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Influence of Remodeled ECM and Co-culture with iPSC-Derived Cardiac Fibroblasts on the Mechanical Function of Micropatterned iPSC-Derived Cardiomyocytes

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Abstract

Introduction

In native heart tissue, functions of cardiac fibroblasts (CFs) include synthesis, remodeling, and degradation of the extracellular matrix (ECM) as well as secreting factors that regulate cardiomyocyte (CM) function. The influence of direct co-culture and CF-derived ECM on CM mechanical function are not fully understood.

Methods

Here we use an engineered culture platform that provides control over ECM geometry and substrate stiffness to evaluate the influence of iPSC-CFs, and the ECM they produce, on the mechanical function of iPSC-CMs. Mechanical analysis was performed using digital image correlation to quantify maximum contractile strain, spontaneous contraction rate, and full-field organization of the contractions.

Results

When cultured alone, iPSC-CFs produce and remodel the ECM into fibers following the underlying 15° chevron patterned ECM. The substrates were decellularized and confirmed to have highly aligned fibers that covered a large fraction of the pattern area before reseeding with iPSC-CMs, alone or in co-culture with iPSC-CFs. When seeded on decellularized ECM, larger maximum contractile strains were observed in the co-culture condition compared to the CM Only condition. No significant difference was found in contractile strain between the Matrigel and decellularized ECM conditions; however, the spontaneous contraction rate was lower in the decellularized ECM condition. A methodology for quantifying alignment of cell contraction across the entire field of view was developed based on trajectories approximating the cell displacements during contraction. Trajectory alignment was unaltered by changes in culture or ECM conditions.

Conclusions

These combined observations highlight the important role CFs play in vivo and the need for models that enable a quantitative approach to examine interactions between the CFs and CMs, as well as the interactions of these cells with the ECM.

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Acknowledgements

The authors would like to thank Jodi Lawson for her assistance in data processing and manuscript preparation. This research was funded by the University of Wisconsin-Madison, through the Karen Thompson Medhi Professorship, Graduate School, and Office of the Vice Chancellor for Research and Graduate Education (WCC). Support was also provided by the National Institutes of Health, under Ruth L. Kirschstein National Research Service Award T32 HL 007936 from the National Heart Lung and Blood Institute to the University of Wisconsin-Madison Cardiovascular Research Center (AS). TJK and and JZ were supported for this work by National Institutes of Health U01HL134764 and National Science Foundation 1648035. This material is based upon work supported by (while serving at) the National Science Foundation (WCC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health nor the National Science Foundation.

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA

    A. Stempien, M. Josvai, J. Notbohm & W. C. Crone

  2. Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA

    A. Stempien, M. Josvai & W. C. Crone

  3. Department of Engineering Physics, University of Wisconsin-Madison, Madison, WI, USA

    J. Notbohm & W. C. Crone

  4. Department of Medicine, Division of Cardiovascular Medicine, University of Wisconsin-Madison, Madison, WI, USA

    J. Zhang & T. J. Kamp

  5. Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA

    T. J. Kamp

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  1. A. Stempien
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13239_2024_711_MOESM1_ESM.tif

(A) Representative 10x objective images of decellularized ECM used in this study on days 0 (prior to CF seeding on micropatterned substrates), 11. 14, and 18 immunolabeled for Collagen Type I (COL1A; teal) and Laminin (green). Laminin present on day 0 originates from Matrigel patterning. Scale bar = 250 µm. (B) Representative 100x objective images of Laminin remodeling at bridge regions from day 0 to 18. Scale bar = 25 µm. (C) Z – stack thickness of 100x images of decellularized scaffolds on days 0 (Matrigel only), 11, 14, and 18. N = 4 for each day. **p < .01, *** p < .001. Supplementary file9 (TIF 19348 kb)

13239_2024_711_MOESM2_ESM.tif

Heatmap images of the maximum state of contraction for CM Only on Matrigel (top left), CM-CF coculture on Matrigel (top right), CM Only on decellularized ECM (bottom left) and CM-CF coculture on decellularized ECM (bottom right). Maximum states of contraction were identified as the maximum average second principal strain calculated by DIC software. Regions of the imaging field not occupied by cells were excluded from calculations and are denoted by the white regions in the heatmap still. The second principal strain values reported in the legend are in percent. Full length heatmap videos, as well as the corresponding raw phase contrast video for each representative image are included as Supplemental Videos 1-8. Supplementary file10 (TIF 5081 kb)

13239_2024_711_MOESM3_ESM.tif

(Left) From a brightfield video of contracting iPSC-CMs, full-field displacements in x and y and computed for the duration of the video. (Middle) The resulting displacement trajectories are shown. The angle of each vector is computed and small trajectories, likely occurring in the regions where cells are not present, are filtered based on the trajectory magnitudes. (Right) A histogram of the trajectory angles is made, and the primary angle used to compute the percent of all trajectories within 20° of the peak angle. Representative histograms of the trajectory angles for (top) chevron, (middle) lane, and (bottom) monolayer pattern. Supplementary file11 (TIF 1231 kb)

13239_2024_711_MOESM4_ESM.tif

Percent of displacement trajectories with 20° alignment for days 6 (top), 12 (center), and 18 (bottom) in both Matrigel and decellularized ECM and culture conditions on chevron patterned substrates (CM Only = black, CM-CF = red). No significant difference exists in overall alignment on any day. Day 6: N= 25 CM Matrigel, N= 22 CM Decellularized, N= 31 CM-CF Matrigel, N = 22 CM-CF Decellularized. Day 12: N= 25 CM Matrigel, N= 19 CM Decellularized, N= 25 CM-CF Matrigel, N = 19 CM-CF Decellularized. Day 18: N= 18 CM Matrigel, N= 16 CM Decellularized, N= 18 CM-CF Matrigel, N = 19 CM-CF Decellularized. Supplementary file12 (TIF 2734 kb)

13239_2024_711_MOESM5_ESM.tif

Effects of micropatterned lane width on displacement trajectory alignment (A), maximum contractile strain (B), and number of neighboring CMs (C). The number of neighboring CMs was determined as the number of nuclei within 100 μm of each individual nucleus. The threshold of 100 μm was chosen as it is the physiological length of adult CMs. For displacement alignment and strain quantification: N = 26 (20 μm), 27 (40 μm), 31 (60 μm), 32 (80 μm), 32 (100 μm), 32 (120 μm), and 29 (140 μm). For the quantification of neighboring CMs, N = 4 biological replicates, with one image of each lane width of interest or chevron pattern taken from each sample. *p < .05, **p < .01 ***p < .001, two-way ANOVA with post hoc Tukey tests. All experiments were performed with CMs only, in the absence of CFs for the duration of culture. Supplementary file13 (TIF 1838 kb)

13239_2024_711_MOESM6_ESM.docx

(A) Quantification of the percent of myofibrils aligned within 10 degrees of the superior angle for the two culture conditions tested determined by SGFT. N = 8 (4 ROIs within a sample, 2 independent samples). (B) α-actinin expression after 18 days of culture for the CM Only (left) and CM-CF (right). α-actinin = green. Scale bars = 50 µm. Supplementary file14 (DOC 6002 kb)

Representative brightfield video recording of a day 12 CM Only sample on Matrigel. Captured on a Nikon Eclipse Ti microscope with a Plan Flour 10x NA 0.3 objective and Nikon DS-QiMc camera with samples maintained at 37°C. Scale bar = 250 µm. Acquisition rate = 18.92 fps (5.55 s). Supplementary file1 (AVI 26955 kb)

Representative brightfield video recording of a day 12 CM-CF sample on Matrigel. Captured on a Nikon Eclipse Ti microscope with a Plan Flour 10x NA 0.3 objective and Nikon DS-QiMc camera with samples maintained at 37°C. Scale bar = 250 µm. Acquisition rate = 18.92 fps (7.45 s). Supplementary file2 (AVI 36264 kb)

Representative brightfield video recording of a day 12 CM Only sample on decellularized ECM. Captured on a Nikon Eclipse Ti microscope with a Plan Flour 10x NA 0.3 objective and Nikon DS-QiMc camera with samples maintained at 37°C. Scale bar = 250 µm. Acquisition rate = 18.92 fps (8.62 s). Supplementary file3 (AVI 45398 kb)

Representative brightfield video recording of a day 12 CM-CF sample on decellularized ECM. Captured on a Nikon Eclipse Ti microscope with a Plan Flour 10x NA 0.3 objective and Nikon DS-QiMc camera with samples maintained at 37°C. Scale bar = 250 µm. Acquisition rate = 18.92 fps (7.93 s). Supplementary file4 (AVI 43510 kb)

Representative heatmap video of day 12 CM Only sample on Matrigel, corresponding to the Supplementary Video 1. Acquisition rate = 18.92 fps (5.55 s). Supplementary file5 (AVI 19269 kb)

Representative heatmap video of a day 12 CM-CF sample on Matrigel, corresponding to the Supplementary Video 2. Acquisition rate = 18.92 fps (7.45 s). Supplementary file6 (AVI 24307 kb)

Representative heatmap video of a day 12 CM Only sample on decellularized ECM, corresponding to the Supplementary Video 3. Acquisition rate = 18.92 fps (8.62 s). Supplementary file7 (AVI 27644 kb)

Representative heatmap video of a day 12 CM-CF sample on decellularized ECM, corresponding to the Supplementary Video 4. Acquisition rate = 18.92 fps (7.93 s). Supplementary file8 (AVI 24139 kb)

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Stempien, A., Josvai, M., Notbohm, J. et al. Influence of Remodeled ECM and Co-culture with iPSC-Derived Cardiac Fibroblasts on the Mechanical Function of Micropatterned iPSC-Derived Cardiomyocytes. Cardiovasc Eng Tech (2024). https://doi.org/10.1007/s13239-024-00711-8

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  • DOI: https://doi.org/10.1007/s13239-024-00711-8

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