Direct Production of Engineered Cardiac Tissue within Hybrid Biomaterials

The human heart contains highly specialized cells that are especially vulnerable to damage because they lack regenerative capabilities. This article describes a novel method for encapsulating and directly differentiating induced pluripotent stem cells in hybrid biomaterials to form cardiac tissue.

Cardiovascular disease is the leading cause of death worldwide, mainly because the heart is unable to regenerate itself after injury. The contracting cells in the heart, called cardiomyocytes, generate the force that propels blood throughout the body. Because cardiomyocytes must continuously meet stringent functional demands, they do not expend resources on cell division. Only 1% of cardiomyocytes turn over (i.e., are replaced by new cells) each year up until age 25; this drops to 0.45% by the age of 75 (1).

Damage to the heart can occur as a result of disease, in response to cardiotoxic compounds, or following cardiac trauma, such as myocardial infarction (i.e., heart attack). Cardiomyocytes that experience trauma undergo apoptosis, or cell death, causing breaches in the tightly aligned muscular wall. Given the low potential of cardiomyocytes to replicate, these breaches in contractile tissue persist long-term.

The heart’s lack of regenerative capacity has serious consequences for patients’ health and quality of life. After a heart attack, more than 1 billion cells can die (2). The heart recruits fibroblasts and growth factors to the injury site for repair. This process is unable to fully reverse damage and it changes the muscular architecture of the heart and increases tissue stiffness, which often leads to chronic heart failure. In addition to trauma caused by ischemia (i.e., inadequate blood supply) during a heart attack, many medications are cardiotoxic, especially those used in chemotherapy. While sometimes the only effective option for treating other very serious diseases, these medications can also have long-term effects on the heart, sometimes causing chronic heart failure.

Tissue engineering provides promise for overcoming the challenges associated with regenerating diseased and damaged heart tissue. Cardiac tissue engineering seeks to create functional human heart tissues with electrophysiological and structural properties as similar as possible to native human heart tissue. Engineered heart tissue has the potential to improve cardiovascular outcomes both through direct use in regenerating the heart, as well as through supporting the discovery and development of new therapeutics.

Because native human cardiomyocytes cannot be cultured in vitro, researchers largely rely on biomimetic materials, biomolecules, and stem cells to form engineered cardiac tissues. Biomimetic materials, or materials that mimic the biological environment, have potential to provide the appropriate mechanical, electrical, and structural cues required for creating functional cardiac tissues.

Cardiac tissue engineering has made significant progress since the first man-made 3D heart tissues were formed in the mid-20th century (3). The first successes in the field involved gyrating dissociated embryonic chicken cardiomyocytes to form spontaneously beating cell aggregates that had better functionality than their 2D cultured counterparts (4). Since then, several cardiac tissue engineering platforms have been developed, including cell sheets (5), engineered matrices (6, 7), decellularized heart tissue (8), and hydrogels (9). Even with significant advancement in the field, some challenges remain, including vascularization, mechanical and electrical stimulation, cell population, and maturation (10, 11).

Current cardiac tissue engineering research primarily employs 2D sheets for the production of cardiomyocytes. Cardiac cell sheets are either studied directly, or the cardiomyocytes are dissociated and combined with a biomaterial and other cell types to create a 3D structure. These models have enabled significant advances in the development of potential regenerative therapies and drug testing platforms and a mechanistic understanding of heart disease.

Cardiac cell sheets have provided substantial information about the responses of interconnected cardiomyocytes to a range of mechanical, electrical, and structural stimuli, including the impact of stimuli on electrophysiological function, which is substantially more challenging to study in 3D models. Because these 2D models do not accurately recapitulate the cell-cell and cell-matrix interactions found in the native human heart, dissociated and reassembled 3D cardiac tissue constructs are employed to better replicate these important aspects of 3D heart structure and make functional tissues.

Nonetheless, this approach of dissociating cardiomyocytes to produce cardiac tissue has inherent drawbacks. This multistep approach is technically complex, making it difficult to automate. Furthermore, the compulsory dissociation of predifferentiated cardiomyocytes disrupts microenvironmental cues that are critical in human cardiac development, destroys critical cell-cell junctions required for action-potential propagation, and results in substantial cell loss. Thus, eliminating the need to dissociate the cardiomyocytes provides multiple advantages for cardiac tissue production.

For commercial production of engineered heart tissues, minimizing the complexity of production is critical. Particularly important to creating a robust production platform is minimizing cell-handling steps to the greatest degree possible. Additionally, significant research in scale-up of production, automation, cell culture media, and reproducibility of 3D cardiac constructs will be required before commercialization. Important design considerations in the process of engineered heart tissue production include the choice of cardiomyocyte cell source, biomimetic materials for recapitulating 3D structure, and methodology for creating 3D cardiac tissues.

Induced pluripotent stem cells: A cell source for generation of cardiomyocytes

Human cardiomyocytes are a highly specialized cell type with limited regenerative capacity. Because these cells cannot be cultured in vitro, alternative cell sources, such as animal cells, must be used for studying cardiac regeneration and disease modeling. Animal cells are often used in cardiac models because they are easy to extract and they can be cultured longer term. Although animal models have provided significant insights into disease processes, they do not accurately recapitulate human disease development and are irrelevant for use in clinical cardiac regeneration.

Pluripotent stem cells (i.e., cells that have the potential to be differentiated into any cell type in the body) can be used to generate cardiomyocytes. Human pluripotent stem cells include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).

The ability to reprogram differentiated somatic cells back to a pluripotent state brought about a major transition in biomedical research and tissue engineering. Since the discovery of hiPSCs by Takahashi and Yamanaka in 2006 (12), significant progress has been made in reprogramming technology. Increased efficiency in reprogramming, as well as more easily accessible somatic cell sources, has enabled the generation of hiPSCs from thousands of patients and facilitated the incorporation of hiPSCs into disease-modeling and drug-screening platforms. The first clinical trials that use pluripotent stem cells for cardiac patch differentiation and implantation into patients with advanced heart failure are currently underway (13).

The discovery and advancement of hiPSCs are bringing personalized therapeutics and regenerative medicines much closer to reality. This novel stem cell technology can be used to model and study diseases in a more robust and accurate way than before. Now, hiPSCs can be derived from patients afflicted with genetic diseases and then differentiated (i.e., transformed into specialized cell types) in order to study disease manifestation in the affected tissues. When somatic cells are reprogrammed to their pluripotent state, their genetic information is maintained. Therefore, the disease-causing genetic mutation will still be present after differentiation into the affected tissue type. Several studies have shown that differentiation of patient-derived hiPSCs produces cells that have characteristics of the patient’s disease in vitro...