Micro Platform for Controlled Cardiac Myocyte Differentiation

Funding Type: 
SEED Grant
Grant Number: 
Award Value: 
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 

Congestive heart failure, the inability of the heart to continue to pump effectively due to damage of its muscle cells, affects approximately 4.8 million Americans and is a leading cause of mortality. Causes of the irreversible damage to the cardiomyocytes that results in congestive heart failure include hypertension, heart attacks, and coronary disease. Because the cadiomyocytes in the adult heart tissue are terminally differentiated and thus cannot regenerate themselves, once they are damaged, they are irreversibly damaged. As a consequence, despite the advances in medical devices and pharmaceuticals, still more than 50% of congestive heart failure patients die within 5 years of initial diagnosis.

The goal therefore must be to restore the heart cells’ functions. This is possible by transplanting fetal and neonatal cardiomyocytes which can then integrate into the host tissue. This approach has demonstrated success in improving heart function. However, the limited availability of fetal donors has prevented its adoption as a viable therapeutic approach.

Embryonic stem cells can overcome this challenge as they proliferate continuously in vitro and can be furthermore stimulated to differentiate. Embryoid bodies are three-dimensional clusters of heterogenous stem cells, some of which contain cardiac myocytes, which demonstrate characteristic spontaneous contractions. Controlled and efficient differentiation of the stem cells into cardiomyocytes and an effective way to characterize/verify these cells is critical. Ensuring a pure population of cardiac myocytes is essential because otherwise there is a high-likelihood of tumor formation when transplanted. Previous studies have shown that a low percentage of all embryoid bodies spontaneously form cardiomyocytes.

Our goal is to therefore develop a self-contained system to grow and controllably differentiate the human embryonic stem cells into cardiomyocytes in high-yields. Few studies have characterized the types of cardiac myocytes in the differentiating human EBs. Our strategy is to use electrical and chemical cues to induce the high-yield differentiation of stem cells into cardiomyocytes and to monitor this process over time both electrically and optically.

Statement of Benefit to California: 

Improvements in differentiating stem cells into homogenous populations of specific cell types are much needed for transplantation therapy in general—and for congestive heart failure patients in particular. The benefits associated with the development of this micro platform have even broader reaching implications beyond biomedical research. After this system is developed, it will serve as a first platform of its kind that can be later commercialized, which would help spur industry growth. To vitalize and enable high-tech/biotech companies to this {REDACTED} area {REDACTED}, engaging industry involvement to this area is necessary. Supporting such activities would furthermore foster the opportunity for student internships with industry and well as afford the students opportunities in entrepreneurship. Our institution is a Hispanic-serving undergraduate institute with almost 50% minority students. Such a proposed system is vital for promoting both the diversity and research culture {REDACTED} and will be leveraged extensively in outreach programs to encourage underrepresented minorities in science education and training. By actively reaching out to specific students who would particularly benefit from our proposed undergraduate internship program, we can attract at-risk students to engage them in research to promote their retention.

Progress Report: 

This year, we have made quite some progress in developing the microtechnology platform. We have developed a new way to form and culture human embryonic stem cells into uniform embryoid bodies in a high throughput fashion. Instead of using the laborious ‘hanging drop method’ or the complicated ‘spinning flask method’, we have developed a way for researchers to easily pipette their cells into standard well plates and increase their throughput by almost 1000x. This is achieved by placing inserts with rounded-bottom microwells into standard well plates. Each one of these inserts that can fit into a standard 24 or 96 well plate can have up to 1000 wells and therefore can create 1000 embryoid bodies, all of uniform size. We can even create wells of various sizes such that we can induce embryoid bodies of predefined sizes and numbers of cells. Many recent publications have demonstrated that the initial size of the embryoid bodies affect differentiation. We have observed this as well. Moreover, this new platform allows researchers to perform real-time microscopy of the cells during this whole process.

In addition to developing this new chip, we have also electrically stimulated at different stages during differentiation. The different stages of differentiation include: 1) during embryoid body development 2) when transferred to gelatin coated dishes 3) after about a week on gelatin and 4) isolated beating areas. Electrical stimulation was accomplished using a C-PACE voltage pulsing device at a 1 Hz frequency, 4.5 V (2.5 V/cm), and a 1 ms duration. Unfortunately, none of the electrical stimulation yielded any exhibited increased expression of cardiac markers. Future studies will examine pacing of differentiated cardiac cells for synchronization and will employ more markers using a PCR super microarray.

We have also worked on custom software development that allows us to automatically identify and track individual cells within the microplatform.

There were a number of factors that caused some unexpected delays in scientific progress this year. Most notably, the PI Michelle Khine and her lab moved to a new university. Therefore, this took quite some time to take down and then re-establish the lab at its new location. Now at UC Irvine, she finally has the ideal infrastructure to make progress quickly on this project. This one year extension to finish this project is therefore much needed and greatly appreciated.

To uniformly control the differentiation of embryoid bodies (EBs), we have developed a very simple to use culture platform the create homogenous-sized EBs.
We have made quite some progress with the EB array culture plate development, described in detail in the last progress report. Since then, we have developed a way to 1) translate to a more transparent material with lower autofluorescence (cyclic olefin copolymer, COC) to be compatible with optical imaging (Figure 1, c) and then 2) mated the microwells to the bottom of 24 well plates for ease of handling. While we have not had success with applying electric fields to induce cardiomyocyte differentiation, we are now working with !) optimizing the EB size to yield the most cardiomyocytes and then 2)perfusing the EBs with soluble factors.