**2. Characteristics of Mature and Immature Cardiomyocytes**

The maturation level of human cardiomyocytes is crucial to clinical therapy. Immature cardiomyocytes fail to maintain full cardiac function and may lead to an aberrant remodeling of cardiac wall and cardiomyocyte hypertrophy because of differences in cell size, myofibrillar switch, conduction velocity, metabolism, and calcium handling between the two statuses. Table 1 briefly summarizes some typical physiological and chemical differences between mature human cardiomyocytes and immature hPSC-CMs and provides criteria for defining mature cardiomyocytes [22–25]. For a review of the details, we refer the reader to the work of Xiulang Yang et al. [23].


**Table 1.** Distinctions between mature human cardiomyocytes (CMs) and immature CMs.

cTnT, Cardiac troponin T2; β-MHC, Myosin heavy β chain; MLC2v, Myosin light chain 2 ventricular isoform; MLC2a, Myosin light chain 2 atrial isoform; ADRA1A, Adrenoceptor α1A; cTnI, Cardiac troponin I3; CSQ, Calsequestrin; PLN, Phospholamban; RYR2, Ryanodine receptor 2; SERCA/ATP2A2, Sarco/endoplasmic reticulum calcium transport ATPases; SRP, Sarcoplasmic Reticulum Proteins; T-tubule, Transverse tubule; RMP, Resting Membrane Potential; LGJ, Location of Gap Junctions; APA, Action Potential Amplitude.

#### **3. Approaches to Obtaining Mature Cardiomyocytes**

In order to obtain more mature and functional hPSC-CMs, the provision of a similar physiological microenvironment in the process of cardiomyocyte development may be a feasible adult direction. In recent years, academics have performed various experiments to stimulate cardiomyocyte maturity, including biophysical, biochemical, electrophysiological, and mechanical experiments.

#### *3.1. Biophysical and Biochemical Factors*

Several practicable methods have been used to promote the maturation of cardiomyocytes, including long-term cultivation, a specified material, a three-dimensional culture, a microfluidic system, a co-culture with other cells following transplantation to model organisms, a dynamic sustainability system, drugs, and metabolic regulation.

Long-term cultivation and stiff matrix have been shown to enhance human pluripotent stem cell-derived cardiomyocyte sarcomere formation, calcium handling, and ion channel protein expression [26,27]. Collagen-coated polyacrylamide gels with an elastic moduli of 10 kPa have been shown to lead to aligned sarcomeres in comparison with a stiffer substrate [28]. Mihic et al. generated human-engineered cardiac tissues from hESC-CMs in a large gelatin. Human-engineered cardiac tissues were subjected to a cyclic stretch and their cell size increased, their Z discs were organized, and the Connexin-43 expression increased significantly [29]. Biohybrids of collagen and pristine graphene increased the metabolic activity of human pluripotent stem cell-derived cardiomyocytes and enhanced sarcomere structures [30].

Three-dimensional (3D) culture systems can mimic the native cardiomyocyte microenvironment in vivo to support the maturation of cardiomyocytes. Tulloch et al. generated 3D human-engineered cardiac tissues from hPSC-CMs in collagen that was seeded into a channel with a silicon floor plus nylon mesh anchors. After seven days, myofibril and Z-disc alignment increased [31]. Lee et al. used 3D bio-print collagen to obtain a human heart tissue model that possessed synchronized contraction and directional action potential propagation [32]. hESC-CMs in 3D patches exhibited more mature characteristics, including significantly faster conduction velocities and longer sarcomeres as compared with two-dimensional (2D) monolayers. The conduction velocities of these cardiac patches increased significantly as the purity of the cardiomyocytes increased [33]. Human cardiac muscle patches transplanted into swine were shown to prominently improve left ventricular function and myocardial stress, promote myocardial hypertrophy, and reduce myocardial apoptosis [34]. It was shown that a 3D culture suppressed smooth muscle -actin content and increased the expression of several cardiac markers [35].

Microfluidic systems can be used to study disease and organoid models. In recent years, combinations of microfluidic systems and functional human myocardium have been developed for drug cardiotoxicity testing [36]. Flow culture systems provide continuous gas and nutrient exchange to induce cardiomyocyte maturation. Dynamic cultures result in an enhancement in sarcomeric protein expression, an increase in size, augmentation of the contraction force, and a higher conduction velocity [37].

In addition, mixtures of human primary or human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts, and endothelial cells have been used to obtain vascularized functional myocardium. This improvement has allowed us to introduce blood flow into cardiomyocyte cultivation systems and paves the way to cardiomyocyte metabolism and maturation [38]. Human cardiomyocyte patches, through several types of cells derived from hPSCs, were shown to enhance the capacity for excitation contraction coupling, calcium handling, and force generation. Moreover, Johannes and co-workers found that co-transplantation of hESC-derived epicardial cells and cardiomyocytes could double the cardiomyocyte proliferation and augmented angiogenesis between the graft and the host simultaneously [39]. Triiodothyronine is essential to myosin heavy chains (MHCs) and Titin isoform switchover in normal cardiac development. Addition of triiodothyronine was shown to compel immature cardiomyocytes to show several maturation characteristics [40]. The co-inhibition of HIF1 (hypoxia-inducible factor 1) and lactate dehydrogenase A promoted the function maturation of hPSC-CM as mitochondria prefer to conduct oxidative phosphorylation rather than aerobic glycolysis and resulted in sarcomere length increase and contraction stress enhance [41].

These biophysical and biomedical approaches promote the growth and proliferation of immature cardiomyocytes and the formation of adult-like cardiac tissue with an organized ultrastructure, longer sarcomeres, more intensively developed mitochondria, more T-tubules, a more mature oxidative metabolism, and more rapid calcium handling.

#### *3.2. Electrophysiological Stimulation*

The spontaneous beating of cardiomyocytes is directly regulated by the cells of atrio-ventricular nodes in vivo. A combination of 3D cultivation with 6 Hz of electrophysiological stimulation was shown to markedly increase myofibril ultrastructural organization and cardiomyocyte size, elevate the conduction velocity, and improve both electrophysiological and calcium ion handling in hPSC-CMs [42]. Electrophysiological stimulation of 2 Hz was used to culture hESC-CMs in a 3D matrix, and significant improvements in contraction and calcium handling were obtained [43]. Chiu et al. found that an electrical field with a symmetric biphasic square and strengths of 2–5 V/cm at a frequency of 1 Hz could enhance the hallmarks of cardiomyocyte maturation in vitro [44]. Thus, the maturation process of cardiomyocytes in vitro progresses more quickly when accompanied by optimized electrophysiological stimulation.

More and more researchers are becoming aware of the fact that electrophysiological stimulation can promote the maturation of hPSC-CMs. However, it remains hard to compare the results from different assays as a universal and gold standard is currently lacking. Besides this, it is obvious that diverse electric field intensities and stimulation frequencies, hPSC cell types, and cultivation conditions can lead to differences in the maturation of cardiomyocytes. Thus, there may be benefits to establish a compatible platform to assess the maturation process, and make comparisons in different stimulation models.
