Improving metabolic and structural maturation of engineered cardiac tissue for the study of diabetic cardiomyopathy

Introduction:: Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) are frequently used for investigating cardiac disease mechanisms and modeling. However, modeling metabolic disorders, such as diabetic cardiomyopathy (DCM), is extremely difficult due to cell’s metabolic and structural immaturity. Previously, we demonstrated hydrogel-encapsulated hiPSCs undergo cardiac differentiation and achieve some aspect of CM maturation including T-tubule formation [1]. Here, we created engineered cardiac tissues (ECTs) by encapsulating hiPSCs in rectangular PEG-fibrinogen (PF) hydrogels followed by in situ cardiac differentiating (Fig.1A). To enhance the metabolic and structural maturation of our ECTs, we cultured them in fully defined fatty-acid-enriches metabolic maturation (MM) media, promoting the use of fatty acid oxidation as the primary energy source. To study the impact of MM media on the metabolic and structural maturation of hiPSC-CMs, we compared them to age-matched RPMI/B27 with insulin (RP)-cultured samples.


Materials and Methods:: ECTs were fabricated by encapsulating hiPSCs within PF hydrogels matrix into a rectangular PDMS mold on acrylated glass coverslips on day -3. The cell density employed in this process was 60 million cells per mL of precursor solutions, which included 1.5 v/v% triethanolamine (TEOA), 0.39 v/v% N-vinyl pyrrolidone (NVP), and 0.1 mM Eosin Y. Rectangular geometry was chosen as it showed less tissue heterogeneity than disc-shaped microisland and square tissues and developed features of maturing cardiomyocytes in our previous work [2]. Cardiac differentiation was initiated by incubating ECTs in RPMI/B27-I supplemented with 12 uM of CHIR on day 0. On day 1, medium was replaced to fresh RPMI/B27-I. On day 3, half of old media was combined half fresh RPMI/B27-I supplemented with 5 uM of IWP2. On day 5, the medium was changed to fresh RPMI/B27-I. On day 7, RPMI/B27-I was replaced with RP medium. ECTs were cultured in MM media [3] on day 9 for one week (until day 16). Age-matched ECTs culturing in RP were used as control. On days 9 and 16, the CM content was determined using flow cytometry (Fig.1B). To investigate hiPSC-CMs intracellular changes cultured in RP and MM medium, dissociated ECTs were cultured on Matrigel-coated coverslips and were stained with alpha sarcomeric actinin, Mitotracker red, Connexin 43, and Hoechst. To assess hiPSC-CM morphology and sarcomere structure, we employed an unbiased algorithm [4], for automatic segmentation and texture analysis (Fig.1D). Contractile functionality and oxygen consumption rate were determined by microscope and seahorse analyzer, respectively.


Results, Conclusions, and Discussions:: ECTs cultured in MM media exhibited a significantly higher CMs (cTnT+) percentage on day 16 (75.6±7.5%) than those cultured in control RP media (65.8±9.8%, n=9 batches, p< 0.05). The percentage of proliferating cells (Ki67+) for ECTs cultured in MM and RP media was 11.4±2.5% and 22.7±3.7%, respectively (n=5 batches, p< 0.01). Higher levels of CX43 were visualized in hiPSC-CMs from ECTs cultured in MM as assessed by immunostaining which may be linked to the creation of functional gap junctions and improved cell-to-cell communication. ECTs cultured in MM media showed a notable increase in cell elongation (88%) and eccentricity (12%), and a significant decrease in cell circularity (31%) after one week, compared to RP-cultured samples. These morphological changes are consistent with a maturation of hiPSC-CMs. In addition, the sarcomere organization score [4] was significantly increased by 527%, and cell-sarcomere misalignment was reduced by 56% (n≥43 cells, p< 0.001). Our results suggested that MM medium promoted hiPSC-CM maturation as shown by changes in cell shape and sarcomere organization. The mitochondrial area to cell area ratio was higher in MM (23.7±6.7) than in RP (15.3±6.9), and the average distance of the mitochondria from the nuclei was also greater in MM (16.7±5.6 µm) compared to RP (13±3.5 µm) (n=34 cells, p< 0.01). These findings suggested that MM media promoted mitochondrial biogenesis and influencing their distribution in hiPSC-CMs. HiPSC-CMs cultured in MM had 73%, 100%, 62% higher maximal respiration rate, spare respiration rate, and ATP production than cultured in RP media, respectively, as quantified by oxygen consumption rates (n=3 wells, p< 0.01) demonstrating enhanced aerobic respiration and ATP production (Fig.1E). Myocardial contractility was 50% higher in MM than in RP media, with a corresponding increase in both maximum contraction (370±127 µm/s versus 245±87 µm/s) and relaxation velocity (308±132 µm/s versus 202±118 µm/s), respectively (n≥2 batches) (Fig.1C). In conclusion, our study indicated that one week of ECT culture in enriched fatty acid media promoted the structural and metabolic maturation of hiPSC-CMs in ECTs. This strategy provides more appropriate mature model for studying cardiac disease, especially for metabolic diseases like DCM.


Acknowledgements (Optional): : I would like to express my sincere gratitude to Dr. Saucerman for generously sharing his MATLAB code for automatic segmentation and texture analysis, as well as Dr. Huebsch for sharing his MM media preparation protocol with our team.


References (Optional): : [1] Kerscher P. Biomaterials. 2016; 83; 383-395. [2] Ellis, M., et al., Tissue Engineering, 2022(ja). [3] Huebsch, N., et al., Nature Biomedical Engineering, 2022. 6(4): p. 372-388. [4] Sutcliffe MD. Sci Rep. 2018 ;8(1);1258.