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 is to create an implantable cardiac patch that can direct the differentiation of induced pluripotent stem cells (iPSC) reprogrammed from human amniotic fluid derived stem cells (AFSC). This differentiation will take place within a closed system, minimizing laboratory handling and maximizing clinical applicability. The resulting cardiac patch will overcome current patch deficiencies associated with arrhythmia, mechanical mismatch, or even heart failure. By creating a 3D hydrogel based system capable of temporally regulating the release of small molecules, autologous induced pluripotent stem cells can 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 are readily isolated from amniotic fluid obtained through routine amniocentesis. These cells have been 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 result in expression of early and late stage cardiac markers, but lack classic cardiomyocyte contractility. Therefore this study will investigate 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.
By encapsulating GSK3/Wnt small molecule inhibitors within porous silicon particles (pSi), reprogrammed AFSC can be differentiated directly to cardiomyocytes with minimal human intervention. The release of inhibitors from pSi can be tuned by varying the thickness of polymer coatings to coincide with the temporal cues for cardiac differentiation. This differentiation system will allow for simple translation to a 3D model. We evaluated nanoparticle size, zeta-potential, and release profile in a 2D transwell culture, as well as cell differentiation efficiency, phenotypic analysis and electrophysiology. Functional beating cardiac like cells were analyzed up to 21 days of differentiation.
Translating this system to a three dimensional space, we plan to incorporate a fibrin/PEG hydrogel with the reprogrammed AFSC and encapsulated inhibitors. The dual stage inhibitor release will direct cardiac differentiation while the fibrin/PEG hydrogel promotes cellular adhesion and nutrient perfusion. This innovative closed system cardiac differentiation platform can lead to functional implantable cardiac tissue. The patch will be evaluated for cell viability, contractile ability, force generation and conduction velocity.
The results of this research could provide a new repair patch 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.