Year 4

Specific Aim 1: To electromechanically condition hESC-derived cardiomyocyte(CM).
Progress: Over the past year, we tested, validated, and published an integrated strain and electrical pacing system that we designed and constructed. As mentioned in our previous reports, one challenge we encountered with our electromechanical devices is maintaining electrical continuity of electrodes as cells are stretched. As an alternative to traditional electrical stimulation, with collaborators at Stanford, we have created a system that optically induces electrical activity in hESC-CM by introducing light activated channelrhodopsin-2 (ChR2), a cationic channel, into undifferentiated hESC. In our initial manuscript we have also demonstrated the effects of light stimulation on a whole heart computational model in which we have virtually injected light-responsive hESC-CM in various areas of the simulated heart.

Specific Aim 2: To engineer a hESC-CM based cardiovascular tissue graft.
Progress: From our first attempt at engineering a cardiovascular tissue graft as we reported in Years 1, 2, and 3 we learned that our grafts would require large populations of relatively pure hESC-CMs. As a result, we’ve continued our efforts in developing a more efficient differentiation method for producing larger yields and quantities of hESC-CM. Our method produces hESC-CM and iPSC-CM in a directed manner under feeder-free, serum-free, and monolayer conditions by controlling TGF-beta/Activin, BMP, Wnt, and FGF pathways. We have used our differentiation protocols to contribute cardiomyocytes to our collaborators, which has resulted in one published manuscript and two submitted publications. Also, with our collaborator at UC Berkeley, we have engineered a novel method for identifying CMs based on their electrical signals and have reported our technology in one accepted manuscript and one under review.

Specific Aim 3: To assess tissue graft viability and function in a small animal model.
Progress: Over the past two years, we created hESC-CM based tissue grafts in linear and circular forms and our now creating grafts that can be optically controlled (see Aim 1 above). As described in our last progress report, in order to quantify the loss of cardiac function between healthy and diseased hearts, we have reported a novel in vitro hybrid experimental/computational system to measure active force generation in healthy ventricular slices of rodent hearts. Quantification of the loss of cardiac function will guide us in determining the numbers of hESC-CM needed for producing grafts with varying force generating capacity. We have also modeled eccentric and concentric cardiac growth through sarcomerogenesis in order to give us insight into how we might terminally mature our hESC-CM grafts. Finally, we have differentiated hESC into CM for one of our collaborators at Stanford and have performed detailed calcium imaging to show engraftment of hESC-CM with human heart tissue. This has given us great insight into how 3D tissue grafts might integrate with human heart tissue.

In summary, in the fourth year of our project we made good progress on all three of our specific aims. Based on our current results, we anticipate we will continue to make significant progress in engineering a robust and functional cardiovascular tissue graft as we originally proposed and we will continue our efforts. Undoubtedly, with the support of the CIRM grant over the past four years, we have made great strides towards creating a 3D tissue graft and believe we will demonstrate functional integration, not only with rodent hearts, but with human tissue, all within the coming year.