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In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells.

ABSTRACT: Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.

SUBMITTER: Jeske R

PROVIDER: S-EPMC7988085 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mesenchymal stromal/stem cells (MSCs) are currently being used in clinical trials as proposed treatments for a large range of genetic, immunological, orthopaedic, cardiovascular, endocrine and neurological disorders. MSCs are potent anti-inflammatory mediators which are considered immune evasive and employ a large range of secreted vesicles to communicate and repair damaged tissue. Despite their prolific use in therapy, sex specific mechanism of action is rarely considered as a potential confounding factor for use. The purpose of this study was to examine the potency and functionality of both female and male adipose derived MSCs in order to gain further insights into donor selection. Methods MSC were expanded to passage 4, secretome was harvested and stored at - 80c. To assess potency MSC were also primed and assessed via functional immune assays, ELISA, multiplex and immunophenotyping. Results Female MSCs (fMSC), consistently suppressed Peripheral blood mononuclear cell (PBMC) proliferation significantly (p < 0.0001) more than male MSC (mMSC). In co-culture mPBMCs, showed 60.7 ± 15.6% suppression with fMSCs compared with 22.5 ± 13.6% suppression with mMSCs. Similarly, fPBMCs were suppressed by 67.9 ± 10.4% with fMSCs compared to 29.4 ± 9.3% with mMSCs. The enhanced immunosuppression of fMSCs was attributed to the production of higher concentrations of the anti-inflammatory mediators such as IDO1 (3301 pg/mL vs 1699 pg/mL) and perhaps others including IL-1RA (1025 pg/mL vs 701 pg/mL), PGE-2 (6142 pg/mL vs 2448 pg/mL) and prolonged expression of VCAM-1 post activation relative to mMSCs. In contrast, mMSCs produces more inflammatory G-CSF than fMSCs (806 pg/mL vs 503 pg/mL). Moreover, IDO1 expression was correlated to immune suppression and fMSCs, but not mMSCs induced downregulation of the IL-2 receptor and sustained expression of the early T cell activation marker, CD69 in PBMCs further highlighting the differences in immunomodulation potentials between the sexes. Conclusion In conclusion, our data shows that female MSC are more potent in vitro than their male counterparts. The inability of male MSC to match female MSC driven immunomodulation and to use the inflammatory microenvironment to their advantage is evident and is likely a red flag when using allogeneic male MSC as a therapeutic for disease states.

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In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells.

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