Massive loss of cardiac tissue as a result of myocardial infarction can create a poorly-conducting substrate with impaired contractility, ultimately leading to heart failure and lethal arrhythmias. Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells that may be transplanted into failing hearts to provide electrical and mechanical support. Experiments in both small and large animal models have shown that standard cell delivery techniques suffer from poor retention and engraftment of cells. In contrast, the transplantation of engineered cardiac tissues may provide improved cell retention at the injury site, creating a more localized paracrine effect and yielding more efficient structural and functional repair. However, tissue engineering methodologies to assemble cardiomyocytes or cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. The objective of this thesis was thus to develop a methodology to generate highly functional engineered cardiac tissues starting from pluripotent stem cells.

To accomplish this goal, we first derived purified populations of cardiac myocytes from mouse embryonic stem cells (mESC-CMs) by antibiotic selection driven by an α-myosin heavy-chain promoter. Culture conditions that yielded robust mESC-CM electrical coupling and fast action potential propagation were optimized in confluent cell monolayers. We then developed a microfabrication-based tissue engineering approach to create engineered cardiac tissues ("patches") with uniform 3D cell alignment. We found that, unlike in monolayers, mESC-CMs required a population of supporting cardiac fibroblasts to enable the formation of 3D engineered tissues. Detailed structural, electrical and mechanical characterization demonstrated that engineered cardiac patches consisted of dense, uniformly aligned, highly differentiated and electromechanically coupled mESC-CMs and supported rapid action potential conduction velocities between 22 - 25cm/s and contractile force amplitudes of up to 2mN.

Next, we sought to circumvent the use of primary cardiac fibroblasts by utilizing a single pluripotent stem cell-derived source, multipotent cardiovascular progenitors (CVPs) capable of differentiating into vascular smooth muscle and endothelial cells in addition to cardiomyocytes. CVPs were derived from mouse embryonic stem cells and induced pluripotent stem (iPS) cells by antibiotic selection driven by an Nkx2-5 enhancer element. Similar to mESC-CMs, CVPs formed highly differentiated cell monolayers with electrophysiological properties that improved with time in culture to levels achieved with pure mESC-CMs. However, unlike mESC-CMs, CVPs formed highly functional 3D engineered cardiac tissues without the addition of cardiac fibroblasts, enabling engineered cardiac tissues to be formed from a single, entirely stem cell-derived source.

Finally, we explored mechanisms of synergistic cardiac fibroblast/myocyte signaling in 3D engineered tissues by using cardiac fibroblasts of different developmental stages in the settings of direct 3D co-culture as well as in conditioned media studies. When co-cultured with fetal cardiac fibroblasts, mESC-CMs were capable of two-fold faster action potential propagation and 1.5-fold higher maximum contractile force generation than when co-cultured with adult cardiac fibroblasts. These functional improvements were associated with enhanced mESC-CM spreading and upregulation of important ion channel, coupling, and contractile proteins. Conditioned medium studies revealed that compared to adult fibroblasts, fetal cardiac fibroblasts secreted distinct paracrine factors that promoted mESC-CM spreading and spontaneous contractility in 3D engineered tissues and acted via the MEK-ERK pathway. Quantitative gene expression analysis revealed paracrine factor candidates that may mediate this action.

In summary, this thesis presents methods and underlying mechanisms for generation of highly functional cardiac tissues from pluripotent stem cell sources. These techniques and findings provide foundation for future engineering of human ES and iPS cell-based cardiac tissues for therapeutic and drug screening applications.