Advancements in Cardiomyocyte Research Using hiPSC-CMs

Cardiovascular diseases are the primary cause of illness and death worldwide, resulting in around 18 million deaths each year. The heart consists of different types of cells, with cardiomyocytes being the most abundant. In adults, these cells are arranged in a way that helps the heart pump blood effectively. Adult cardiomyocytes have a specific shape and structure that allows them to function properly. However, studying these cells directly from human hearts has its challenges, such as limited availability and difficulties in maintaining them in the lab.

To overcome these challenges, scientists use a type of cell called HiPSC-CMs, which are derived from human stem cells. These cells can be grown in large amounts and provide valuable insights into heart diseases and potential new treatments. However, hiPSC-CMs are less mature than adult cardiomyocytes and have different properties. Researchers are working on creating better environments for these cells to grow in the lab, which resemble the conditions in a healthy heart.

#Creating the Right Environment

An ideal setup for growing cardiomyocytes involves using a substrate that mimics the texture and Stiffness of heart tissue. This substrate should support the attachment and growth of cardiomyocytes, help them align properly, and accommodate the natural movement of the heart. The stiffness of the substrate should match that of healthy heart tissue, which varies between 1-6 kPa in young hearts and 10-15 kPa in healthy adult hearts.

To produce such Substrates, researchers have employed various techniques, including soft lithography and microfabrication. One popular material for making these substrates is polydimethylsiloxane (PDMS), known for its stability, biocompatibility, and low cost. While PDMS has many benefits, it can be challenging for cardiomyocytes to stick to its surface because PDMS is naturally hydrophobic.

To improve cell attachment, researchers have developed methods to modify the surface of PDMS, making it more suitable for cell growth. These modifications include coating the surface with proteins that help cells adhere better.

#Methods of Fabrication

The process of creating the soft micropatterned substrate involves several steps. First, a mold is made using PDMS, which requires careful etching to form grooves. These grooves guide the cells to grow in the desired orientation. Once the mold is ready, a layer of a material called polyvinyl alcohol (PVA) is applied to facilitate the removal of the PDMS substrate later.

Afterward, a softer version of PDMS is poured onto the mold. Following this, the substrate is treated to enhance its surface properties before it is used for cell culture. Researchers utilize various techniques to ensure that the depth and spacing of the grooves are consistent, as these features influence how the cells grow and behave.

#Characterizing the Substrate

To ensure the substrates are suitable for culturing cardiomyocytes, researchers measure aspects like groove depth and surface texture. These characterizations help in understanding how the substrate might affect cell growth and function.

They also conduct tests to measure the stiffness of the substrates, as different stiffness levels can create varied responses in the cells. This mechanical characterization is essential because it helps predict how the cells will respond to the substrate during experiments.

#Improving Cell Attachment

Cell attachment to the substrate is critical for successful studies of cardiomyocytes. Researchers measure how well cells stick to different substrate types. They find that substrates treated with specific coatings, like proteins, significantly improve the cell’s ability to attach and grow.

One successful approach was to apply a combination of PD and extracellular matrix (ECM) proteins. Treating the substrate in this way allows cardiomyocytes to form a stable and functional monolayer. This layer is crucial for investigating their electrical activity and contractile behavior.

#Differentiating Stem Cells into Cardiomyocytes

To produce cardiomyocytes, researchers derive them from hiPSCs. These stem cells undergo a series of steps, first being plated on a surface coated with special proteins to support their growth. After some time, the medium is changed to encourage the cells to start differentiating into cardiomyocytes. The cells reach a state of maturity after several days, showcasing characteristics similar to those found in adult heart cells.

During this differentiation process, scientists closely monitor the cells to ensure they develop properly. The aim is to achieve a high purity of cardiomyocytes, which enables more reliable results in subsequent experiments.

#Effects of Substrate Features on Cell Behavior

Researchers investigate how features of the micropatterned substrate, like groove dimensions, influence the cardiomyocytes. They examine how different pattern sizes affect the alignment of the cells and their nuclei. A crucial finding is that specific parameters, such as groove depth and spacing, promote better alignment and functionality of cardiomyocytes.

In their experiments, researchers observe that the best performance occurs with certain micropattern dimensions. The cells cultured on these optimal patterns exhibit improved organization and function, reflecting more closely the behavior of adult cardiomyocytes.

#Maturation of Cardiomyocytes

Culturing hiPSC-derived cardiomyocytes on specially designed substrates allows researchers to drive maturation further. They observe changes over time in properties such as sarcomere organization, contractile force, and calcium handling.

After a few weeks on the soft micropatterned substrate, the cardiomyocytes show enhanced structural organization. This maturation is indicated by improved sarcomere lengths and orientations, which are essential for effective heart function. Researchers also note that the cells' ability to handle calcium improves, leading to better contractility.

#Evaluating Functionality

The functionality of cultured cardiomyocytes is assessed using various techniques. By analyzing their contraction and relaxation rates, researchers determine how well these cells mimic the behavior of adult heart cells. Tests measure parameters like sarcomere shortening and calcium transient dynamics, providing insights into the cells' overall performance.

These assessments are crucial for understanding how well the cultured cells can be used for studying heart diseases or for testing new drugs.

#Long-Term Culturing

An essential aspect of the research is the ability to culture cells over extended periods, sometimes exceeding three months. Long-term observations help scientists understand how cardiomyocytes change over time and how their functions might be affected by the microenvironment.

During long-term studies, researchers monitor changes in cell structure and function, identifying points of maturation and any signs of dedifferentiation. This knowledge is vital for establishing stable models for studying heart diseases and drug responses.

#Developing Useful Techniques

Throughout the study, various techniques and procedures are developed to create effective environments for culturing cardiomyocytes. These methods allow researchers to produce stable, functional cell models that can provide insights into heart development, diseases, and potential therapies.

One key advancement is the ability to create micropatterned substrates in a straightforward manner without the need for complex cleanroom facilities. This accessibility encourages further experimentation and innovation in the field.

#Future Directions

Going forward, researchers plan to expand their understanding by examining how different environmental factors impact cardiomyocyte behavior. There is a focus on exploring 3D structures, which may better mimic the conditions found in actual heart tissue.

Additionally, investigating protein expression in stem cells and cardiomyocytes may offer insights into how these cells adapt to various environments. Such studies are essential for advancing the field of regenerative medicine and improving treatment for heart conditions.

#Conclusion

This research highlights the importance of creating optimal environments for hiPSC-CMs to enhance their maturation and functionality. By developing novel micropatterned substrates with specific mechanical properties, scientists have paved the way for new techniques in studying heart diseases and testing potential treatments.

The ability to culture cells over extended periods while maintaining their functionality is a significant advancement. This work holds promise for future applications in cardiac tissue engineering and drug discovery, ultimately contributing to better outcomes for patients with heart conditions.