Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density

Abstract

Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.

Fig. 1

Schematic of ELP hydrogel formation. ELP consists of 4 repeats of a cassette containing 1 bioactive domain and 3 identical elastin-like domains, which include lysine residues within the elastin-like domain to provide amine reactive crosslinking sites that form covalent bonds with THPC (A). Elastic moduli of 5 wt% ELP hydrogels (n = 4-5) with varying THPC:ELP reactive group stoichiometry (B). Effective diffusion coefficient for 70 kDa FITC-dextran in 5 wt% ELP hydrogels of varying crosslink density (C). All values are reported as mean ± SEM, where * denotes significant different p < 0.05.

Fig. 2

Representative fluorescent images of EGFP expression within hydrogels after 2, 4, and 8 days of culture for crosslink densities of 0.5:1, 0.75:1, and 1:1 THPC to ELP reactive groups (A). Yield of EGFP positive hydrogels (n = 15-18), where * denotes significant difference among groups (p<0.05) by χ² test for binomial distributions (B).

Fig. 3

Yield of contracting hydrogels (A) and contractility rate (B) plotted for each hydrogel-encapsulated embryoid body exhibiting contractile behavior for up to 8 days. Data are displayed as the mean and SEM for each group (n = 8-14). Significant differences among groups (p<0.05) using χ² test for binomial distributions is denoted by *. No significant differences in contractility rates were observed among all groups.

Fig. 4

Functional pathway enrichment analysis of genes differentially regulated (p<0.05) between 0.5:1 and 1:1 crosslinking groups. Top five up and down regulated categories are presented, where the magnitude of functional increase (green) or decrease (red) of 0.5:1 group over 1:1 group for respective processes are indicated by activation z-score.

Fig. 5

Metabolic activity (n = 5-6) (A) and DNA content (n = 11-12) (B) for murine EBs encapsulated in 5 wt% ELP hydrogels of 0.5:1 and 1:1 crosslink density, with 5.3 or 0 mM RGD ligand density, at days 1, 4, and 8. DAPI coverage (n = 7-11) of murine EBs encapsulated after 8 days of culture (C). Significant difference (p<0.05) compared to 1:1 counterparts determined by Tukey's posthoc test is denoted by *. No significant differences were observed between 0 (using scrambled RDG ELP variant) and 5.3 mM RGD hydrogels at the same crosslink density.

Fig. 6

MMP activity within 5 wt% ELP hydrogels with 5.3 or 0 mM RGD ligand density at 0.5:1 and 1:1 crosslink density (n = 3).

Fig. 7

Cardiomyocyte differentiation yield within 5wt% ELP hydrogels with 0.5:1 crosslink density and 5.3 mM RGD ligand density quantified by EGFP expression (A) and contractility (B) with or without the addition of MMP inhibitors, PD166793 and doxycycline (n=11-14). Significant differences among groups (p<0.05) using χ² test for binomial distributions is denoted by *.