Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O₂ concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEF^Mfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEF^Mfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEF^Mfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEF^Mfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.