2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

Abstract

Human mesenchymal stem cells (hMSCs) fuel discrete biosynthetic pathways to multiply and differentiate into specific cell lineages; with undifferentiated hMSCs showing reliance on glycolysis. hMSCs differentiating towards an osteogenic phenotype rely on oxidative phosphorylation as an energy source. Here, the metabolic profile of hMSCs was profiled during osteogenic differentiation over 14 days using a non-invasive live-cell imaging platform- two-photon fluorescence lifetime imaging microscopy (2P-FLIM) which images and measures NADH fluorescence. During osteogenesis, we observe a higher dependence on oxidative phosphorylation for cellular energy; concomitant with an increased reliance on anabolic pathways. We validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time-points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further during osteogenic differentiation. An indirect method of promoting glutaminolysis was explored so as to not impact cellular differentiation. As Lactate has been shown to promote glutamine uptake via c-Myc activation triggering expression of glutamine transmembrane transporters and glutaminase 1; we chose to increase extracellular lactate concentrations to drive increased glutaminolysis rates leading to higher levels of mineral deposition and osteogenic gene expression. Lactate supplementation of osteogenic medium also promoted upregulation of lactate metabolism and increased the expression of transmembrane cellular lactate transporters. Higher rates of lactate dehydrogenase gene expression coupled with higher NADH fluorescence intensity demonstrate a conversion of lactate to pyruvate. During this conversion, NADH is formed by the reverse enzymatic reaction of lactate dehydrogenase resulting in increased NADH fluorescence intensity. In order to evaluate the importance of glutaminolysis and lactate metabolism in osteogenic differentiation, these metabolic pathways were shut down using BPTES and -CHC respectively which led to reduced hMSC mineralisation. In summary, we demonstrate that hMSCs osteogenic differentiation has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture. Osteogenic differentiation was demonstrated to be directly dependent on OxPhos and on glutaminolysis and validated using biochemical assays. Furthermore, extracellular lactate is an essential metabolite to ensure osteogenic differentiation as a metabolic fuel and signalling molecule to promote glutaminolysis. These findings have significant impact in generating potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite driven osteogenesis.