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. 2018 Oct 28:2018:9502451.
doi: 10.1155/2018/9502451. eCollection 2018.

Oxygen Tension Strongly Influences Metabolic Parameters and the Release of Interleukin-6 of Human Amniotic Mesenchymal Stromal Cells In Vitro

Asmita Banerjee  1   2 , Andrea Lindenmair  2   3 , Ralf Steinborn  4 , Sergiu Dan Dumitrescu  1   2 , Simone Hennerbichler  5 , Andrey V Kozlov  1   2 , Heinz Redl  1   2 , Susanne Wolbank  1   2 , Adelheid Weidinger  1   2
Affiliations

Oxygen Tension Strongly Influences Metabolic Parameters and the Release of Interleukin-6 of Human Amniotic Mesenchymal Stromal Cells In Vitro

Asmita Banerjee et al. Stem Cells Int. .

Abstract

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. In vivo (in utero), cells of the hAM are exposed to low oxygen tension (1-4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.

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Figures

Figure 1
Figure 1
Mitochondrial activity and glycolysis. Mitochondrial respiration was measured in freshly isolated hAMSCs (day 0) and after 4-day incubation at 5% and 20% oxygen (a)–(e). Lactate in the cell culture supernatant was measured at day 4 (5% and 20% oxygen) (f). Cultivation of hAMSCs at 5% and 20% oxygen for 4 days led to elevated ROUTINE respiration (a), LEAK (b), and ETS capacity (c) for both oxygen concentrations but no difference in lactate production (f). Phosphorylation-related respiration was significantly increased only in samples incubated at 20% oxygen (d). This switch to oxidative phosphorylation in hAMSCs incubated at 20% oxygen was confirmed by the netROUTINE control ratio (e) which increases upon stimulation of oxidative phosporylation. n = 5–7 (biological replicates), mean ± SD. Abbreviations: ETS: electron transfer system; hAMSCs: human amniotic mesenchymal stromal cells; O2: oxygen.
Figure 2
Figure 2
Cellular mitochondrial DNA copy number. The mtDNA copy number per cell was measured by digital PCR in freshly isolated human amniotic mesenchymal stromal cells (day 0) and after 4 days (5% and 20% oxygen) (a) and (b). We observed a trend to an increasing mtDNA copy number in samples incubated at 20% oxygen, but this increase was not significant. n = 5 (biological replicates), mean ± SD.
Figure 3
Figure 3
Reactive oxygen and nitrogen species. Intracellular reactive oxygen species levels and nitric oxide levels were measured after 4 days incubation at 5% and 20% oxygen (a)–(c). No significant differences in ROS levels between 5% and 20% oxygen samples were observed (a). hAMSCs from placental amnion showed significantly lower levels of intracellular ROS (a). Calculation of correlation coefficient showed a strong negative association (r = −0.9039) between intracellular ROS levels and phosphorylation-related respiration (b). Increased nitric oxide release was detected in cell culture supernatants of samples incubated at 20% oxygen (c). n = 5 (biological replicates), mean ± SD. Abbreviations: hAMSCs: human amniotic mesenchymal stromal cells; ROS: reactive oxygen species; RA: reflected amnion; P: placental amnion.
Figure 4
Figure 4
Release of immunoactive substances. Release of immunoactive substances in the cell culture supernatant of hAMSCs of reflected and placental amnion was measured after 4 days (5% and 20% oxygen) incubation (a)–(d). Higher oxygen tension (20%) lead to significantly higher secretion of IL-6 (a) and a trend to higher IL-10 release (b) in hAMSCs from placental amnion and had no effect on the release of hepatocyte growth factor (c) and IL1-beta release (d) compared to lower oxygen tension (5%). Cells from placental amnion released more IL-6 (a), IL-10 (b) and hepatocyte growth factor (c) when compared within cultures at same oxygen concentrations (5% and 20% oxygen). n = 6 (biological replicates), mean ± SD. Abbreviations: hAMSCs: human amniotic mesenchymal stromal cells; IL: interleukin.
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