ^ϕp < 0.05 vs Wk-4.

Figure 7

RT-qPCR analysis showing the expression of cell cycle genes in hiPSC-CMs. Cells were cultured for Wk-1, Wk-2, and Wk-4 and the results show expression of cell cycle genes. Data expressed as mean ± SD, n = 4, *p < 0.05 vs Wk-1, ^#p < 0.05 vs Wk-2.

Figure 8

Identification of maturation-associated miRNAs in hiPSC-CMs during time-course culturing. hiPSC-CMs were cultured for Wk-1, Wk-2, and Wk-4, and the expression profiles of 800 miRNAs were analyzed. (A) miRNAs comparison of Wk-1 vs Wk-2 hiPSC-CMs; 4 miRNAs were differentially regulated. (B) miRNAs comparison of Wk-1 vs Wk-4 hiPSC-CMs; 12 miRNAs were differentially regulated. (C) Venn diagram showing that 3 miRNAs were common between Wk-1 vs Wk-2 hiPSC-CMs and Wk-1 vs Wk-4 hiPSC-CMs.