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

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.

Critical Impact of Human Amniotic Membrane Tension on Mitochondrial Function and Cell Viability In Vitro

“…In that study, responsiveness of placental amnion to inhibition of ATP synthase was much more pronounced. Furthermore, human amniotic mesenchymal stromal cells of placental amnion were found to be more susceptible to changes in oxygen tension [30]. As expected, ROUTINE respiration of both regions (reflected and placental) dramatically decreased in floating biopsies after 21 days compared to day 0.…”

Sub-Regional Differences of the Human Amniotic Membrane and Their Potential Impact on Tissue Regeneration Application

“…Beside the aforementioned TGFB family ( Han et al, 2008 ), higher protein levels of epidermal growth factor could also be detected in reflected hAM tissue (mid-zone and apical amnion) compared to placental hAM ( Gicquel et al, 2009 ). In contrast, other growth factors which are important for keratinocyte migration ( Seeger and Paller, 2015 ), such as keratinocyte growth factor ( Han et al, 2008 ), insulin-like growth factor 1 ( Han et al, 2008 ), insulin-like growth factor-binding protein ( Litwiniuk et al, 2017 ), and hepatocyte growth factor ( Banerjee et al, 2018b ) were higher in placental compared to reflected amnion. In addition, PTHrP, a hormone which enhances differentiation of keratinocytes ( Kaiser et al, 1994 ) and promotes fracture healing [reviewed in Kostenuik and Mirza (2017) ], showed higher protein expression levels ( Germain et al, 1992 ; Curtis et al, 1997 ; Han et al, 2008 ) and higher in vitro secretion ( Germain et al, 1992 ; Farrugia et al, 2000 ) in placental amnion compared to reflected amnion.…”

“…Another example on how differently cells of the amniotic sub-regions react to their micro-environment was shown with different cultivation conditions. Four days of culture of hAMSCs at 20% oxygen strongly increased the rate of mitochondrial oxidative phosphorylation in cells of the placental region but not in cells of the reflected region ( Banerjee et al, 2018b ). This is important to note, since, as mentioned before, the rate of oxidative phosphorylation can influence the readiness of a cell to proliferate or differentiate.…”

“…This can be seen by the higher expression of proteins, mostly known for their pro-inflammatory nature, such as C-X-C motif chemokine 6 ( Han et al, 2008 ), and prostaglandin-endoperoxide synthase 2 (PTGS2) or cyclooxygenase 2 (COX2) ( Lee et al, 2010b ) in placental hAM tissue. Furthermore, placental hAMSCs incubated without lipopolysaccharide (LPS) ( Banerjee et al, 2018b ), and placental hAM explants incubated with LPS ( Han et al, 2008 ) showed a much stronger pro-inflammatory response in terms of interleukin (IL)-6 release ( Banerjee et al, 2018b ) and mitogen-activated protein kinase (MAPK) 3/MAPK1 activation and IL1B mRNA expression ( Han et al, 2008 ) compared to reflected amnion samples. These observations are particularly interesting considering the fact that therapeutic cells are often transplanted in a microenvironment with a high degree of inflammatory factors.…”

Human Amniotic Mesenchymal Stem Cells Promote Endogenous Bone Regeneration

“…The production of some cytokines by hAMSCs vary under different conditions of different oxygen tension. Consequent to exposure to 20% oxygen culture condition, hAMSCs secrete abundant IL-6 as a response to changes in the mitochondrial function, but the content of intracellular reactive oxygen species (ROS) remained unaltered ( 63 ). These properties of hAMSCs rendered the stem cell-based therapy applicable and also provided valuable guidance for bone regeneration.…”