Year 2

Specific Aim 1: To electromechanically condition hESC-derived cardiomyocytes.
Progress: Over the past year, we have designed and constructed a computer controlled integrated stretch system and electrical pacing system for applying mechanical and electrical stimulation. This system was used in conjunction with a stretchable microelectrode array (sMEA) and shown to successfully support, stretch, and pace primary murine cardiomyocytes (CM). We also have developed a strain array device for cell culture that effectively interfaces the desirable properties of high-throughput microscale fluidic devices with macroscale user-friendly features. One challenge we have encountered with our sMEAs is maintaining electrical continuity of electrodes as cells are stretched. As an alternative to traditional electrical stimulation we have created a system that optically induces electrical activity in hESC-CM. We are now able to optically and non-invasively pace cardiomyocytes differentiated from our modified hESC line.

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 Year 1, we learned that our grafts would require large populatoins of relatively pure hESC-CMs. As a result, we concentrated our efforts over the past year in developing a more efficient differentiation method for producing larger yields and quantities of hESC-CM. Our method produces hESC-CM in a directed manner under feeder-free and serum-free conditions by controlling multiple cardiomyogenic developmental pathways. Also, in a collaborative effort, we are engineering a novel method for sorting cardiomyocytes. In order to promote improved viability of hESC-CM in our tissue grafts, co-transplantation with hESC-derived endothelial cells (hESC-EC), as opposed to VEGF alone, will likely be needed as shown recently by others. Over the past year, we have shown that we can produce hESC-EC and that their survival in the heart is enhanced by activation of acetylcholine receptors that lead to activation of pro-survival and anti-apoptosis pathways. Finally, in order to control spatial orientation of hESC-CM within our tissue grafts, we have demonstrated on-demand micropatterning of matrix proteins for cell localization and stem cell fate determination. We have illustrated the utility of a cantilever-based nano-contact printing technology for cellular patterning, mESC renewal, and mESC fate specification. We are currently extending our results to undifferentiated hESC and hESC-CM.

Specific Aim 3: To assess tissue graft viability and function in a small animal model.
Progress: Over the past year, we created hESC-CM based tissue grafts in linear form. In order to quantify the loss of cardiac function between healthy and diseased hearts, we have recently developed a novel in vitro hybrid experimental/computational system to measure active force generation in 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. Finally, as outlined in our original proposal, we will first implant our tissue grafts in rat aortas as a novel test-bed to assess the graft’s inherent function while minimizing the confounding effects of underlying cardiac contractions. Over the past year we have successfully implanted decellularized aortic patches in rat aortas and are currently working on adding hESC-CM and hESC-EC to the patches to assess their viability and function.

In summary, in the second year of our project we have made strong 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.