Cellular senescence is a form of permanent growth arrest due to consistent stress stimuli. While the decline of regenerative potential is a hallmark of aging, stem cell decline due to cellular senescence can disrupt tissue homeostasis and repair. As a primary cell source in cell therapy, human mesenchymal stem cells (hMSCs) isolated from elderly donor have reduced regenerative capacity associated with increased levels of senescence and senescence-associated changes. Moreover, this age related changes of hMSCs appear to be similar or even more pronounced during in vitro expansion. The lost of stem cell properties due to senescence/aging may reduce the therapeutic outcome, but more importantly may have deleterious effect on recipients after transplantation. Recent studies has suggested that in vitro senescence of stem cell is resulted from hyper-stimulation of nutrient sensing pathway from growth factors and nutrient that force cells into premature, and loss of quiescence. Understanding the metabolic regulation governing in vitro proliferation and quiescence has important implication in optimizing hMSCs properties while preserving their multipotent phenotype. Our prior studies have shown that glycolytic energy metabolism significantly regulates clonogenic MSCs proliferation and maintenance of multi-potency. The objective of this study is to investigate the metabolic regulation between hMSCs proliferation and quiescence during in vitro expansion, with the aim to develop a metabolic approach to preserve the multipotent state of hMSCs and consequently maintain their regenerative capacity.
Materials and Methods
Standard hMSCs culture protocol was used to maintain hMSCs in continuous culture from passage 5 to passage 9. Metabolic modulators such as dehydroepiandrosterone (DHEA), 2-deoxyglucose (2-DG), and antimycin A (AmA), were used to selectively modulate the specific metabolic pathways and evaluate their regulatory roles.
The results revealed that cellular senescence of high passage hMSCs is significantly attenuated by inhibiting the rate-limiting enzyme of pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) via treatment of inhibitor DHEA at a concentration of 200uM for a period of 4 days. hMSCs treated with DHEA at passage 9 have significantly lowered staining and enzyme activity of senescent marker β-gal compared to hMSCs at the same passage without DHEA treatment. DHEA treated hMSCs retain regenerative capacity after continuous passaging, indicated by higher proliferation rate than cells without treatment, after being re-expanded into DHEA free culture medium. The attenuation effect of DHEA on hMSCs senescence is associated with cellular quiescence that DHEA treatment significantly decreased cell proliferation and growth, indicated by lower cell number fold increase and smaller cell size. The senescent attenuation effect by inhibiting PPP enzyme G6PDH is further associated with metabolic reconfigure towards a more oxidative status. DHEA decreased the mRNA level of glycolytic enzymes, including LDHA, and PKM2, while increased oxidative phosphorylation indicated by lower mRNA level of PDK1, higher cellular ATP content, and ROS level. Together, the results revealed the mechanistic connection between metabolic regulation and hMSCs in vitro quiescent state, and demonstrate that regulation of hMSCs metabolism as a strategy in preserving stem cell properties during expansion.
The accumulative changes of hMSCs metabolic phenotype during expansion significantly influence hMSCs phenotypic properties, such as proliferation, quiescence, and multi-lineage differentiation. Understanding the metabolic mechanisms governing hMSCs fate decision can contribute to a better strategy to maintain stem cell properties in hMSCs expansion and application.
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