Development of a High-Throughput Human iPSC Chondrogenesis Platform and Applications for Arthritis Disease Modeling

Abstract

The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. In this dissertation, we demonstrate robust cartilaginous matrix production in multiple hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations, improved matrix homogeneity, and reduced variability between tissues. We next demonstrated the ability of the system to serve as a high-throughput system for arthritis disease modeling using cytokine stimuli. Finally, we used this platform to screen for transcription factors whose activation might be involved in chondrogenic lineage specification of hiPSCs. Taken together, these studies describe the generation of a high-throughput system for chondrogenesis and its application for screens and arthritis disease modeling. Future applications of this platform may be useful for identifying pathways regulating cartilage regeneration and novel therapeutics for arthritis.