CREATING A 3D STEM CELL MICROENVIRONMENT TO PRODUCE HUMAN DEVELOPING CARDIAC TISSUES FOR SCALE-UP AND DISEASE MODELING
Type of DegreePhD Dissertation
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In the field of tissue engineering, the use of human induced pluripotent stem cells (hiPSCs) has proven extremely valuable based on their ability to differentiate into almost any cell type within the human body, but keep their inherent biological origin. Until now, human physiology, disease modeling and regeneration, as well as organ development are not well understood due to the lack of available human-based tissues for experimentation and cell therapy. These challenges are used as the ultimate goal in cardiac tissue engineering, and are sought to be overcome by using hiPSCs to create authentic 3D developing heart tissue in vitro. HiPSCs can be differentiated into contracting cardiomyocytes, following natural pathways of human heart development. Here we created a highly-reproducible, single-cell handling step procedure to encapsulate hiPSCs in PEG fibrinogen hydrogels to generate functional and maturing 3D developing human engineered cardiac tissues (3D-dhECTs) in the geometry of immobilized microislands. This single-cell handling procedure not only reduced processing steps, but also allowed cells to form important cell-cell and cell-material interactions. The feasibility of differentiating hiPSCs within PEG-fibrinogen to create 3D-dhECT microislands was compared to an already published, highly efficient cardiac differentiation procedure which produced 2D cardiac monolayers. After validation of our novel 3D model, 3D-dhECTs were exposed to the known teratogen thalidomide, which caused congenital defects in more than 10,000 newborns in the 1950s. 3D-dhECT microislands were used to detect differences in tissue growth, frequency of contraction, total cell number and percent CMs, as well as CM size, sarcomere distance, and mitochondria distribution between control and thalidomide-treated CMs. Next, 3D cardiac microspheres were produced by applying our single-cell handling approach. Microspheres were created by encapsulating hiPSCs within PEG-fibrinogen and formed by a water-in-oil emulsion technique in a custom microfluidic device. Once encapsulated, hiPSCs grew and differentiated into contracting cardiac microspheres with CMs responding to drug treatment and outside electrical pacing. Finally, we wanted to evaluate the suitability of other biomaterials to create a favorable microenvironment for hiPSC encapsulation and cardiac differentiation. Gelatin methacryloyl (GelMA) was used due to its successful implementation in other tissue engineering and bioprinting applications. GelMA was synthesized and characterized using NMR; acellular GelMA hydrogels successfully degraded in the presence of collagenase. Once hiPSCs were encapsulated, cells grew and degraded the hydrogel over time. HiPSCs differentiated into CMs to produce GelMA developing human engineered cardiac tissues (GEhECTs), with frequency and velocity of contraction increasing over time. GEhECT CMs developed defined and aligned sarcomeres; CMs also responded to outside pacing and drug treatment.
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