Highly efficient chondrogenic differentiation of human iPSCs and purification via a reporter allele generated by CRISPR-Cas9 genome editing

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. Here we demonstrate robust cartilaginous matrix production in three unique hiPSC lines using a highly efficient 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. The ability to enrich for chrondroprogenitors and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications.