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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. 

Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

 

Protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted proteins. Human mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive abilities to secrete inflammatory modulators, angiogenic, and regenerative cytokines. However, there exists the problem of human MSC expansion with compromised therapeutic quality. Duringin vitro expansion, human MSCs are plated on stiff plastics and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that three‐dimensional (3D) aggregation can reverse some of these alterations by heightening autophagy and recovering the metabolic state back to a naïve phenotype. To further understand the proteostasis in human MSC culture, this study investigated the effects of 3D aggregation on the human MSC proteome to determine the specific pathways altered by aggregation. The 3D aggregates and 2D cultures of human MSCs derived from bone marrow (bMSC) and adipose tissue (ASC) were analyzed along with differentiated human dermal fibroblasts (FB). The proteomics analysis showed the elevated eukaryotic initiation factor 2 pathway and the upregulated activity of the integrated stress response (ISR) in 3D aggregates. Specific protein quantification further determined that bMSC and ASC responded to ISR, while FB did not. 3D aggregation significantly increased the ischemic survival of bMSCs and ASCs. Perturbation of ISR with small molecules salubrinal and GSK2606414 resulted in differential responses of bMSC, ASC, and FB. This study indicates that aggregation‐based preconditioning culture holds the potential for improving the therapeutic efficacy of expanded human MSCs via the establishment of ISR and homeostasis.