Kaitlyn McEntire and Dr. Alonzo Cook, Chemical Engineering Department
Introduction
If a heartbeat connotes life, then cardiomyocytes are the life givers. When heart disease results in cardiomyocyte death, however, these life-giving, beating cells are unable to reproduce, and portions of the heart irreversibly die. Despite modern technology and developments in heart disease treatment and prevention, heart disease remains the number one cause of death in America. With an inability to meet an ever-increasing demand for heart transplants, any potential alternative must be pursued.
Here at Brigham Young University, the members of Dr. Cook’s “heart team” are developing a process to engineer patient-specific hearts for transplant. This process utilizes porcine hearts as the scaffold, removing the native cells in a process called decellularization, and subsequently recellularizing the heart by differentiating human pluripotent stem cells (or, IPS cells) into cardiomyocytes, fibroblasts, and other such cells required for heart function. Already, our team has successfully developed the heart decellularization process, as well as succeeded in producing beating IPS-differentiated cardiomyocytes on a 1 cm disc of ECM. Unfortunately, these beating cardiomyocytes are comparably weak per physiological standards (our IPS-differentiated cardiomyocytes currently beat at about 10 beats/minute, whereas the cardiomyocytes in the typical human heart are beating at a rate of about 60 beats/minute).
Our project seeks to progress towards our goal of engineering patient-specific, transplantable hearts. We determined, through extensive research, that two different methods of stimulation have the highest potential for increasing the strength and frequency of cardiomyocyte contraction towards physiological standards: mechanical and electrical. This project focuses on the investigation of these two methods by testing their individual effects on cardiomyocyte contraction, and their effect in combination. We hypothesize that both methods will increase both contractile force and rate of our IPS-differentiated cardiomyocytes. The end goal of this research is to develop a process that produces beating cardiomyocytes on a porcine cardiac extracellular matrix (ECM) that beat at the rate and with the strength of typical adult cardiomyocytes in the heart.
Methodology
In order to determine the efficacy of mechanical and electrical stimulation, we would use the following steps:
1. Obtain a porcine heart
2. Culture IPS cells
3. Decellularize and dissect the porcine heart
4. Cryosection the ECM into 300 μm thick slices
5. Begin differentiation of the IPS cells for 3 days (to the progenitor stage)
6. Transfer the differentiating IPS cells onto the ECM slice
a. As a control, allow the IPS cells to complete differentiation without stimulus
b. Apply mechanical and electrical stimulation to the remaining samples until complete differentiation of the cardiomyocytes (approximately 14 days)
7. Measure the contractile force and rate of contraction of each sample using a force transducer and stopwatch, respectively.
Completion of these steps would allow us to compare the contractile force and rate of IPSdifferentiated cardiomyocytes without stimulation (the control) to that of IPS-differentiated cardiomyocytes with stimulation.
Mechanical stimulation is to be delivered to the differentiating IPS cells using an Arduino controlled actuator, shown in Figure 1.
Results
Complications with the decellularization and cell culture processes prevented consistent progress past step 4 of the above described procedure. Therefore, a majority of our testing and research remains in verifying these two processes, as well as developing an appropriate mechanical stimulation device.
Our team was able to verify and train new team members in the decellularization and cryosectioning procedures, as well as verify that successfully differentiated IPS cells on ECM were able to maintain a ≥8 beats per minute rate of contraction for 90 days. (The maximum rate of contraction recorded was 12 beats per minute.) The developed mechanical stimulator, shown in Figure 1, was successfully constructed. It can attain stretching at a rate of 60 beats per minute.
Discussion & Conclusions
Moving forward, our team seeks to consistently attain differentiation of the IPS cells on a ECM structure before continuing on to test the effects of mechanical and electrical stimulation on the cardiomyocytes’ rate and force of contraction. We expect to be able to also provide electrical stimulation using a square wave function, with a voltage range of 2-5 volts and current of .5-5 mA will be applied to our cells constantly through the carbon electrodes.
We expect to see both improved strength and contractile rate of the cardiomyocytes after stimulation and to be able to determine the most effective method of mechanical and electrical stimulation. Data collected in the future will be statistically compared between each trial and the control to determine if the stimulation was effective, and results are anticipated to be utilized in determining a combined mechanical and electrical stimulation system to reach our goal of physiologically comparable cardiomyocytes.
