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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.

Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.