Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture

Abstract

Chondrogenic differentiation of human embryonic (hESCs) or induced pluripotent stem cells (hiPSCs) has been achieved in embryoid bodies (EBs) by adding selected growth factors to the medium. Also chondrocyte-secreted factors have been considered to promote the chondrogenic differentiation. Hence, we studied whether co-culture with primary chondrocytes can induce hESCs or hiPSCs to differentiate into chondrocyte lineage. Co-culture of hESCs or hiPSCs was established in a transwell insert system in feeder-free culture conditions, while hESCs or hiPSCs grown alone in the wells were used as controls. After 3-week co-culture with weekly replenished chondrocytes, the chondrogenically committed cells (hCCCs) were evaluated by morphology, immunocytochemistry, quantitative real-time RT-PCR, and analysis of chondrogenic, osteogenic and adipogenic differentiation markers. The expressions of chondrocyte- and pluripotency-associated genes were frequently measured during the monolayer expansion of hCCCs from passage 1 to 10. Human CCCs displayed morphology similar to chondrocytes, and expressed chondrocyte-associated genes, which were declined following passaging, similarly to passaged chondrocytes. They also formed a chondrogenic cell pellet, and differentiated into chondrocytic cells, which secreted abundant extracellular matrix. Human CCCs also proliferated rapidly. However, they did not show osteogenic or adipogenic differentiation capacity. Our results show that co-culture of hESCs or hiPSCs with primary chondrocytes could induce specific chondrogenic differentiation.