RESUMEN
CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.
Asunto(s)
Nucléolo Celular/fisiología , Senescencia Celular/fisiología , Homeostasis , Ligasas/fisiología , Células Madre Mesenquimatosas/fisiología , Osteoartritis/terapia , Proteínas del Grupo Polycomb/fisiología , Animales , Proteínas Cromosómicas no Histona/metabolismo , Técnicas de Inactivación de Genes , Terapia Genética , Células HEK293 , Humanos , Ligasas/genética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas del Grupo Polycomb/genéticaRESUMEN
FOXO3 is an evolutionarily conserved transcription factor that has been linked to longevity. Here we wanted to find out whether human vascular cells could be functionally enhanced by engineering them to express an activated form of FOXO3. This was accomplished via genome editing at two nucleotides in human embryonic stem cells, followed by differentiation into a range of vascular cell types. FOXO3-activated vascular cells exhibited delayed aging and increased resistance to oxidative injury compared with wild-type cells. When tested in a therapeutic context, FOXO3-enhanced vascular cells promoted vascular regeneration in a mouse model of ischemic injury and were resistant to tumorigenic transformation both in vitro and in vivo. Mechanistically, constitutively active FOXO3 conferred cytoprotection by transcriptionally downregulating CSRP1. Taken together, our findings provide mechanistic insights into FOXO3-mediated vascular protection and indicate that FOXO3 activation may provide a means for generating more effective and safe biomaterials for cell replacement therapies.
Asunto(s)
Células Madre Embrionarias/citología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Ingeniería Genética , Regeneración , Adulto , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Células Madre Embrionarias/metabolismo , Proteína Forkhead Box O3/deficiencia , Humanos , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCIDRESUMEN
Protein separation using hydrodynamic countercurrent chromatography (CCC), where low backpressure is inherent, is more challenging, more time consuming and more costly when compared with separating small molecules. The most hopeful approach is to rationally design suitable columns for already commercialized J-type CCC machinery. By comparing 3 column geometries (3D helix, 2D spiral and 3D cone), we firstly constructed the mechanical model tailored to the conical column on J-type CCC using aqueous two-phase system (ATPS) on protein separation. Aimed at mechanistically understanding hydrodynamic CCC, we then developed a semi-quantitative model to account for contributions of both hydrodynamic and hydrostatic forces to stationary phase retention, and have subsequently compared the modelling outcomes with experimental results. We practiced a methodology to delineate both phase mixing and stationary phase retention before committing to physically constructing CCC columns. Following theoretical analyses, we finally constructed conical columns for J-type CCC. Using model proteins (myoglobin and lysozyme) and with 2 ATPSs containing PEG1000 and phosphate, sound protein separation has been achieved (resolution reaches 1.5-2.0 and stationary phase retention also exceeds 40%) for the selected ATPSs and under a varied level of sample volumes and loadings.