Shaking culture attenuates circadian rhythms in induced pluripotent stem cells during osteogenic differentiation through the TEAD-Fbxl3-CRY axis.

Circadian rhythms, which synchronize cellular and organismal activities with the Earth's 24-hour light-dark cycle, are controlled by clock genes. These genes not only regulate metabolic and physiological processes but also influence osteogenesis. Despite extensive research on the genetic control of circadian rhythms, little is known about the mechanisms by which mechanical factors in the extracellular environment affect these rhythms during the osteogenic differentiation of induced pluripotent stem cells (iPSCs). Shaking culture, which promotes the formation of three-dimensional organoid-like constructs from iPSC embryoid bodies (iPSC-EBs), introduces distinct biomechanical forces compared with static adherent culture. This raises the question of how these forces affect the circadian gene expression during osteogenic differentiation. In this study, we investigated the effects of shaking cultures on the circadian rhythm of key clock genes (Clock, Bmal1, and Npas2) in iPSC-EBs. In the adherent culture, iPSC-EBs displayed rhythmic oscillations of the clock genes, which were attenuated in the shaking culture. RNA-seq analysis revealed that the yes-associated protein (YAP)-transcriptional enhanced associate domain (TEAD) transcriptional cascade was activated in the shaking culture. Further investigations using assay for transposase-accessible chromatin with sequencing and chromatin immunoprecipitation assays identified Fbxl3 as a direct target of this transcriptional cascade. Fbxl3 upregulation in the shaking culture enhanced the degradation of CRY proteins, which are essential components of the circadian feedback loop, thereby suppressing clock gene oscillations. In addition, treatment with verteporfin, a YAP-TEAD inhibitor, restored circadian gene oscillations and increased the expression of osteogenic markers in shaking culture. These findings highlight a novel mechanistic link between biomechanical cues and circadian regulation and offer potential insights for optimizing tissue engineering strategies in regenerative medicine.