Year 2

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.