Integration of co-culture conditions and 3D gelatin methacryloyl hydrogels to improve human-induced pluripotent stem cells-derived cardiomyocytes maturation.

Abstract

<h4>Introduction</h4>Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an excellent alternative to animals for <i>in vitro</i> cardiac studies. However, their immature fetal phenotype represents an important limit to consider. Approaches proposed to overcome this issue are based on better reproducing the <i>in vivo</i> native CMs microenvironment. In the present work, a biomimetic environment to enhance hiPSC-CMs maturation was developed by combining a 14-day co-culture of hiPSC-CMs and Human Coronary Artery Endothelial cells (HCAECs) in a 3D Gelatin Methacryloyl (GelMA) hydrogel system.<h4>Methods</h4>Chemical characterization of custom-synthesized GelMA was performed through Attenuated Total Reflectance Fourier Transformed Infrared (ATR-FTIR) and proton Nuclear Magnetic Resonance (¹H NMR) spectroscopies. GelMA degree of methacryloylation (DoM) was estimated through the ninhydrin colorimetric assay. Then, hydrogels were prepared by solubilizing GelMA in presence of phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP) as photoinitiator (0.05% w/v) and photo-rheological tests were carried out to investigate the photo-polymerization process (at 365 nm, 10 mW/cm²) and the mechanical properties of the resulting gels. Hydrogel swelling ratio was also monitored up to 5 days of incubation in aqueous medium at 37°C. The maturation phenotype was achieved by co-culturing hiPSC-CMs with HCAECs in the 3D model composed of GelMA with around 96% DoM, solubilized at 5% w/v concentration in cell culture medium, added with LAP and crosslinked by UV light (40 s). The expression of specific cardiac maturation markers was investigated through Real-Time PCR (RT-PCR). Omics analyses were carried out to compare terms of biological processes, cellular components, and molecular functions between the 3D model here presented and a classical 2D monoculture of hiPSC-CMs.<h4>Results</h4>GelMA was successfully synthesized with two different DoMs (i.e., 30%-40% and 96%-97%) and used to prepare hydrogels at 5%, 7.5% and 10% w/v concentrations. Both GelMA DoM and hydrogel concentration appeared as tuning parameters of gel behavior in aqueous environment at 37°C and mechanical properties, with Young's Modulus of photo-cured gels ranging between <i>ca.</i> 4 and 55 kPa. Within this plethora, photo-cured gels prepared from GelMA with <i>ca</i>. 96% DoM solubilized at 5% w/v concentration showed prolonged stability over time and E value (8.70 ± 0.12 kPa) similar to the native cardiac tissue and were thus selected to design bioengineered cardiac tissue models upon hiPSC-CMs and HCAECs loading. A direct comparison with the classical 2D monoculture of hiPSC-CMs highlighted the improved maturation profile achieved by hiPSC-CMs in the 3D GelMA system, as demonstrated by the higher expression of cardiac maturation markers (TNNT2, ACTN2, Myl2, MYH 7, CX43 and PPAR-α), in association with proteomics and transcriptomics data, that showed the modulation of specific biological pathways related to cardiac differentiation and contraction processes in the 3D system. A more in-depth investigation of cell health and function also suggested a higher viability and less suffering condition for cells co-cultured in the 3D hydrogel.<h4>Conclusion</h4>Our results demonstrated that the 3D bioengineered model proposed here represents a good replica of the native cardiac tissue environment, improving the hiPSC-CMs maturation profile, thus opening the opportunity for its application in disease modeling and toxicological screening studies.