<i>Psme1</i> gene knockout reduces proliferation of mouse embryonic stem cells without affecting reactive oxygen species levels

BACKGROUND: Embryonic stem cells are a unique type of cells derived from the pre-implantation epiblast of mammalian embryos. These cells demonstrate the ability to undergo indefinite division and maintain an undifferentiated state. The potential for differentiation into any cell type renders embryonic stem cells a significant tool for regenerative medicine. Furthermore, they may be used in investigating the mechanisms of embryogenesis and disease modeling. The maintenance of pluripotency and the directed differentiation of embryonic stem cells rely on strictly regulated cell processes, including ubiquitin-proteasome-mediated protein degradation. Aberrant functions of the ubiquitin-proteasome pathway are linked to loss of embryonic stem cell pluripotency and apoptosis initiation. These processes are driven, particularly under oxidative stress, by the regulatory complex PA28αβ, which consists of Psme1- and Psme2-encoded α- and β-subunits. The early stages of mouse embryonic stem cell differentiation are accompanied by an increase in the expression of PA28αβ, and the knockdown of the α-subunit results in the accumulation of carbonylated proteins. This suggests that PA28αβ plays a role in the early stages of embryonic stem cell differentiation. However, the functions of PA28αβ in maintaining embryonic stem cell pluripotency remain insufficiently studied. AIM: To determine the effects of knockout of the Psme1 gene, which encodes the α-subunit of the PA28αβ regulator, on mouse embryonic stem cell proliferation and accumulation of reactive oxygen species (ROS). METHODS: In the study, a Psme1 knockout mouse embryonic stem cell line was generated through the use of genome editing. Teratoma assay was used to assess the ability to differentiate in vivo. The expression of pluripotency markers in embryonic stem cells was determined by real-time polymerase chain reaction and western blotting. The experimental stage also included flow cytometry to assess cell proliferation and production of ROS. RESULTS: A Psme1 knockout mouse embryonic stem cell line was successfully obtained. The analysis of the expression of key pluripotency markers did not show statistically significant differences between the control and mutant embryonic stem cells. The teratoma assay confirmed the maintenance of pluripotency in Psme1 knockout embryonic stem cells, demonstrating their ability to differentiate into derivatives of all three germ layers (i.e., ectoderm, mesoderm, and endoderm). The mutant embryonic stem cells demonstrated a lower proliferation rate compared to the control cells, whereas the ROS level remained unchanged. CONCLUSION: Psme1 knockout mouse embryonic stem cells have been demonstrated to maintain pluripotency and the ability to differentiate into derivatives of all three germ layers in vivo. Although the ROS level remained unchanged, the knockout of the α-subunit of the PA28αβ regulator caused a reduction in the proliferation rate, thereby emphasizing the critical role of PA28αβ in maintaining the proliferative potential of embryonic stem cells.