2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified
Authors: Neto Nuno GB , Suku Meenakshi , Hoey David A , Monaghan Michael G 
Published: April 2023

Abstract
I. Background: Human mesenchymal stem cells (hMSCs) fuel discrete biosynthetic pathways to multiply and differentiate into specific cell lineages; with undifferentiated hMSCs showing reliance on glycolytic respiration. hMSCs differentiating towards an osteogenic phenotype rely on oxidative phosphorylation as an energy source. Two-photon fluorescence lifetime imaging (2P-FLIM) is a powerful technique for non-invasive probing and monitoring of cellular metabolism; and we hypothesize that we can use this approach to monitor the osteogenic differentiation of hMSCs to uncover potential routes to boost hMSC differentiation based on their metabolic behaviour.
II. Methods: The metabolic profile of hMSCs was profiled during osteogenic differentiation over 14 days using 2P-FLIM to image and measure NADH fluorescence. We validated this metabolic profile using qPCR and extracellular metabolite analysis. Upon revealing a higher dependence on glutaminolysis; we sought to drive glutaminolysis further during using the metabolite lactate. In order to establish the importance of glutaminolysis and lactate metabolism in osteogenic differentiation these metabolic pathways were shut down using Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulphide (BPTES) and α-cyano-4-hydroxycinnamic acid (α-CHC) respectively.
III. Results: During osteogenesis, we observe a higher dependence on oxidative phosphorylation for cellular energy; and a higher reliance on glutaminolysis in the earlier time-points of osteogenic differentiation. Driving glutaminolysis further using lactate supplementation led to higher levels of mineral deposition and osteogenic gene expression. This supplementation 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. The significance of glutaminolysis and lactate metabolism in osteogenic differentiation was highlighted when these metabolic pathways were shut down using BPTES and α-CHC respectively which led to reduced hMSC mineralisation.
IV. Conclusions: In summary, we demonstrate using a unique non-invasive imaging approach 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.