Transcriptomic Landscape of Human iPSCs-derived Osteogenic Differentiation Identifies a Key Regulator, KLF16

The osteogenic differentiation process is essential for bone development and metabolism, but the underlying gene regulatory networks have not been well investigated. In this study, we performed RNA-seq analysis of mesenchymal stem cell differentiation to preosteoblasts and osteoblasts, derived from normal human induced pluripotent stem cell (iPSC) lines. Systematic analyses revealed potential transcription factor (TF) - TF regulatory networks, regulatory roles of long noncoding RNAs (lncRNAs), and differential splicing of extracellular matrix, TF, lncRNA, and splicing factor genes. TF-TF regulatory and gene co-expression network analyses predicted a key role for KLF16 in osteogenesis. In vitro and in vivo biological functional studies further established the inhibitory role of Klf16 in osteogenesis. Our model system and transcriptomic approach identify novel osteogenic genes and highlight the regulatory complexity of osteogenesis. Overall design: Cells at three osteogenic differentiation stages of mesenchymal stem cell (MSC) at day 0, preosteoblast (preOB) at day 7 and osteoblast (OB) at day 21 were harvested. Total RNA was isolated, converted to RNA-seq libraries, and sequenced on the Illumina HiSeq 2500 platform. Comparisons between MSC to preOB, and preOB to OB stages were performed to identify differentially expressed genes. Global TF-TF regulatory network and gene co-expression network was used to indentify key regulators of osteogenesis.