Abstract
Upon withdrawal of leukemia inhibitory factor (LIF) self-renewing pluripotent mouse embryonic cells (mESC) undergo reversible commitment, followed by irreversible commitment and heterogeneous cell differentiation or apoptosis, within three days. In this study we investigated bioenergetics profile of these different maturation stages. For this purpose mESC were cultivated in the presence or absence of LIF for 24, 48 and 72h and then analysed. Mitotracker green staining showed greater mitochondrial mass in the pluripotent than in the differentiated cells. To determine if this different mitochondrial content provides bioenergetics differences between this cells populations, we measured oxygen consumption rate (OCR) as indicator of principal mitochondrial respiration parameters and extracellular acidification rate as an indicator of glycolysis using XF24 analyzer. We found that basal OCR and ECAR declined in the order: pluripotent cells (+LIF) > reversible committed cells (-LIF 24h)> irreversible committed cells (-LIF 48h) > differentiated cells (-LIF72h). This was associated with mitochondrial ATP output, estimated as coupled rate (OCR linked to ATP synthesis), showing the same downtrend. Likewise, + LIF cells possessed the highest maximal respiratory capacity indicating their higher resistance to oxidative stress comparing to maturating stages. In addition, inhibition of mitochondrial respiration impaired proliferation of +LIF, but not –LIF 72h cells. Also, suppression of mitochondrial ATP synthesis, provoked compensatory increase of glycolysis in the + LIF cells which is impaired in the course of the maturation. Bioluminescent measurement of ATP content showed the highest level in the –LIF 72h cells, implying lessen ATP turnover in the more differentiated cells. Our results demonstrated that self-renewing pluripotent mESC have bioenergetics advantage reflecting in the high OXPHOS and glycolytic activity, which declines throughout their maturation.
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