PMID- 38087380 OWN - NLM STAT- MEDLINE DCOM- 20231216 LR - 20240125 IS - 1757-6512 (Electronic) IS - 1757-6512 (Linking) VI - 14 IP - 1 DP - 2023 Dec 12 TI - 2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified. PG - 364 LID - 10.1186/s13287-023-03606-y [doi] LID - 364 AB - BACKGROUND: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. METHODS: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. RESULTS: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, 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 by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and alpha-CHC respectively. A reduction in mineralization was found after treatment with BPTES and alpha-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. CONCLUSION: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis. CI - (c) 2023. The Author(s). FAU - Neto, Nuno G B AU - Neto NGB AD - Department of Mechanical, Manufacturing and Biomedical Engineering, Trinity College Dublin, Parsons Building, Dublin 2, Ireland. AD - Trinity Centre for Biomedical Engineering, Trinity College Dublin, Dublin, Ireland. FAU - Suku, Meenakshi AU - Suku M AD - Department of Mechanical, Manufacturing and Biomedical Engineering, Trinity College Dublin, Parsons Building, Dublin 2, Ireland. AD - CURAM SFI Research Centre for Medical Devices, National University of Ireland, Galway, Ireland. AD - Trinity Centre for Biomedical Engineering, Trinity College Dublin, Dublin, Ireland. FAU - Hoey, David A AU - Hoey DA AD - Department of Mechanical, Manufacturing and Biomedical Engineering, Trinity College Dublin, Parsons Building, Dublin 2, Ireland. AD - CURAM SFI Research Centre for Medical Devices, National University of Ireland, Galway, Ireland. AD - Advanced Materials for Bioengineering Research (AMBER), Centre, Trinity College Dublin and Royal College of Surgeons in Ireland, Dublin, Ireland. AD - Trinity Centre for Biomedical Engineering, Trinity College Dublin, Dublin, Ireland. FAU - Monaghan, Michael G AU - Monaghan MG AUID- ORCID: 0000-0002-5530-4998 AD - Department of Mechanical, Manufacturing and Biomedical Engineering, Trinity College Dublin, Parsons Building, Dublin 2, Ireland. monaghmi@tcd.ie. AD - CURAM SFI Research Centre for Medical Devices, National University of Ireland, Galway, Ireland. monaghmi@tcd.ie. AD - Advanced Materials for Bioengineering Research (AMBER), Centre, Trinity College Dublin and Royal College of Surgeons in Ireland, Dublin, Ireland. monaghmi@tcd.ie. AD - Trinity Centre for Biomedical Engineering, Trinity College Dublin, Dublin, Ireland. monaghmi@tcd.ie. LA - eng GR - 16/RI/3403/SFI_/Science Foundation Ireland/Ireland GR - 13/RC/2073_P2/SFI_/Science Foundation Ireland/Ireland GR - 19/FFP/6533/SFI_/Science Foundation Ireland/Ireland PT - Journal Article DEP - 20231212 PL - England TA - Stem Cell Res Ther JT - Stem cell research & therapy JID - 101527581 RN - 33X04XA5AT (Lactic Acid) SB - IM MH - Humans MH - *Osteogenesis/genetics MH - Lactic Acid/metabolism MH - *Mesenchymal Stem Cells/metabolism MH - Cell Differentiation/physiology MH - Bone and Bones MH - Cells, Cultured PMC - PMC10717614 OTO - NOTNLM OT - Differentiation OT - Glutaminolysis OT - Imaging OT - Mesenchymal stem cells OT - Metabolism OT - Non-invasive characterization OT - Osteogenesis OT - Tissue engineering COIS- The authors declare that they have no competing interests. EDAT- 2023/12/13 06:42 MHDA- 2023/12/17 13:19 PMCR- 2023/12/12 CRDT- 2023/12/13 00:14 PHST- 2023/08/01 00:00 [received] PHST- 2023/12/06 00:00 [accepted] PHST- 2023/12/17 13:19 [medline] PHST- 2023/12/13 06:42 [pubmed] PHST- 2023/12/13 00:14 [entrez] PHST- 2023/12/12 00:00 [pmc-release] AID - 10.1186/s13287-023-03606-y [pii] AID - 3606 [pii] AID - 10.1186/s13287-023-03606-y [doi] PST - epublish SO - Stem Cell Res Ther. 2023 Dec 12;14(1):364. doi: 10.1186/s13287-023-03606-y.