Characterisation of human mesenchymal stem cell metabolism during proliferation and differentiation.

Abstract

Mesenchymal stem cells (MSCs) have the ability to differentiate towards cartilage, bone, fat and muscle, and therefore have great therapeutic potential. Human MSCs reside under hypoxia (4-7% oxygen) in vivo and recent investigations have described an increase in population doublings and maintenance of differentiation capacity upon culture under these conditions. The reason for these differences may be related to their cellular metabolism. This thesis examines MSC metabolism during proliferation, chondrogenic and osteogenic differentiation. Furthermore, MSCs were cultured under uninterrupted and controlled hypoxia (5% or 2% oxygen) to observe its effect on proliferation and differentiation. The production of lactate and consumption of oxygen by MSCs indicated a mixed metabolism, with the cells utilising both oxidative phosphorylation and glycolysis under 20% oxygen or normoxic conditions. The majority of cellular ATP production was through glycolysis, whilst the full oxidative capacity of the mitochondria was not fully utilised. During chondrogenic differentiation, oxygen consumption was significantly reduced with time in culture and the cells became highly glycolytic, whilst osteogenic differentiation maintained the mixed metabolism of expanded MSCs. Fewer colonies were formed at 5% oxygen, compared to 2% and 20% oxygen. Hypoxic culture induced fewer cells per colony compared with normoxia. However, MSCs expanded under hypoxia had reduced cellular senescence after five passages, potentially due to the reduced utilisation of oxidative phosphorylation that has been shown to lead to the production of reactive oxygen species (ROS). The oxygen levels during expansion did not affect chondrogenic potential but expansion under hypoxia prevented ostoegenic differentiation. Osteogenesis was also inhibited for MSCs expanded at normoxia and differentiated under hypoxia. These results may be related to the initial colony formation under hypoxia and the changes in cellular metabolism during osteogenic differentiation.