Programmable Assembly of Genetically Engineered Human Tissues

Abstract

Genetically engineered cells promise to revolutionize our ability to pattern human tissues. Genetic approaches to guiding cell differentiation provide rapid and efficient methods to control cell phenotype. Transcription factor-driven differentiation can directly reprogram human induced pluripotent stem cells (hiPSCs) into specific cell types such as endothelium, neurons, fibroblasts, and cardiomyocytes. Tissues assembled from multiple genetically engineered cell types open new avenues to creating multicellular tissues for drug discovery, disease modeling, and regenerative medicine. My Ph.D. dissertation focuses on new methods to program cell composition and phenotype in hiPSC-derived organoids and tissues. By coupling genetic engineering and biomanufacturing, one can control the local cell phenotype and organization of multiple cell populations during human tissue fabrication. As a first demonstration, we developed selective transfection via electroporative printing (STEP), which combines continuous flow electroporation with 3D bioprinting to transfect hiPSCs on-the-fly during tissue printing. To enable STEP printing, we developed viscoelastic, shear-thinning agarose microparticle bioinks that support both 3D printing and electroporation. Next, we created custom electroporative printheads capable of transfecting hiPSCs with mRNA in a voxelated manner. We then demonstrated that bioinks containing agarose microparticles and 100×106 hiPSCs/ml could be transfected on-the-fly with greater than 90% efficiency using STEP, while maintaining high cell viability (> 80%). We further demonstrated the ability to program the relative number of transfected cells per printed voxel by adjusting the electric field strength, providing precise control of cell composition in STEP tissues. To genetically program multiple distinct cell types within human tissues, we developed a second method referred to as orthogonally induced differentiation (OID. Most transcription factor (TF) overexpression protocols produce a single cell type of interest, yet a multitude of cell types and structural organization is needed to recapitulate native human tissues. Using OID, hiPSCs are simultaneously co-differentiated into distinct cell populations in the form of organoids and bioprinted tissues with controlled composition and organization. To demonstrate this platform, we differentiated endothelial cells and neurons from hiPSCs in a one-pot system containing either neural or endothelial stem cell-specifying media. By aggregating inducible-TF and wild type hiPSCs into pooled and multicore-shell embryoid bodies, vascularized and patterned cortical organoids could be produced within days. By combining OID with multimaterial 3D bioprinting, we patterned 3D neural tissues from densely cellular, matrix-free stem cell inks that underwent orthogonal induced differentiation to generate distinct layered regions composed of neural stem cells, endothelium, and neurons, respectively. Given the high proliferative capacity and patient-specificity of hiPSCs, our platform provides a facile route for programming multicellular brain and other human tissues. In summary, we developed multiple methods to control stem cell differentiation by integrating genetic engineering and bioprinting. Transfecting cells on-the-fly can produce patterns of gene expression throughout printed tissues, while multiple inducible hiPSC lines can be assembled into a single tissue to program organoid and tissue composition. Both STEP and OID open new avenues for creating multicellular human tissues and organoid building blocks for drug screening, disease modeling, and therapeutic use.