Replicative Senescence Of Mesenchymal Stem Cells Research Articles

Ginsenoside Rg2 Promotes the Proliferation and Stemness Maintenance of Porcine Mesenchymal Stem Cells through Autophagy Induction

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

Effects of long-term culture on the biological characteristics and RNA profiles of human bone-marrow-derived mesenchymal stem cells

Expansion in vitro prior to mesenchymal stem cells (MSCs) application is a necessary process. Functional and genomic stability has a crucial role in stem-cell-based therapies. However, the exact expression and co-expressed profiles of coding and non-coding RNAs in human bone marrow (BM)-MSCs in vitro aging are still lacking. In the present studies, the change of morphology, immunophenotype, and capacity of proliferation, differentiation, and immunoregulation of MSCs at passage (P) 4, P6, P8, P10, and P12 were investigated. RNA sequencing identified that 439 mRNAs, 65 long noncoding RNAs (lncRNAs), 59 microRNAs (miRNAs), and 229 circular RNAs (circRNAs) were differentially expressed (DE) in P12 compared with P4, with a similar trend in P6. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) identified several significant biological processes and pathways, including binding, ossification, and Wnt and PPAR signaling pathways. Interaction and co-expression/localization analyses were performed for DE mRNAs and lncRNAs, and several key lncRNAs, circRNAs, and important pathways like autophagy and mitophagy were identified in the competing endogenous RNA (ceRNA) network. Some key RNAs found in the bioinformatics analysis were validated. Our studies indicate that replicative senescence of MSCs is a continuous process, including widespread alterations in biological characteristics and global gene expression patterns that need to be considered before therapeutic applications of MSCs.

Comparative evaluation of proliferative potential and replicative senescence associated changes in mesenchymal stem cells derived from dental pulp and umbilical cord.

Mesenchymal stem cells (MSC) have been widely studied for tissue regeneration and cell-based therapy. MSC can be isolated from different body tissues while several biological waste sources like dental pulp, umbilical cord, cord derived blood, amniotic fluid or urine have also emerged as potential sources of MSCs. Specifically, isolation of MSCs from such non-conventional sources show promising outcomes due to the non-invasiveness of the extraction process and high proliferation capacity of the isolated MSC. However, these stem cells also exhibit the limitation of replicative senescence in long-term culture condition. Inter-cellular reactive oxygen species is an important contributor for inducing cellular senescence under long-term culture conditions. For translational application, it becomes imperative to compare the stem cells isolated from these sources for their senescence and proliferative properties. In this study, MSC were extracted from two different sources of biological waste materials-dental pulp and umbilical cord, and compared for their proliferation capacity and replicative senescence at different passage numbers (i.e. P2 and P6). Intracellular ROS production was significantly (p < 0.001) less in dental pulp stem cells culture in comparison to umbilical cord-derived stem cells at P6. The β-gal expression also showed significantly (p < 0.001) low expression in DPSC culture compared to that of UCSC at P6. The study indicates the source of stem cells influences the proliferation capacity as well as replicative senescence of MSCs. This study will thus pave the path of future research in selecting appropriate stem cell source for regenerative medicine application.

Culturing on Wharton's Jelly Extract Delays Mesenchymal Stem Cell Senescence through p53 and p16INK4a/pRb Pathways

Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.

Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.