ENHANCED 3D CULTURE OF HUMAN IPSCS AND THEIR APPLICATION FOR HUMAN TROPHOBLAST MODELS

Abstract

Implantation is one of the most critical processes in early human embryonic development, as it establishes the first physical and molecular interaction between the embryo and the maternal endometrium. Understanding this process is essential for birth defects prevention, drug discovery, and regenerative medicine. However, direct investigation of human implantation remains challenging due to ethical constraints and the significant interspecies differences between humans and model organisms, which limit the accuracy of these models. Human induced pluripotent stem cells (hiPSCs), reprogrammed from somatic cells, offer a promising platform for in vitro modeling of human implantation, as they can be differentiated into trophoblasts, the cell lineage responsible for embryo attachment to the maternal endometrium. However, current methods primarily generate trophoblasts in two-dimensional (2D) culture systems, failing to recapitulate key aspects of early embryonic development, including cell-cell and cell-matrix interactions. In addition, generating three-dimensional (3D) hiPSC spheroids in large-scale and uniform size remains a challenge. Existing methods fail to produce homogeneous spheroids, require enzymatic detachment that yields apoptotic single cells, and rely on high concentrations of Rho-associated kinase inhibitor (RI), which can compromise cell quality. This dissertation addresses these limitations by developing: (1) a platform for producing high-quality, homogeneous hiPSC spheroids without RI, and (2) a strategy for differentiating these spheroids into functional 3D trophoblast spheroids. To achieve this, cold-responsive, micropatterned culture dishes (crMPDs) were fabricated to enable the detachment of intact 2D hiPSC colonies simply by ice cooling, thus eliminating the need for enzymatic treatments. The detached micropatterned Matrigel islands guided uniform colony formation, which subsequently self-assembled into homogeneous 3D spheroids in RI-free medium. These spheroids were then differentiated into trophoblast structures exhibiting controlled size, sustained culture stability, and robust functional characteristics. Collectively, this work establishes a high-throughput, high-fidelity platform for 3D hiPSC culture and a reliable differentiation method for generating 3D trophoblast model, providing valuable tools for studying early human development and advancing in vitro modeling for regenerative medicine and developmental biology.