Anastasiya Lukacheva

Oct4 is well established as a core regulator of pluripotency, yet emerging evidence points to an additional role in lineage specification during the exit from the pluripotent state. Although Oct4 expression has been observed in early mesodermal progenitors, its precise function in this developmental context remains unclear. To investigate this, we employed embryoid bodies (EBs) as a model of spontaneous differentiation that recapitulates key aspects of early embryonic development in vitro. In accordance with previous studies, reporter assay revealed a distinct temporal pattern characterized by the strong, transient co-expression of Oct4 and the early mesoderm-specifying marker gene Brachyury within a narrow developmental window, consistent with the Oct4 role in early mesodermal progenitors. We further examined the consequences of the Oct4 loss at early stages of this differentiation. Conditional knockout of the Oct4 gene resulted in a significant reduction in EB size and accumulation of cells in the G0/G1 phase, indicating a critical requirement for Oct4 in maintaining cell proliferation. Despite this defect, cells retained the ability to initiate multilineage differentiation, albeit with reduced expression of Brachyury and elevated expression of endodermal markers FoxA2 and Sox17. Interestingly, the formation of beating cardiomyocyte-like structures was also diminished following Oct4 loss and could not be rescued by simply increasing cell numbers. Taken together, these findings highlight an important Oct4 function in mesodermal differentiation, mediated through the maintenance of proliferative capacity of early mesodermal progenitors.

Background/Objectives: A comparative study of silver (Ag), titanium nitride (TiN), zirconium nitride (ZrN), and copper (Cu) coatings on titanium (Ti) disks, considering the specifications of a microporous skin- and bone-integrated titanium pylon (SBIP), was performed to assess their biocompatibility, osseointegration, and mechanical properties. Methods: To assess cytotoxicity and biocompatibility, Ti disks with various metal coatings were co-cultured with FetMSCs and MG-63 cells for 1, 3, 7, and 14 days and subsequently evaluated using a cell viability assay, as supported by SEM and confocal microscopy studies. The antimicrobial activity of the selected four materials coating the implants was tested against S. aureus by mounting Ti disks onto the surface of LB agar dishes spread with a bacterial suspension and measuring the diameter of the growth inhibition zones. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) analysis of the relative gene expression of biomarkers that are associated with extracellular matrix components (fibronectin, vitronectin, type I collagen) and cell adhesion (α2, α5, αV integrins), as well as of osteogenic markers (osteopontin, osteonectin, TGF-β1, SMAD), was performed during the 14-day follow-up period. Additionally, the activity of matrix metalloproteinases (MMP-1, -2, -8, -9) was assessed. Results: All samples with metal coatings, except the copper coating, demonstrated a good cytotoxicity profile, as evidenced by the presence of a cellular monolayer on the sample surface on the 14th day of the follow-up period (as shown by SEM and inverted confocal microscopy). All metal coatings enhanced MMP activity, as well as cellular adhesion and osteogenic marker expression; however, TiN showed the highest values of these parameters. Significant inhibition of bacterial growth was observed only in the Ag-coated Ti disks, and it persisted for over 35 days. Conclusions: The silver-based coating, due to its high antibacterial activity, low cytotoxicity, and biointegrative capacity, can be recommended as the coating of choice for microporous titanium implants for further preclinical studies.

Mesenchymal stem cells, cultured in 2D, are motile fibroblast-like cells with a well-developed actin cytoskeleton. In this study, we analyzed changes in cell motility during long-term cultivation accompanied by replicative senescence (RS) for DF2 and MSCWJ-1 cell lines derived from various sources and donors of different age under both normal and inflammatory conditions, the latter obtained by treatment with lysophosphatidic acid (LPA). Our results indicate that RS is associated with non-stationary alterations in the average migration speed: while the median speed derived from single-cell tracking is unaffected by the senescence stage, the average speed in young cells is enhanced due to the contribution of a subpopulation of fast-moving cells. The sensitivity of cell motility metrics to the impact of LPA varied depending on their origin, with the most pronounced effects observed during the initial passages. Using multivariate statistical analysis, we have shown explicitly that the common motility metrics (average and maximum speed, distance, sinuosity of trajectories, etc.) are associated with the passage, thus clearly reflecting senescence effect. Altogether, our results indicate that cell motility exhibits complex alterations with RS, with multiple metrics besides the average speed being affected and associated with their RS stage. Graphical Abstract

Cells of the TMS cell line in the well with the addition of a PES inhibitor on the matrigel matrix.

Analysis of the mass-spectrometry data from isolated lipid rafts.

Preparing a tumor sample for confocal microscopy.

Motility of Hsp70-positive cells in a tumor sample.

List of proteins detected by mass spectrometric analysis of lipid rafts isolated from normal brain tissue (obtained from a patient with epilepsy).

Effect of Hsp70 inhibitors PES and JG-98 on cell viability analyzed using the MTT assay.

Gating of mHsp70High cells (field R2) and mHsp70Low cells (field R3) by FACS.