Measuring the contractile forces of human induced pluripotent stem cell-derived cardiomyocytes with arrays of microposts

Abstract

Human stem cell-derived cardiomyocytes hold promise for heart repair, disease modeling, drug screening, and for studies of developmental biology. All of these applications can be improved by assessing the contractility of cardiomyocytes at the single cell level. We have developed an in vitro platform for assessing the contractile performance of stem cell-derived cardiomyocytes that is compatible with other common endpoints such as microscopy and molecular biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded onto elastomeric micropost arrays in order to characterize the contractile force, velocity, and power produced by these cells. We assessed contractile function by tracking the deflection of microposts beneath an individual hiPSC-CM with optical microscopy. Immunofluorescent staining of these cells was employed to assess their spread area, nucleation, and sarcomeric structure on the microposts. Following seeding of hiPSC-CMs onto microposts coated with fibronectin, laminin, and collagen IV, we found that hiPSC-CMs on laminin coatings demonstrated higher attachment, spread area, and contractile velocity than those seeded on fibronectin or collagen IV coatings. Under optimized conditions, hiPSC-CMs spread to an area of approximately 420 μm2, generated systolic forces of approximately 15 nN/cell, showed contraction and relaxation rates of 1.74 μm/s and 1.46 μm/s, respectively, and had a peak contraction power of 29 fW. Thus, elastomeric micropost arrays can be used to study the contractile strength and kinetics of hiPSC-CMs. This system should facilitate studies of hiPSC-CM maturation, disease modeling, and drug screens as well as fundamental studies of human cardiac contraction.

Fig. 1

Technique the twitch force, velocity, and power of a single hiPSC-CM is determined by seeding cells onto a micropost array, selecting a beating cell of interest, and then taking a high-speed video of the post tips, as well as a reference image of the post bases (a). This video is taken in the phase contrast setting (b), while the reference image is taken in the red fluorescent channel (c). A custom matlab code is then used to determine the location of each post’s centroid in the reference plane (plus sign), as well as it is used to track the location of the post’s centroid in the video plane (d). The difference in location between these two centroids over time gives the deflection of the post over multiple twitch events, which can be multiplied by the post stiffness to yield the twitch force (e). Here, the dashed line represents the passive tension measured for this post. This deflection data can then be used to determine the twitch velocity (f), and power (g) of that post. Scale bars represent 6 μm.

Fig. 2

Twitch force representative images of hiPSC-CMs on fibronectin (a), laminin (b), and collagen IV (c) at peak twitch force indicate that the highest forces are present along the edges of the cell, and that the majority of the cell’s force is directed towards the cell center. Here, the arrows indicate the direction and magnitude of the force at each post. When the magnitude of these individual force vectors are summed, the total force produced by the cell can be plotted over multiple twitch cycles. Representative force traces for hiPSC-CMs on fibronectin (d), laminin (e), and collagen IV (f) demonstrate that the micropost array platform is capable of capturing these twitch cycles with high temporal resolution. Here, the text on the left of the graph indicates the twitch force, while that on the right indicates total force, and the dashed line indicates the passive force produced by the cell. Quantification of the passive force (g), maximum twitch force (h), and force per area (i), for hiPSC-CMs on different ECM proteins revealed that the quantities are statistically similar across all three conditions. Additionally, there was no significant difference in the spontaneous beating rate of the cells on the three different ECM proteins (j). Scale bar represents 6 μm and scale arrow indicates 6 nN.

Fig. 3

Twitch velocity representative maximum velocity traces for hiPSC-CMs on fibronectin (a), laminin (b), and collagen IV (c) indicate that the micropost platforms is capable of capturing both the contraction and relaxation velocity produced by spontaneously beating cells. Quantification of these traces revealed higher overall contraction and relaxation velocities for cells on collagen IV, but no significant difference between any of the treatments (d) and (e).

Fig. 4

Twitch power representative power traces for cells seeded on fibronectin (a), laminin (b), and collagen IV (c) demonstrate the ability of this technique to effectively resolve the contraction and relaxation power produced by hiPSC-CMs. Quantification of these power traces revealed higher overall contraction and relaxation velocities for cells on collagen IV, but no significant difference between any of the treatments (d) and (e).

Fig. 5

Maturation Immunofluorescent imaging of hiPSC-CMs seeded onto fibronectin (a), laminin (b), and collagen IV (c) coated microposts revealed punctate, unorganized sarcomeres within cells on fibronectin and collagen IV post, and more highly organized sarcomere structure within cells on laminin posts. Here, α-actinin is indicated by green, the cell nuclei is blue, and the microposts are red. The figure inset demonstrates how measurements of sarcomere length (dark dashed line) and Z-band width (light dashed line) were performed, Cell attachment (d) and spread area (e) are significantly higher on laminin posts than fibronectin posts, as well as attachment is significantly higher on laminin when compared to collagen IV. The circularity (f), Z-band widths (g), sarcomere lengths (h), and percentage of multinucleated cells (i) of the cells was not significantly different based on treatment. Color figures are available in the online version of this publication. Scale bar indicates 6 μm.