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Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

ABSTRACT: Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated ?-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.

SUBMITTER: Fernandez-Rebollo E

PROVIDER: S-EPMC7013233 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

Fernandez-Rebollo Eduardo E Franzen Julia J Goetzke Roman R Hollmann Jonathan J Ostrowska Alina A Oliverio Matteo M Sieben Torsten T Rath Björn B Kornfeld Jan-Wilhelm JW Wagner Wolfgang W

Stem cell reports 20200123 2

Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired simila ...[more]

PMID: 31983656

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Project description:BACKGROUND:The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. METHODS:In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. RESULTS:Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. CONCLUSIONS:Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

Project description:BackgroundHuman multipotent progenitor cells (hiMPCs) created from induced pluripotent stem cells (iPSCs) represent a new cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) are needed to enhance transforming growth factor-β (TGFβ)-induced hiMPC chondrogenesis. In contrast, TGFβ alone is sufficient to result in robust chondrogenesis of human primary mesenchymal stromal cells (hMSCs). Currently, the mechanism underlying this difference between hiMPCs and hMSCs has not been fully understood.MethodsIn this study, we first tested different growth factors alone or in combination in stimulating hiMPC chondrogenesis, with a special focus on chondrocytic hypertrophy. The reparative capacity of hiMPCs-derived cartilage was assessed in an osteochondral defect model created in rats. hMSCs isolated from bone marrow were included in all studies as the control. Lastly, a mechanistic study was conducted to understand why hiMPCs and hMSCs behave differently in responding to TGFβ.ResultsChondrogenic medium supplemented with TGFβ3 and BMP6 led to robust in vitro cartilage formation from hiMPCs with minimal hypertrophy. Cartilage tissue generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and resulted in a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFβ3 induced phosphorylation of both Smad2/3 and Smad1/5 in hMSCs, but only activated Smad2/3 in hiMPCs. Supplementing BMP6 activated Smad1/5 and significantly enhanced TGFβ's compacity in inducing hiMPC chondrogenesis. The chondro-promoting function of BMP6 was abolished by the treatment of a BMP pathway inhibitor.ConclusionsThis study describes a robust method to generate chondrocytes from hiMPCs with low hypertrophy for hyaline cartilage repair, as well as elucidates the difference between hMSCs and hiMPCs in response to TGFβ. Our results also indicated the importance of activating both Smad2/3 and Smad1/5 in the initiation of chondrogenesis.

Dataset Information

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

Publications

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

Similar Datasets

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