Metabolism and Redox Cycle in Human Mesenchymal Stem Cell with Culture Induced Senescence

Human mesenchymal stem cells (hMSCs) isolated from various adult tissues are primary candidates in cell therapy and being tested in clinical trials for a wide range of diseases. The pro-regenerative and therapeutic properties of hMSCs are largely attributed to their trophic effects that coordinately modulate the progression of inflammation and enhance the endogenous tissue repair by host progenitor cells. However, immediately after isolation and upon in vitro culture expansion, hMSCs lose their in vivo quiescent state and start to accumulate genetic and phenotypic changes that significantly alter their phenotypic properties, with increased heterogeneity and reduced therapeutic potential. The proliferation of hMSCs is limited and long-term culture-induced changes lead to cellular senescence and metabolic alteration, resulting in reduced therapeutic outcome. Since clinical application requires large-scale production of hMSCs with defined cellular properties, preserving cellular homeostasis during hMSCs in vitro expansion is a major barrier for hMSCs based industrial production. Once viewed as a mere consequence of the state of a cell, metabolism is now acknowledged to play regulatory roles in cellular events and signaling pathways that govern stem cell phenotype and functional properties. Regulation of hMSC metabolism via preconditioning strategies have been proposed to enhance hMSC stem cell properties. However, the mechanistic details of metabolic and redox alterations in hMSC replicative senescence are not well understood. The current study is to understand the role of energy metabolism in regulating hMSC cell fate during in vitro culture expansion to develop metabolic strategies to augment hMSCs therapeutic outcome. We studied therapeutic relevant properties of hMSC such as immune modulation with regards to the energy metabolism and cellular signaling networks in Chapter 2. Moreover, preconditioning of hMSCs via 3D aggregation regulated energy metabolism and redox cycle, further activated PI3K/Akt survival pathways. The therapeutic potentials of 3D aggregate-derived hMSCs were studied in a rat MCAO stroke model in Chapter 3. To address the scale-up production of hMSC aggregates for potential pre-clinical applications, a novel microcarrier-based bioreactor was developed with thermal-response materials. Non-invasive and non-enzymatic procedures can be achieved for hMSC expansion and 3D aggregates production as demonstrated in Chapter 4. At last, we reported the breakdown of cellular homeostasis in hMSCs with culture-induced senescence. Basic cellular characteristics including proliferation, regenerative potential, cell cycle, and mitochondrial function were disrupted during culture expansion of hMSCs. Culture-induced senescence of hMSCs also induced impairment of migratory ability and immunomodulation. Decrease of basal autophagy and mitophagy indicated the breakdown of cellular homeostasis in hMSCs with replicative senescence. GC-MS metabolomics and proteomics revealed the loss of glycolytic phenotype and energy homeostasis with replicative expansion of hMSCs, which reconfigured hMSCs to an insufficient energy production state from glycolysis towards OXPHOS following senescence. Rapid production of energy required for maintaining cellular properties of hMSCs induced mitochondrial dysfunction and redox imbalance. We also found that nicotinamide adenine dinucleotide (NAD+) plays a central role in regulating senescent response along with hMSC expansion. It has been shown that NAD+ repletion restored mitochondrial and stemness in improving longevity of rodent. Our results show a significant decline of NAD+ during rapid expansion of hMSCs. NAD+/Sirtuin axis plays a crucial role in restoring mitochondrial function, including mitochondrial biogenesis, membrane potential and electron transport ability. By repletion of NAD+ to senescent hMSCs, various stem cell properties were recovered. Together, the results revealed the mechanistic connection between metabolic regulation and hMSC fate and therapeutic potentials, and provided metabolic and redox target to maintain hMSC cellular homeostasis for cell therapy applications in manufacturing.