Mechanically and Chemically Defined PEG Hydrogels Improve Reproducibility in Human Cardioid Development

Abstract

Cardioids are 3D self-organized heart organoids directly derived from induced pluripotent stem cells (hiPSCs) aggregates. The growth and culture of cardioids is either conducted in suspension culture or heavily relies on Matrigel encapsulation. Despite the significant advancements in cardioid technology, reproducibility remains a major challenge, limiting their widespread use in both basic research and translational applications. Here, for the first time, we employed synthetic, matrix metalloproteinase (MMP)-degradable polyethylene glycol (PEG)-based hydrogels to define the effect of mechanical and biochemical cues on cardioid development. Successful cardiac differentiation is demonstrated in all the hydrogel conditions, while cardioid cultured in optimized PEG hydrogel (3 wt.% PEG-2mM RGD) underwent similar morphological development and comparable tissue functions to those cultured in Matrigel. Matrix stiffness and cell adhesion motif play a critical role in cardioid development, nascent chamber formation, contractile physiology, and endothelial cell gene enrichment. More importantly, synthetic hydrogel improved the reproducibility in cardioid properties compared to traditional suspension culture and Matrigel encapsulation. Therefore, PEG-based hydrogel has the potential to be used as an alternative to Matrigel for human cardioid culture in a variety of clinical applications including cell therapy and tissue engineering.

Keywords: cardioids; human induced pluripotent stem cells; mechanobiology; organoids; synthetic hydrogel.

Figure 1

PEG hydrogels with synthetic peptides replicated key ECM cues. A) Schematic representation of hydrogel composition using 8‐arm PEG‐norbornene macromers. B) Hydrogel network incorporating MMP‐degradable peptide (blue) as crosslinkers and RGD peptide (green) for cell adhesion. C) Hydrogel polymerization process using the photo‐initiator LAP and long‐wave ultraviolet (UV) light (≈5 mW cm^{− 2}, 365 nm, 3 min) for cardioid encapsulation. D) Storage modulus and E) loss modulus of PEG‐based hydrogels. F) Swelling ratio and G) mesh size calculations of the hydrogels. Statistical analysis was performed using one‐way ANOVA with Tukey post‐hoc correction (*p < 0.05, n = 3).

Figure 2

Impact of hydrogel properties on cardioid viability and growth behaviors. A) Timeline of cardioid encapsulation and differentiation. Phase‐contrast images of cardioids encapsulated in different hydrogels on B) day 10 and C) day 20. D) Percentage of beating organoids under different culture conditions, indicating cardiac differentiation efficacy. Comparison of E) organoid size and F) outward cell migration across different culture conditions. Matrigel resulted in the largest organoids and highest cell migration, while increasing hydrogel stiffness reduced organoid size and differentiation efficiency. Statistical significance was determined using one‐way ANOVA with Tukey post‐hoc correction (**p < 0.01, n ≥ 15).

Figure 3

Impact of hydrogel properties on chamber formation within cardioids. A) Immunostaining of cardiomyocyte‐specific protein cardiac troponin T (cTnT) and smooth muscle marker calponin 1. B) Immunostaining of sarcomere α‐actinin to demonstrate sarcomere structures of cardioids. C) Quantification of the cavity area ratio and D) cavity size across different culture conditions. E) Measurement of sarcomere distance in cardioids. Cardioids cultured in 3 wt.% PEG‐2 mM RGD hydrogels exhibited comparable cavity formation to those in Matrigel, both of which were higher than in other conditions. Statistical significance was determined using one‐way ANOVA with Tukey post‐hoc correction (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant, n ≥ 15).

Figure 4

Impact of hydrogel properties on cardiac contractile function. A) Fluorescent calcium flux imaging and B) brightfield video‐based contraction motion analysis of cardioids. C) Motion heatmaps visualizing contraction dynamics across different hydrogel conditions. D) Quantitative analysis of contractile function, including beat rate, maximum relaxation and contraction velocities, peak fluorescence intensity, upstroke duration (T0), peak duration, and decay times (T30, T50, and T75). Statistical significance was determined using one‐way ANOVA with Tukey post‐hoc correction (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n ≥ 15).

Figure 5

Transcriptomic profile of cardioids across different culture conditions. A) Principal component analysis (PCA) plot showing distinct clustering of cardioids cultured in 3 wt.% PEG hydrogels (with and without 2 mM RGD), Matrigel, and suspension culture (No Gel). B) Volcano plot depicting differentially expressed genes between Matrigel / 3 wt.% PEG‐2 mM RGD hydrogels and 3 wt.% PEG‐0 mM RGD / No Gel groups. C) Gene ontology (GO), Reactome, and KEGG pathway analyses highlighting distinct biological processes and signaling pathways enriched in different conditions. Averaged gene expression levels related to D) cell‐ECM interaction and mechanotransduction, E) vasculature development, F) heart development, and G) ion channels across the different culture groups.

Figure 6

Analysis of top 1000 genes with highest variances. A) Heatmap and hierarchical clustering showed distinct transcriptome profiles among different groups for the top 1000 genes with highest variance. B–D) Gene ontology, Reactome analysis and KEGG pathway showed 3%‐2mm RGD and Matrigel derived organoids highly enriched the genes associated with heart development, calcium signaling and contractile functions, while 3%‐0mM RGD and No gel cultured organoids expressed the genes associated with embryonic development. The genes enriched in PEG hydrogel groups are related to metabolic activity and NOTCH signaling.