Identification and Functional Characterization of CDK1, CCNB1, CCNB2, RRM2, and GTSE1 Associated with Replicative Senescence of Human Umbilical Cord-Derived Mesenchymal Stem Cells.

INTRODUCTION

Replicative senescence limits the expansion and therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). This study aimed to identify passage-associated candidate genes and assess their relationships with senescence-associated phenotypes.

METHODS

The RNA-seq dataset GSE233634 was reanalyzed using 12 planar-culture samples from Donor 1 at p3, p6, p9, and p12. Candidate expression was examined by qRT-PCR in Donors 2-4, and siRNA experiments were performed in Donor 4-derived p12 cells. Senescence-associated features were evaluated using morphology, SA-β-galactosidase staining, CCK-8-derived metabolic activity, cell-cycle distribution, and reactive oxygen species (ROS).

RESULTS

Compared with p6 cells, p12 cells showed enlarged and flattened morphology, a higher SA- β-galactosidase-positive area fraction, lower CCK-8-derived metabolic activity, reduced S-phase representation, G0/G1 accumulation, and increased ROS. Transcriptomic screening identified 405 genes shared among three p12 comparisons, of which 205 progressively increased with passage. CDK1, CCNB1, CCNB2, RRM2, and GTSE1 were selected as senescence-associated candidates and showed consistent passage-associated mRNA increases in Donors 2-4. CDK1, CCNB1, CCNB2, and RRM2 were also supported at the protein level, whereas GTSE1 was supported at the mRNA level only. Candidate-gene knockdown was associated with differential changes in selected senescenceassociated readouts, with CCNB1 knockdown showing the broadest pattern.

DISCUSSION: These findings link the five candidates to passage-associated phenotypes but do not establish direct regulatory causality or reversal of senescence.

CONCLUSION

The five genes represent candidates for further mechanistic investigation in hUC-MSC replicative senescence.