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

Abstract

Introduction: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an excellent alternative to animals for in vitro 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 in vivo 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.

Methods: 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.

Results: 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 ca. 4 and 55 kPa. Within this plethora, photo-cured gels prepared from GelMA with ca. 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.

Conclusion: 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.

Keywords: 3D model; gelatin methacryloyl; hiPSC-CMs culture; hiPSCs differentiation; hydrogels.

FIGURE 1

ATR-FTIR spectra of gelatin (black), GelMA_LOW (red), and GelMA_HIGH (blue). Dashed lines identify the characteristic bands of gelatin and GelMA.

FIGURE 2

¹H NMR spectra of gelatin (black), GelMA_LOW (red) and GelMA_HIGH (blue). The blue rectangle underlines the region of the spectra containing the signals due to the acrylic protons (CH₂ = C(CH₃)CONH) of methacrylamide groups; the purple rectangle highlights the chemical shift range where the free lysine methylene signal (NH₂CH₂CH₂CH₂CH₂-) appears; and the orange rectangle underlines the region of the spectra with the signal at 1.85 ppm due to the methyl protons (CH₂ = C(CH₃)CO-) of methacryloyl groups.

FIGURE 3

DoM values estimated through the Ninhydrin assay for different GelMA batches (i.e., 3 batches for both GelMA_LOW and GelMA_HIGH) (means compared using Student’s independent t-test. *p from 0.01 to 0.05; **p from 0.001 to 0.01, n = 3, data reported as mean ± SD).

FIGURE 4

Results of the photo-rheological characterization performed on GelMA formulations. (A) Trend of storage modulus (G′) as a function of time, as registered during photo-rheological tests (60s light OFF + 90s light ON + 60s light OFF) carried out on GelMA_LOW_5%, GelMA_LOW_7.5%, GelMA_LOW_10%, GelMA_HIGH_5%, GelMA_HIGH_7.5%, and GelMA_HIGH_10% (red and blue tones for GelMA_LOW and GelMA_HIGH formulations, respectively); (B) Value of storage modulus registered at the end of the photo-rheological time sweep tests for all the investigated GelMA formulations (red and blue for GelMA_LOW and GelMA_HIGH formulations, respectively) (means were statistically compared using Student’s independent t-test. ***p from 0.0001 to 0.001, n = 3, data reported as mean ± SD).

FIGURE 5

Young’s modulus of photo-crosslinked gels calculated based on G′ and G″ values, as measured upon photo-curing of GelMA-HIGH and GelMA-LOW aqueous solutions. In the figure, red and blue colors are used for GelMA_LOW and GelMA_HIGH formulations, respectively. (means were statistically compared using Student’s independent t-test. ***p from 0.0001 to 0.001, n = 3, data reported as mean ± SD).

FIGURE 6

Swelling ratio of photo-crosslinked GelMA_LOW and GelMA_HIGH gels after 1, 3 and 5 days of incubation in PBS at 37°C. The initial gel swelling ratio (i.e., relaxed swelling ratio, Q_mr), is also reported (data at 0 days incubation time). In the figure, red and blue tones are used for GelMA_LOW and GelMA_HIGH formulations, respectively (dotted, dashed and plain colors identify gels prepared at 5, 7.5 and 10% w/v concentration, respectively). (data were statistically compared using ANOVA followed by Bonferroni post-test. ***p from 0.0001 to 0.001, **p from 0.001 to 0.01, *p from 0.01 to 0.05, ns p ≥ 0.05, n = 3, data reported as mean ± SD). (GelMA_LOW_5: 0 days vs. 1 day ***; 1 day vs. 3 days *; 3 days vs. 5 days ***; GelMA_LOW_7.5: 0 days vs. 1 day ***; 1 day vs. 3 days ns; 3 days vs. 5 days **; GelMA_LOW_10: 0 days vs. 1 day **; 1 day vs. 3 days **; 3 days vs. 5 days ns; GelMA_HIGH_5: 0 days vs. 1 day ns; 1 day vs. 3 days ns; 3 days vs. 5 days ns; GelMA_HIGH_7.5: 0 days vs. 1 day ns; 1 day vs. 3 days ns; 3 days vs. 5 days ns; GelMA_HIGH_10 0 days vs. 1 day ns; 1 day vs. 3 days ns; 3 days vs. 5 days ns).

FIGURE 7

(A) hiPSCs differentiation process: starting from undifferentiated clusters of hiPSCs (A1-A4, scale bar 100 μM) to beating hiPSC-CMs (A5, scale bar 100 μM) using a 12-day differentiation protocol; (B) Immunofluorescence staining of hiPSCs (B1, scale bar 20 μM) and hiPSC-CMs (B2-B3, scale bar 20 μM): nucleus in blue (DAPI), TNNT2 or ACTN2 in green (Alexa Fluor 488); (C) Real-Time PCR of TNNT2 in hiPSCs (yellow bar) and hiPSC-CMs culture (blue bar).

FIGURE 8

Relative mRNA expression levels of: cardiac maturation structure genes (A) TNNT2, (B) ACTN2, (C) MYL2, (D) MYH7; Gap junction gene (E) CX43 or GJA1; gene of metabolism (F) PPAR-α in hiPSC-CMs monoculture (100% hiPSC- CMs) and co-culture with HCEACs (90% hiPSC-CMs + 10% HCAECs and 80% hiPSC-CMs + 20% HCAECs) in 2D (blue bars) and 3D (red bars) microenvironment. The mRNA expression data were normalized by the geometric mean of the most stably expressed genes (RPL13a, RPS4X, and PPIA), and the relative quantification was performed by the ∆∆Ct method. The Fisher’s test was used after ANOVA and the results were expressed as mean ± SEM (p-value <0.05 was considered significant). (N = 2, n = 3).

FIGURE 9

(A) ROS-GLO H₂O₂ assay, (B) LDH assay, and (C) high sensitive Troponin I detection immunoassay on hiPSC-CMs monoculture in the 2D system (blue bars) versus 80% hiPSC-CMs + 20% HCAECs co-culture condition in the 3D system (red bars) (at least N = 3, n = 3). Parametric t-test was used in (A,B), non-parametric t-test (Mann-Whitney test) was used in (C) (*p < 0.05; **p < 0.01).

FIGURE 10

(A) Live-dead assay on 80% hiPSC-CMs + 20% HCAECs co-culture in the 3D microenvironment for 14 days. Live cells are stained green (Calcein), dead cells red (Propidium iodide), and nuclei blue (Hoechst). (B) Statistical analysis (Unpaired t-test, **p < 0.01) for quantification of green (calcein AM) and red (propidium iodide) fluorescence staining, indicating live and dead cells, respectively. Green and red fluorescence values were normalized to Hoechst fluorescence.