RESUMEN
The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.
Asunto(s)
Aterosclerosis , Inflamación , Interferón Tipo I , Macrófagos , Retroelementos , Síndrome de Werner , Interferón Tipo I/metabolismo , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Humanos , Aterosclerosis/metabolismo , Aterosclerosis/inmunología , Aterosclerosis/genética , Aterosclerosis/patología , Macrófagos/metabolismo , Macrófagos/inmunología , Retroelementos/genética , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Técnicas de Cocultivo , Miocitos del Músculo Liso/metabolismo , Células Endoteliales/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Senescencia Celular , Proliferación CelularRESUMEN
Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Silenciador del Gen , Células Madre Pluripotentes Inducidas/metabolismo , Células Progenitoras de Megacariocitos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Plaquetas/metabolismo , Línea Celular , Proliferación Celular , Células Clonales , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Regulación hacia Arriba , Proteína bcl-X/metabolismoRESUMEN
Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.