COMMITTEE:
Todd McDevitt, Ph.D. (Advisor)
Thomas Barker, Ph.D.
Andrés Garcia, Ph.D. 
Manu Platt, Ph.D.
Chunhui Xu, Ph.D. (Emory University)

Embryonic stem cells (ESCs) hold tremendous promise for tissue regeneration, due to their capacity to differentiate into cells of all three germ layers. In particular, differentiation of ESCs into cell types with limited intrinsic renewal capabilities, such as cardiomyocytes, remains a key goal of regenerative medicine and is currently being explored to generate clinically viable cardiac tissue constructs for in vivo transplantation. Cardiomyocytes have been efficiently generated from both mouse and human ESCs via temporally sensitive differentiation protocols consisting of brief soluble Activin A supplementation followed by more sustained delivery of BMP4. However, pluripotent cell differentiation into cardiomyocytes is currently performed extensively on monolayer or on dissociated aggregates to monolayer cultures to increase cardiotropic factor-cell contact for improvement of cardiomyocyte efficiency, despite previous studies demonstrating higher engraftment of aggregates over single cell delivery to the damaged myocardium.4 Additionally, the ability to directly differentiate cell aggregates into cardiomyocytes would eliminate the requirement for disruptive aggregate dissociation, potentially enabling generation of large cardiomyocyte yields with minimal manipulation. Cardiomyocyte differentiation within aggregates has proven challenging due to limited understanding of the complex temporal and spatial patterns of growth factor concentration gradients necessary to promote more homogeneous differentiation towards cardiomyocyte phenotypes.

Our lab has previously demonstrated that incorporation of biomolecule-laden microparticles (MPs) within ESC aggregates, or embryoid bodies (EBs), facilitates spatial control over differentiation, overcomes transport limitations, and enables more homogenous differentiation compared to traditional soluble delivery of molecules from the culture media. Furthermore, proteolytic degradation rate can be modulated by tuning the cross-linking density of the MPs, offering a platform for the controlled release of morphogens for directed differentiation approaches. Thus, delivery of morphogens in a temporal manner from MPs incorporated within the EB may provide a novel means to engineer 3D aggregates of functional cardiomyocyte tissue constructs, which can be further developed for clinical implantation. The objective of this proposal is to efficiently differentiate 3D ESC aggregates into cardiomyocytes via enzymatically degradable MP-delivery of cardiotropic factors. The central hypothesis is that MP-mediated temporal release of BMP4 will enhance mature cardiomyocyte differentiation within ESC aggregates compared to soluble delivery to aggregates and monolayer cultures.