3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids

Abstract

Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

Keywords: 3D-culture; cardiomyocyte; co-culture; fibroblast; induced pluripotent stem cells; microtissue; myofibroblast; scaffold-free.

Figure 1

SEM of spheroids made of hiPSC-CMs and CF placed on gelatin-coated glass coverslips thereby allowing outgrowth of fibroblasts. (A) Two spheroids and out-growing CF on the glass surface. (B) Spheroid cut by scalpel after critical point drying and re-sputtering with gold. (C,D) Details of the spheroid surface (enlarged in D).

Figure 2

Immunohistochemical characterization of tissue, 2D-, and 3D-cultures. Nuclei are labeled with DAPI (blue) in all images. (A) Cryosection of an adult mouse heart immunostained for myomesin (red) and vimentin (green). (B) Confocal optical section on the substrate level of a spheroid made of hiPSC-CM and CF cultured for 1 month. Immunostaining for myosin heavy chain (red), and vimentin (green). (C) Confocal optical section above the substrate level of the same spheroid as shown in (B). (D) 2D-cultured cardiac fibroblasts immunostained for alpha-SMA (red) and all actin (green) shown as a maximum intensity projection of optical sections. (E) Single confocal optical sections perpendicular to the substrate through a CF-only, small spheroid (above) cultured for 5 days and immunostained for all actin (red) and alpha-SMA (green), and an optical section at midlevel (below). (F) Cryosection of fibroblast-only spheroid cultured for 3 weeks immunostained for vimentin (red) and alpha-SMA (green). (G) Western blot experiments and quantitative assessment of the ratio of alpha-SMA/vimentin (n = 4, ***p < 0.001) demonstrating the difference of alpha-SMA content in 2D vs. 3D cultures of pure CF. (H) Whole-mount immunostaining of a cardiomyocyte-only spheroid 1 month in culture, N-cadherin (red) and EH-myomesin (green). (I) Cardiomyocyte-only spheroid 1 month in culture, whole-mount immunostained for all-actin (red) and laminin (green).

Figure 3

Spheroids made of hiPSC-CMs and CF were cultured for 1 month, then processed for TEM. (A) Low magnification image of a region close to the surface of a spheroid. (B) High magnification of a cell-cell contact region. Arrowhead points to a well-formed intercalated disc-like structure. Z-disc densities are found around the intercalated disc-like structure. (C) Inner layer of a spheroid that to shows the contact-zone between a cardiomyocyte (on the left) and non-myocytes (middle, marked by an asterisk). (D) Perinuclear mitochondria and myofibrils in a cardiomyocyte.

Figure 4

qPCR of 2D- and 3D-cultured hiPSC-CMs and CF. Shown are fold-changes of expression normalized to GAPDH expression. (A) Comparison of gene expression of pure hiPSC-CMs (CM) in 3D vs. 2D culture. (B) Comparison of co-cultured hiPSC-CMs and CF in 3D vs. pure hiPSC-CMs in 3D. (C) Comparison of pure CF in 3D vs. CF monolayer culture in 2D. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Electrophysiological measurements in 2D- and 3D-cultured hiPSC-CMs (CM) with and without CF showing spontaneous APs. (A–D) Patch-clamp assessments of hiPSC-CMs cultured in 2D culture for 1 week (n = 10 cells). (A) Representative AP tracings of 2D-cultured cardiomyocytes. (B) Amplitude (AMP) and resting membrane potential (RMP). (C) Action potential duration at 20, 50, and 90% of the amplitude. (D) Maximum upstroke velocity. (E) Representative AP tracing measured in pure hiPSC-CM spheroids. (F) Representative AP tracing from co-cultured spheroids of hiPSC-CM and CF. (G) Amplitude and resting membrane potential in spheroids without (black) and with CF (gray). (H) Action potential duration at 20, 50, and 90% of the amplitude in spheroids without and with CF. (I) Maximum upstroke velocity in spheroids without and with CF. N = 6–10 spheroids per group.

Figure 6

(A) Contraction features in co-culture spheroids 10 days in culture at different pacing frequencies. The spheroids followed pacing frequencies of 2–4 Hz reflected by the time of the “p-p” parameter since 2 Hz = 500 ms, 3 Hz = 333 ms, and 4 Hz = 250 ms. (B) Spontaneous contractions with spheroid of pure hiPSC-CMs or co-culture spheroids at 1 month in culture. (C) Spontaneous contractions in 2D cultures of hiPSC-CMS or cultures with added CF. N = 4–6 spheroids per group or fields in 2D-cultures, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Contraction features in small and large multicellular aggregates, and effects of cardiotoxic substances. (A) Features of spontaneous contractions of small and large aggregates (50 ± 8 and 400 ± 47 μm in diameter) of hiPSC-CMs and CF were investigated. (B) The cardiotoxic cancer therapy doxorubicin (Doxo) was added to co-culture spheroids. The spheroids were electrically paced at 3 Hz (the corresponding value is indicated by an arrow in the y-axis for the p-p parameter) (C) The effect of Doxo on 2D cultured hiPSC-CMs. N = 4–6 spheroids per group or fields in 2D-cultures, *p < 0.05, **p < 0.01.