Leveraging single cell multiomic analyses to identify gene regulatory networks that drive human articular cartilage cell fate

Cartilage plays a crucial role in skeletal development and function, and abnormal development contributes to genetic and age-related skeletal disease. Compromises to the integrity of joint-lining articular cartilage in particular lead to debilitating chronic degenerative diseases including osteoarthritis. To better understand how human articular cartilage develops in vivo, we jointly profiled the transcriptome and open chromatin regions in individual nuclei recovered from distal femurs at 2 fetal timepoints. We used these multiomic data to predict transcription factor-based regulatory networks that are important for articular chondrocyte differentiation. We developed a human pluripotent stem cell platform for interrogating the function of predicted transcription factors during chondrocyte differentiation and identified new biological roles for CREB5. We have identified genes whose expression is regulated by over-expression of CREB5 in either human iPS-derived cartilage or in bovine deep zone articular chondrocytes. Overall design: We have identified genes expressed in bovine deep zone articular chondrocytes whose expression was altered following forced overexpression of the transcription factor, CREB5. We performed RNA-Seq analysis of bovine deep zone articular chondrocytes that had been infected with lentivirus programmed to express doxycycline-inducible bovine CREB5 lentivirus. We cultured the infected deep zone articular chondrocytes for three days with TGFb2 (20 ng/ml) and TGFa (100 ng/ml) either in the absence (DZCiCreb5-L, Dox-) or in the presence (DZCiCreb5-L, Dox+) of doxycycline (1mg/ml)