Human mesenchymal stem cells (hMSCs) are primary cell source in cell therapy for a wide range of diseases. However, immediately after isolation and upon culture expansion, MSCs acquire and accumulate genetic and phenotypic changes in culture and that many proliferating cells progressively enlarge, exit the cell cycle, and become senescent. Emerging studies have shown that in vitro self-assembled 3D MSCs have intriguing biological properties, including enhanced multi-lineage differentiation potential and more primitive stem cell phenotype. It has been shown that Wnt signaling and Notch signaling play significant roles in hMSCs aggregate functional activity, including anti-inflammation, anti-apoptosis, and endothelia cell-supporting function. The mechanism of hMSCs stemness enhancement during cellular aggregation is however remains uncertain. Moreover, as 3D culture of MSC been proposed as preconditioning strategy to enhance MSCs function, understanding the adaptive changes of MSCs metabolism during self-assembly has important implication in optimizing aggregate properties and function activation. The objective of present study is to investigate energy metabolism in regulating MSCs phenotype changes in 3D aggregates of hMSCs cells with aim to develop approachable strategy for MSCs expansion with enhanced stem cell phenotype.
Materials and Methods
hMSC aggregates were formed by seeding cells into ultra-low adherent surface plates. To assess the role of specific metabolic pathway in regulating MSCs phenotype enhancement under aggregation, several metabolic modulators, including glycolysis inhibitor (2-DG), pentose phosphate pathway (PPP) inhibitor (DHEA), were used to treat hMCs in aggregate and adherent conditions, and their metabolic and cellular impacts were discussed.
The results revealed that hMSCs in self-assembled 3D cell aggregates have improved “stemness” compared to hMSCs grown in adherent condition, indicated by up-regulation of three pluripotent stem cell genes, including Nanog, Oct4, and Sox2. This phenotypic change of hMSCs in cell aggregates is associated with metabolic reprogramming towards increased glycolytic and decreased pyruvate oxidation metabolism pattern. mRNA level of glycolysis enzymes, including HK2, PKM2, and LDHA are up-regulated in hMSCs aggregates, with mRNA level of PDK1 down-regulated, an enzyme involved in pyruvate oxidation within TCA cycle. Among the reprogrammed metabolic pathways involved in hMSCs aggregate, pentose phosphate pathway (PPP) was identified playing an significant role as inhibiting the rate limiting enzyme in PPP, G6PDH, by DHEA dramatically decreased mRNA level of Nanog, Oct4, Sox2, HK2, PKM2, and LDHA, thus abolished the phenotypic enhancement and metabolic reprogramming of hMSCs aggregates. The regulatory role of PPP during stemness enhancement of hMSCs is attributed to its stress sensing ability during cell aggregation. The redox balancing substrate NADPH and stress-response genes, including HIF-1a, NFkB and Notch were all increased in hMSCs aggregates, and were decreased dramatically upon DHEA treatment. Together, the results revealed the mechanistic connection between metabolic reconfiguration and hMSCs potency under 3D aggregation, and identified the novel role of PPP as stress sensor during hMSCs phenotypic enhancement.
The results of present study demonstrate that energy metabolic transition plays an important role in hMSC stemness improvement under 3D culture. Understanding the metabolism transition associated with hMSCs phenotypic changes has important implication for adapting 3D aggregation culture into as a non-genetic preconditioning method to potentiate hMSC properties for cell therapy.
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