Vol 56, No 6 (2025)

Cockayne syndrome (CS) is an autosomal recessive disorder. Mutations in one of two genes, CSA/ERCC8 and CSB/ERCC6, in the DNA repair mechanism are the cause of the hereditary progeroid disorder. In this report, we aimed to generate the iPSC line from fibroblasts, which carries the compound heterozygous mutations of c.2566C>T (p.Gln856Ter) and c.1821+1G>A in the CSB/ERCC6 gene. Dermal fibroblasts derived from a CS patient with compound heterozygous mutations in the CSB/ERCC6 gene were reprogrammed into an iPSC line (iPSC-CS-1), using episomal-based plasmid expressing OCT4, MYC, LIN28, shRNA against p53, SOX2, KLF4, and EBNA1. The IPSC-CS‑1 line demonstrated pluripotency characteristics by expressing pluripotency markers and differentiating into three germinal layers. The spontaneous differentiation of the iPSC-CS-1 line revealed reduced expression of the neuroectodermal marker OTX2.

Many objects of developmental biology are structurally heterogeneous, being composed of diverse tissues that present unique challenges for sample preparation and microscopic analysis. Adapting protocols to these structural peculiarities is, therefore, a crucial step for investigations in this field. A prime example is the hatching gemmule of the freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759), which comprises a central cell mass surrounded by a rigid outer shell with silicious spicules, and a delicate migratory front composed of cells leaving the gemmule and spreading along the substrate. The objective of our study was to optimize sample preparation protocols to investigate the migratory behavior of cells during early E. fluviatilis development using histology, scanning electron microscopy, and transmission electron microscopy.

Modern achievements in the field of genomic medicine and biobanking open up new prospects for improving reproductive medicine. The integration of high-performance genetic technologies with biobanking systems for the storage of biological samples makes it possible to fundamentally change approaches to the prediction and prevention of reproductive pathologies. The key advantage of such an integrative approach is the possibility of conducting a large-scale analysis of genetic risks at the population level, followed by the development of personalized preventive strategies, such as the creation of complex genetic panels for preconceptional screening of hereditary pathologies, the improvement of algorithms for predicting reproductive complications based on multifactorial analysis, and the development of test systems and drugs for targeted therapy.

This study aimed to evaluate the long-term stability and cryopreservation resilience of human induced regulatory T-Cells (iTregs) in culture. Methods: CD4⁺ T-Cells were isolated from human peripheral blood mononuclear cells (PBMCs) using magnetic separation. iTreg differentiation was induced by culturing the cells in RPMI‑1640 medium supplemented with TGF-β, IL‑2, and anti-CD3 antibodies. The proportion of cells with a CD4⁺CD25⁺CD127^low phenotype was assessed by flow cytometry at isolation and on days 7, 14, and 21 of culture, as well as post-cryopreservation. Furthermore, the expression of key Treg markers (CD4, IL2RA, FOXP3, IKZF2) was analyzed at the gene level via total RNA sequencing. Results: A significant increase in the CD4⁺CD25⁺CD127^low population was observed following induction, reaching a high purity of 96.3±2.5% by day 7, which was maintained throughout the 21-day culture period. This phenotype remained stable after cryopreservation, with no significant loss in cell numbers. RNA sequencing confirmed stable transcriptional upregulation of canonical Treg markers, including FOXP3 and IL2RA. Conclusion: Human iTregs demonstrate remarkable phenotypic and transcriptional stability over 21 days in culture and maintain their defining characteristics following cryopreservation. This confirms their suitability for long-term studies and potential therapeutic applications.

The key processes determining cell differentiation are the synthesis of new proteins and restructuring of the extracellular matrix. The production of matrix and cytosolic proteins, in turn, requires the constant involvement of the heat shock protein system — chaperones that ensure the proper folding of newly synthesized protein molecules and maintain their functional state. The aim of this study was to evaluate the effect of the heat shock protein inducer geranylgeranylacetone (GGA) on the differentiation of human mesenchymal stem cells. The study found that pretreating cells with GGA for 24 hours altered the pattern of calcium responses to the pro- osteogenic stimulus, parathyroid hormone. There was an increase in the number of cells responding to anti- osteogenic calcium oscillations associated with a decrease in the osteogenic potential of MSCs. Induction of osteogenic differentiation demonstrated a decrease in calcium deposition and RUNX2 and OSX gene expression. However, the addition of GGA had no significant effect on adipogenic differentiation. In search of a possible anti- osteogenic mechanism for the action of GGA, we analyzed the MSC proteomes treated with GGA or DMSO as controls. We found that incubation with GGA resulted in the absence of several proteins. Among these, calmodulin proteins (CALM1, CALM2, and CALM3), coactivators of adenylate cyclases, and proteins that regulate the actin cytoskeleton play a key role in osteogenesis. Thus, we demonstrated that the effect of GGA leads to a significant reduction in the capacity of MSCs for osteogenic, but not adipogenic, differentiation. A more detailed study of the mechanism of GGA action on embryonic and postnatal stem cells will further elucidate key features of the formation of new osteoblasts during embryogenesis and postnatal tissue renewal.