Genes & Cells

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare genetic disorder with a prevalence of approximately 1 case per 1.5–2.0 million individuals. The primary cause of the disease lies in mutations of the ACVR1 gene, which encodes the homonymous receptor involved in the regulation of bone histogenesis. The most common genetic variant is the 617G>A single nucleotide substitution (R206H, rs121912678), resulting in the replacement of arginine with histidine. This variant is observed in over 95% of all registered patients with fibrodysplasia ossificans progressiva. The mutation disrupts intracellular signaling, leading to the formation of abnormal heterotopic bone tissue in muscles, tendons, and ligaments. The clinical presentation may vary: some patients experience additional symptoms presumably associated with rare mutations in other domains of the ACVR1 receptor. Despite active research, the exact precursor cells responsible for pathological osteogenesis remain unidentified. Scientists hypothesize the involvement of multiple cell types, including mesenchymal stem cells and endothelial cells. Currently, no effective treatment exists for fibrodysplasia ossificans progressiva, underscoring the urgent need for novel therapeutic targets. Gene therapy, successfully employed in other monogenic disorders, represents a promising direction. Several experimental approaches, including targeted inhibitors of the ACVR1 signaling pathway, are undergoing preclinical and clinical trials.

BACKGROUND: Wilson disease is a rare autosomal recessive disorder involving mutations in the ATP7B gene, which encodes the copper-transporting ATPase. The dysfunctional protein disrupts biliary copper excretion, which results in copper accumulation in hepatocytes. The available range of cell models to investigate the molecular mechanisms of this disease and to identify novel therapeutic approaches is currently limited.

AIM: To develop an in vitro induced human pluripotent stem cell-derived model for investigating the molecular mechanisms of Wilson disease and evaluating therapeutic strategies.

METHODS: A 2D cell model has been developed using validated induced pluripotent definitive endoderm cells obtained from a healthy donor and differentiated by activin A and CHIR99021. The copper overload that is a hallmark of the pathogenesis of Wilson disease was simulated by introducing exogenous copper into the growth medium. Relative cell viability was measured by the Alamar Blue assay.

RESULTS: The induced pluripotent stem cells demonstrate a normal karyotype. Their morphology is typical of embryonic stem cells. They express pluripotency markers (SOX2, OCT4, TRA-1-60, and SSEA-4) and form tissues of all three germ layers during spontaneous differentiation in embryoid bodies. The differentiation of these induced pluripotent stem cells using the suggested procedure produces definitive endoderm cells that exhibit the expected morphology and express the markers SOX17, FOXA2, and ATP7B. The obtained model demonstrates sensitivity to exogenous copper overload at IC₅₀ 197 μM.

CONCLUSION: The developed platform of definitive endoderm cells obtained from healthy donor’s induced pluripotent stem cells can be used to model the copper overload in vitro, simulating a cellular metabolic dysfunction associated with Wilson disease. Therefore, the proposed model can be used for both fundamental research and the development of novel therapeutic approaches.

BACKGROUND: Lipopolysaccharide (LPS) administration in mice is a widely used model for studying inflammation-associated depression. However, the mechanisms underlying LPS-induced changes in the brain remain unclear.

AIM: This study is aimed to investigate behavioral, cellular, and molecular changes induced by chronic-interval LPS treatment in two brain regions implicated in depression, the hippocampus and the prefrontal cortex.

METHODS: The study involved adult wild-type male mice (2–3 months old, 25–35 g, n = 28) and glial cell primary cultures.

The experimental design included a two-phase LPS administration protocol (1 mg/kg, 3 injections intraperitoneally) with a 7-day interval. During the first phase, behavioral assessments were performed; whereas in the second phase, tissue samples (prefrontal cortex and whole hippocampus) were collected for molecular and histological analyses. Behavioral assessment included the Open Field Test (general activity and anxiety-like behavior), the Tail Suspension Test, the Sucrose Preference Test (anhedonia), and the Y-Maze Test (spatial working memory). Glial cell primary cultures were incubated in the presence of LPS to induce neuroinflammation and fibroblast growth factor 2 (FGF2) to assess changes in the microglial phenotype.

Molecular and cellular changes in vivo and in vitro were analyzed using real-time polymerase chain reaction and immunohistochemistry assays.

RESULTS: LPS-treated mice exhibited depression-like behavior, including decreased interest in hedonic stimulus, increased immobility, reduced locomotor activity, and memory deficits. The inflammatory reaction was associated with the elevated expression of proinflammatory cytokines (TNF-α, IL-1β) in both the spleen and brain with distinct regional patterns of astrocytic and microglial activation. LPS increased the expression of tight junction protein 1 (TJP1), vascular endothelial growth factor A (VEGFA), and E-selectin, decreased the expression of claudin 3, occludin, FGF2, and significantly increased the number of mast cells. Microglial activation was observed in both regions with a shift towards the amoeboid phenotype. Glutamatergic signaling was altered with downregulation of glutamate transporters (GLT-1) and glutamine synthetase in the hippocampus, suggesting the impaired glutamate buffering. In vitro, LPS induced microglial activation, which was reversed by FGF2.

CONCLUSION: LPS-induced neuroinflammation differentially affected the hippocampus and prefrontal cortex with the hippocampus appearing to be more vulnerable. FGF2 reversed LPS-induced microglial activation, indicating its potential as a therapeutic target for neuroinflammation-associated depression.

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.