hiPSC Extract Attenuates Senescence-Associated Phenotypes in AB-MSCs In Vitro

Background: Bone marrow mesenchymal stromal cells (BMSCs) are widely used as seed cells in bone tissue engineering. However, sufficient cell quantities for effective engineering typically require prolonged in vitro expansion of BMSCs, which can lead to cellular senescence after excessive subculturing. Objective: This study aimed to investigate the effects of human induced pluripotent stem cell (hiPSC) extract on replicative senescence, clonogenic ability, proliferation-associated characteristics, and osteogenic differentiation capacity of human alveolar bone-derived mesenchymal stromal cells (AB-MSCs). Methods: AB-MSCs were isolated from alveolar bone fragments discarded during dental implant surgery. Cells were divided into three experimental groups: P3 AB-MSCs (Young), P10 AB-MSCs (senescence-associated, YRS), and P10 AB-MSCs treated with hiPSC extract (YRSiE). Senescence markers, clonogenic ability, proliferation-associated characteristics, osteogenic potential, mRNA sequencing and pathway-related expression changes were compared among groups. Small RNA sequencing on hiPSCs and AB-MSCs with qRT-PCR validation were performed to identify candidate miRNAs in hiPSC extract that were differentially expressed in this setting. Results: In the presence of hiPSC extract, senescent AB-MSCs showed altered morphology, a lower proportion of β-galactosidase-positive cells, higher Ki67 expression, and lower expression of the senescence-associated genes and proteins P16 and P21. Treated senescent AB-MSCs also showed higher proliferation-associated metabolic activity, clonogenicity, and osteogenic differentiation than untreated senescent cells. Pluripotency-related genes (NANOG, OCT-4) and osteogenic markers (ALP, RUNX2, OCN) were higher in the treated group. qRT-PCR validation of small RNA sequencing data showed that miR-514a-3p, miR-582-5p and miR-520c-3p were upregulated in senescent AB-MSCs after hiPSC extract treatment. Concurrently, pathway enrichment and pathway-related expression changes were consistent with an association with the NF-κB pathway. Conclusion: In this in vitro study, hiPSC extract attenuates senescence-associated phenotypes in AB-MSCs, and the observed transcriptional and pathway-related expression changes were consistent with NF-κB pathway enrichment, with the identified miRNAs upregulated in treated cells and previously reported to target NF-κB pathway components. However, these mechanisms require further validation.