Introducing cellular senescence in human induced pluripotent stem cells and differentiated neural lineage for modeling of age-associated diseases
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenetic landscapes that mark the aging clock, and consequently cells differentiated from iPSCs resemble fetal cells rather than adult or aged cells. The lack of proper cellular aging in cells differentiated from iPSCs presents a significant problem in iPSC-based disease models to investigate the pathological progression of age-associated diseases such as neurodegeneration. To address this caveat, we introduced cellular senescence into iPSC-based cell models in this study, as senescence has been shown to play a critical role not only in aging but also in neurodegeneration. We created an inducible CRISPR interference (CRISPRi) targeting TERF2, a major component of the telomere protecting Shelterin complex. We demonstrated that suppression of TERF2 robustly activated DNA damage response, the p53/p21 signaling, cellular senescence and inflammatory response in iPSCs. The inducible approach allows the temporal control of senescence activation over the course of differentiation of iPSCs to desired cell types. We applied the CRISPRi-TERF2 system to differentiation of iPSCs into neural progenitor cells (NPCs) and showed that suppression of TERF2 efficiently activated DNA damage response, the p53/p21 signaling and senescence in differentiated NPCs. The inducible cell model of cellular senescence generated in this study has broad application in investigation of cellular senescence in the progression of age-related diseases and improvement of disease modeling with a proper cellular aging context to facilitate drug discovery. Overall design: To investigate the consequence of TERF2 suppression in activation of senescence, we established human iPSC lines with an inducible CRISPRi targeting TERF2. Two iPSC lines and two gRNAs were used. Cells were treated with or without doxycycline (DOX) for 4 or 6 days. We performed gene expression profiling analysis of RNA-seq data obtained from untreated and Dox-treated iPSCs at two different treatment points with 16 samples in total.