PhD Scientific Days 2026

Theoretical and Translational Medicine 3.

Impact of Genetic Background, Passage Number, and Confluency on the Transcriptome of Mesenchymal Stem Cells

Name of the presenter

Demeter, Flóra

Institute/workplace of the presenter

Semmelweis University, Department of Internal Medicine and Hematology, Research Laboratory

Authors

Flóra Demeter¹, Erika Kajdácsi¹, Dorina Vadicsku¹, György Bihari¹, László Cervenak¹
1: Semmelweis University, Department of Internal Medicine and Hematology, Research Laboratory

Text of the abstract

Mesenchymal stem cells (MSCs), due to their immunomodulatory and tissue regenerative properties, are promising tools for cell-based therapies; however, their clinical efficacy shows considerable variability. This variability may arise from several factors, including donor heterogeneity, as well as differences in passage number and confluency—culture parameters that are critical for the bioreactor-based production of therapeutic quantities of MSCs. Our aim was to investigate how donor variability, passage number, and confluency influence the transcriptomic and functional state of MSCs. Two well-characterized human MSC lines derived from individual donors were cultured under identical conditions at different passage numbers (4 and 8) and confluencies (30% and 100%). Global gene expression profiling of the eight resulting samples was performed using the Agilent microarray platform. Following preprocessing of the raw data, 13,474 expressed protein-coding genes (“whole MSC transcriptome”) were further analyzed using bioinformatic approaches. Hierarchical clustering of the whole MSC transcriptome primarily separated samples according to confluency and secondarily by passage number. Differential gene expression analysis identified 225 genes associated with passage number, 212 with confluency, and 156 genes reflecting inter-donor genetic differences, each showing at least a twofold statistically significant difference in expression. Correlation and principal component analyses revealed that sample segregation was mainly driven by confluency and passage number, whereas donor effects were less pronounced. Confluency predominantly affected cellular functions related to inflammatory responses, chemotaxis, and responses to growth factors, while passage number influenced processes associated with stem cell fate determination, extracellular matrix organization, cell migration, and tissue regeneration. Our results indicate that MSC gene expression and function are more strongly influenced by culture conditions than by donor variability, which may impact their immunosuppressive and regenerative potential. Therefore, strict standardization of cell culture protocols is essential to improve the efficacy of MSC-based therapies. Supported by the University Research Scholarship Programme (EKÖP),Ministry of Culture and Innovation, National Research, Development and Innovation Fund.