Hemodynamic Stimulation Using the Biomimetic Cardiac Tissue Model (BCTM) Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Human induced pluripotent stem cell (hiPSC)-derived cardio-myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally -immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo-dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga-nization and molecular signaling consistent with organ growth and functional maturation.

Keywords: Cardiovascular tissue engineering; Hemodynamics; Hypertrophy; Stem cell differentiation.

Fig 1:

(A) BCTM cell culture chamber with agarose mold prior to addition of cells within the fibrin gel. (B) BCTM cell culture chamber with the cell laden fibrin gel suspended between the two posts. This image was taken 2 days after removing the agarose mold. The gel originally takes the shape of the agarose mold but contracts over time as shown in the image. (C) The position of the posts within the cell culture chamber at the end of systole. The fiber experiences no strain at this point as the posts are at the initial resting position. (D) Position of posts within the cell culture chamber at the end of diastole. The thin membrane contacts with the insert below and deforms along its surface thus pulling the posts away from each other and applying uniaxial strain to the fiber. (E) Diagram representing how the BCTM reproduces the cardiac cycle. The BCTM imposes uniaxial strain onto the cells by using a flexible membrane that is stretched over an arched surface placed below the membrane. The yellow in this diagram represents the cell laden fibrin fiber.

Fig. 2:

Pressure-volume loops that hiPSC-CMs were subjected to during the course of the 7 day study. The systolic/diastolic ratio was maintained at 40/60% and the frequency was maintained at 1 Hz throughout the duration of the experiment.

Fig. 3:. (Left)

Static Controls and (Right) BCTM Stimulated samples with the entire fiber stained with cardiac troponin T (cTnT), alpha sarcomeric actin (α-SA), and DAPI with magnified images of α-SA (bottom) cTnT (middle), and combined image (top row).

Fig. 4:

Static controls (Left) and stimulated samples (Right). (Top) Visualization of ECM. (Middle) Representative images of sarcomeres and associated structures within cardiomyocytes. Arrows indicate lipid deposits (black) and an example of unorganized sarcomeres (white) both of which were common throughout the static controls. (Bottom) High magnification images of representative sarcomeres from the static controls and stimulated samples. The black arrow on the stimulated sample marks a white area along the middle of the sarcomere that was found consistently in the stimulated sarcomeres and what could be the formation of the M-line.

Fig. 5:

Static controls vs. BCTM stimulated samples normalized to GAPDH expression levels. Statistically significant upregulation was observed in genes GAT4, MYL2, CAM2KB, and MYH7 in stimulated samples (N=5) vs. static samples (N=4).

Fig. 6:

Data collected from videos made of the fibers undergoing electrical stimulation at frequencies: 1 Hz, 1.3 Hz, 1.5 Hz, and 2.0 Hz. Videos were also made of the intrinsic (spontaneous) fractional shortening of each fiber (N=4). The stimulated samples showed significantly higher fractional shortening at 1.3, 1.5, and 2.0 Hz. The intrinsic contractions were not significantly different.