ZIM1 Combined with Hydrogel Inhibits Senescence of Primary PαS Cells during In Vitro Expansion

Bone marrow stem cells (BMSCs) are a promising source of seed cells in bone tissue engineering, which needs a great quantity of cells. Cell senescence occurs as they are passaged, which could affect the therapeutic effects of cells. Therefore, this study aims to explore the transcriptomic differences among the uncultured and passaged cells, finding a practical target gene for anti-aging. We sorted PαS (PDGFR-α+SCA-1+CD45-TER119-) cells as BMSCs by flow cytometry analysis. The changes in cellular senescence phenotype (Counting Kit-8 (CCK-8) assay, reactive oxygen species (ROS) test, senescence-associated β-galactosidase (SA-β-Gal) activity staining, expression of aging-related genes, telomere-related changes and in vivo differentiation potential) and associated transcriptional alterations during three important cell culture processes (in vivo, first adherence in vitro, first passage, and serial passage in vitro) were studied. Overexpression plasmids of potential target genes were made and examed. Gelatin methacryloyl (GelMA) was applied to explore the anti-aging effects combined with the target gene. Aging-related genes and ROS levels increased, telomerase activity and average telomere length decreased, and SA-β-Gal activities increased as cells were passaged. RNA-seq offered that imprinted zinc-finger gene 1 (Zim1) played a critical role in anti-aging during cell culture. Further,Zim1combined with GelMA reduced the expression of P16/P53 and ROS levels with doubled telomerase activities. Few SA-β-Gal positive cells were found in the above state. These effects are achieved at least by the activation of Wnt/β-catenin signaling through the regulation ofWnt2. The combined application ofZim1and hydrogel could inhibit the senescence of BMSCs during in vitro expansion, which may benefit clinical application.Keywords:stem cells;cellular senescence;transcriptomics;Wnt pathway;hydrogel

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