Micropattern platform promotes extracellular matrix remodeling by human PSC-derived cardiac fibroblasts and enhances contractility of co-cultured cardiomyocytes

Abstract

In native heart tissue, cardiac fibroblasts provide the structural framework of extracellular matrix (ECM) while also influencing the electrical and mechanical properties of cardiomyocytes. Recent advances in the field of stem cell differentiation have led to the availability of human pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) in addition to cardiomyocytes (iPSC-CMs). Here we use a novel 2D in vitro micropatterned platform that provides control over ECM geometry and substrate stiffness. When cultured alone on soft micropatterned substrates, iPSC-CFs are confined to the micropatterned features and remodel the ECM into anisotropic fibers. Similar remodeling and ECM production occurs when cultured with iPSC-CMs in a co-culture model. In addition to modifications in the ECM, our results show that iPSC-CFs influence iPSC-CM function with accelerated Ca²⁺ transient rise-up time and greater contractile strains in the co-culture conditions compared to when iPSC-CMs are cultured alone. These combined observations highlight the important role cardiac fibroblasts play in vivo and the need for co-culture models like the one presented here to provide more representative in vitro cardiac constructs.

Keywords: cardiac fibroblast; cardiomyocyte; extracellular matrix; human pluripotent stem cell; micropattern.

FIGURE 1

Decellularized fibronectin from (a) monolayers and (b) micropatterned lanes of varying widths after 2, 5, and 8 days of culture with iPSC‐CFs. (c) Quantification of the amount of fibronectin fibers aligned within ±10% of the superior angle using SGFT software. White = Fibronectin. Scale bar = 100 µm. *p < 0.05, **p < 0.001, two‐way ANOVA with post hoc Tukey tests. n = 8 (ROIs within a sample) for each day and lane width tested

FIGURE 2

The 10 kPa PDMS substrates patterned with Matrigel using the 15° chevron pattern and stained for fibronectin (white). (a) Day 0 control sample with no iPSC‐CFs attached. Decellularized scaffolds after iPSC‐CFs were cultured for (b) 2 days and (c) 18 days. Scale bars = 25 µm. (d) Quantification of the amount of fibronectin present over the available micropattern area. *p < 0.001, one‐way ANOVA with post hoc Tukey tests. n = 6 (ROIs within a sample) for all conditions

FIGURE 3

iPSC‐CFs attached to 10 kPa PDMS substrates for 1 day prior to 13 h time‐lapse imaging. Available ECM for cell migration on the 10 kPa PDMS (a) Monolayer, (b) 30 µm lanes and (c) 15° chevron pattern (Laminin = red). (a’–c’) Representative bright field images, (a’’–c’’) migration patterns of individual iPSC‐CFs and (a’’’–c’’’) angle measurements for each of the three conditions tested: monolayer, 30 µm Lanes and 15° chevron pattern. Scale bars = 100 µm for a–c. Units are in µm for a’’–c’’. 90° indicates an upward movement and 270° a downward movement from the last measurement for a’’’–c’’’. Migration patterns of 30–50 iPSC‐CFs were tracked over multiple samples for each condition

FIGURE 4

(a) Schematic of the experimental timeline and iPSC differentiation protocols. Red numbers denote days after initiation of iPSC‐CM differentiation, blue numbers are days after initiation of iPSC‐CF differentiation and black numbers represent experimental time points. Co‐culture conditions consist of iPSC‐CF addition on either Day 0 (CF Day 0) or Day 4 (CF Day 4) and are co‐cultured with iPSC‐CMs until Day 18. (b) Bright field images of patterned iPSC‐CMs at the start and end of co‐culture. Bright field locations are the same for the start and end of co‐culture. (c) ROIs of areas not occupied by cells highlighted in red for the start and end of co‐culture. (d) Quantification of area of cell coverage for the start and end of co‐culture. Scale bars = 250 µm. *p < 0.001, two‐way ANOVA with post hoc Tukey tests. n = 9 for CM Only, n = 11 for Day 0 CF, and n = 11 for Day 4 CF (2–3 images per sample, 4 independent experiments)

FIGURE 5

(a) Migration patterns of individual iPSC‐CFs when co‐cultured with iPSC‐CMs on the 15° chevron pattern. 90° indicates an upward movement and 270° a downward movement from the last measurement. Migration patterns of 33 iPSC‐CFs were tracked over two independent experiments. (b) Quantification of the percent of myofibrils aligned within 10° of the superior angle for the three conditions tested determined by SGFT. n = 4 (2 ROIs within a sample, 2 independent samples). Collagen and fibronectin expression after 18 days of culture for the CM only (c–e), Day 0 CF (f–h) and Day 4 CF conditions (i–k). Collagen = green, Fibronectin = white, Actin = red, DAPI = blue. Scale bars = 50 µm

FIGURE 6

Optical mapping results for (a) CaT duration (CaTD), (b) CaT rise‐up time, (c) conduction velocity (CV) and (d–e) anisotropic conduction velocity for the different conditions tested. Black arrows in optical mapping images represent the major axis of the 15° chevron pattern, scale bar = 2.5 mm. *p < 0.001, one‐way ANOVA with post hoc Tukey tests. n = 7 for all conditions (2–3 samples per experiment, three independent experiments)

FIGURE 7

(a) Heat maps of the second principal strain for each of the three conditions during peak contraction on Day 12. (b) The maximum contractile strain for the three patterned conditions tested on Day 6, 12, and 18 of culture. *p < 0.001, **p < 0.05, two‐way ANOVA with post hoc Tukey tests. n = 19 for CM Only, n = 19 for CF Day 0, and n = 22 for CF Day 4. (two ROIs within a sample, 2–4 samples per experiment, three independent experiments)