Self-contained 3D Differentiation of Reprogrammed Amniotic Fluid Derived Stem Cells for Congenital Heart Repair

Self-contained 3D Differentiation of Reprogrammed Amniotic Fluid Derived Stem Cells for Congenital Heart Repair by Christopher J.M. Tsao Congenital heart defects (CHD) are the most common type of birth defect and the leading cause of infant death. The most severe defects, such as Tetralogy of Fallot and hypoplastic left heart syndrome, can require immediate surgical intervention soon after birth. Current repair strategies involve surgically implanting inactive patch materials which often require repeat surgeries. Since congenital heart defects can be detected as early as the first trimester, the time between diagnosis and surgery can effectively be used to engineer functioning cardiac tissue. The goal of this study was to create an implantable cardiac patch that could direct the differentiation of induced pluripotent stem cells (iPSC) reprogrammed from human amniotic fluid derived stem cells (AFSC). This differentiation would take place within a closed system, minimizing laboratory handling and maximizing clinical applicability. The resulting cardiac patch would overcome current patch deficiencies associated with arrhythmia, mechanical mismatch, or even heart failure. By creating a three-dimensional system capable of temporally regulating the release of small molecules, autologous induced pluripotent stem cells could be directed to functional cardiomyocytes for use as an implantable cardiac patch for congenital heart defect repair. Further development of this system could also be used to develop repair strategies for ischemic heart repair. In order to obtain an autologous cardiomyocyte cell source for CHD, AFSC were readily isolated from amniotic fluid obtained through routine amniocentesis. These cells were classified by previous members in our lab as broadly multipotent, though not sharing the same pluripotency as embryonic stem cells. Attempts to directly differentiate AFSC into cardiac cells resulted in expression of early and late stage cardiac markers, but lack of classic cardiomyocyte contractility. Therefore this study investigated the reprogramming of AFSC to iPSC by modified mRNA transfection and the differentiation of these reprogrammed cells into cardiomyocytes through small molecule inhibitors of the GSK3 and Wnt signaling pathways. Reprogrammed cells were shown to express markers of pluripotency and formed teratomas in vivo. Cardiac differentiation resulted in immature spontaneously beating cells which were characterized through genetic expression, immunohistochemistry and electrophysiology. By encapsulating GSK3/Wnt small molecule inhibitors within porous silica particles (pSi), reprogrammed AFSC were differentiated into to cardiomyocytes with minimal intervention. The release of inhibitors from pSi was tuned by varying the thickness of polymer coatings to coincide with the temporal cues for cardiac differentiation. We evaluated the nanoparticle size, zeta-potential, and release profile in a 2D culture, as well as cell differentiation efficiency, phenotypic analysis and electrophysiology. Before translating the iPSC-derived cardiomyocyte (CM) differentiation into a three dimensional space, we first investigated an electrospun (ES) gelatin biomaterial and evaluated it for cardiac cell toxicity and the promotion of neovascularization in vivo. pSi containing vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) were conjugated to the ES gelatin and shown to have a sequential sustained release in vitro. Results showed a decrease in cellular toxicity in vitro due to reduced particle internalization and increased neovascularization in vivo. The results of this research could provide new insights into repair strategies for CHD that would be functional and able to grow with the patient. It can also provide an innovative platform for future tissue engineering constructs as well as help develop cardiac specific toxicity platforms.