*3.3. Mechanical Stress*

Functional cardiomyocytes are linked to varieties of cells and structure in vivo. These structures provide the cells with anchors to contract and contribute to physiological hypertrophy. Mechanical loading may be the efficient factor with the most potential when considering the explosion of research in this area in recent years. Mechanical stress increases cells' size and improves the contraction force that is associated with hypertrophic growth. Jianzhong et al. devised a system for assembling muscle-powered microdevices based on precise manipulation of materials to monitor muscle tissue function [45]. Furthermore, periodic stretching of hPSC-CMs in a 3D structure mixture was shown to cause faster force production, higher calcium influxes, an increased expression of β-MHC and cTnT [35]. Schmelter et al. demonstrated that cyclic mechanical stretching activated the Reactive Oxygen Species (ROS) signaling pathway and enhanced the differentiation of ESCs into cardiomyocytes [46]. Ronaldson et al. formed cardiac grafts from early stage iPSC-derived cardiomyocytes and trained them via cyclic mechanical stress for several weeks. After one month, the grafted cardiomyocytes showed adult-like gene expression profiles, increased sarcomere length, enhanced density of mitochondria, the presence of T-tubules, metabolism switch, and functional calcium handling [47]. Table 2 summarizes some typical engineered approaches to the maturation of human and rodent cardiomyocytes.




**Table 2.** *Cont*.

Mechanical loading can improve the rate of maturation of hPSC-CMs and contractile properties. All these characteristics reflect the state of maturity of these cells. However, there is little published data about the real-time monitoring of cardiomyocyte development; to date, the shortage of clinical feedback has slowed its application.

#### **4. Conclusions**

In this review, we summarized the approaches that have been adopted to improve the maturity of hPSC-CMs under different conditions. Human embryonic cardiac development and postnatal physiological hypertrophy processes are difficult to study because of species specificity and the lack of availability of human heart tissues. Mature hPSC-CMs may reflect the pathological state in adults more accurately and serve as preferential disease models for clinical use.

With the rapid development in this multidisciplinary field, our understanding of the maturation of human cardiomyocytes has been growing in recent years. Many studies on the maturation of cardiomyocytes have been published in multiple journals in the last decade. Some methods have been applied to successfully produce adult-like cardiomyocytes with respect to the biochemical indicators; however, the remaining methods still need improvement. Current hurdles include achieving adult-like cardiomyocytes with angiogenesis and organized, mixed assemblies of multi-layer 3D heart tissues. A real-time assessment system is required to compare different approaches and obtain an optimized maturation status in hPSC-CMs. Besides this, in order to monitor feedback from cell and tissue signals, we may need an electrophysiological surveillance system that can regulate the differentiation and maturation of cardiomyocytes in real time in clinical practices. A mature and functional human cardiac model in vitro could play a role in myocardial tissue development research, cardiotoxicity drug screening, and clinical therapies.

**Author Contributions:** F.G. wrote original draft. F.G., Z.W. and J.J.X. reviewed and edited manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Special thanks are given to Smina Mukhtar and Martyn Rittman for excellent scientific secretary assistance and English editing service.

**Conflicts of Interest:** The authors declare no conflict of interest.
