Аннотация:Multipotent mesenchymal stem/stromal cells (MSCs) of adults are considered as a perspective
tool in cell therapy and regenerative medicine. Unfortunately, autologous cell therapy does not
always provide positive outcomes in elder donors, perhaps as a result of the alterations of stem
cell compartments. Ex vivo expansion is an essential step of MSC proceeding tor cell therapy.
Long-term cultivation leads to MSC replicative senscence and lost of quality. The mechanisms of
stem and progenitor cell senescence and the factors engaged are investigated intensively.
Modification of cell culture conditions is a promising approach to improve the quality of expanded
cells. According to the current data, reduction in O2 to the tissue-related level in cellular
microenvironment (“physiologic” hypoxia) can affect the features of MSCs. Recently we have
demonstrated an increase in the proliferative activity and CFU-F number of adipose tissuederived stromal cells (ASCs), while, on the contrary, the adipogenic and osteogenic differentiation
decelerated. Global microarray analysis of ASCs expanded at 5% O2 revealed the differential
gene expression of 558 genes in comparison with ASCs at 20% O2. Wherein, the expression of
genes involved in proliferation, cell metabolism and signaling was upregulated, while that of the
ones responsible for the connective tissue matrix and intracellular cytoskeleton components
decreased.
Here we elucidated the effects of tissue-related O2 on ASCs functions in replicative senescence
in vitro model. ASCs at ambient (20%) O2 (12-21 passages) demonstrated an increased average
cell size, granularity, reactive oxygen species level, including anion superoxide, lysosomal
compartment activity, and IL-6 production сompared to 5% O2. Long-term expansion at both O2
levels did not lead to immortalization that was evidenced by the low level of hTERT transcription
and encoded enzyme activity. Decreased ASC viability and proliferation, as well as the change
of more than ten senescence-associated gene expression were detected. Among them the
upregulation of several genes was noted: CCND1 SERPINE1, PAI1. CD44. Downregulation of
CDKN1C, NOX4, and CREG1, ID1, was determined. In long-term ASCs the expression of only
11 genes changed with 5% O2, compared to 16 ones at 20% O2. CCND1, CD44, SERPINE1,
NOX4, ID1, and CDKN1C were upregulated as well as under 20% O2, but less intensively.
Expansion of ASCs at 5% O2 resulted in an attenuation of the most of detected senescenceassociated changes, which may be considered as in vitro cellular senescence deceleration. Thus,
the O2 level of in vitro milieu affects the cellular senescence events via the fed-back loop of
mitochondria-ROS-mitochondria-lysosomes and can decelerate it. The data on the effect of
oxygen level on cellular replicative senescence are definitely of interest to modern gerontology.
At the same time, standard cell culture condition under atmospheric O2 (20%) may be associated
with an increase in intracellular disorders and are less favorable compared to the “physiologic
“hypoxia (5% O2).
We concluded that the in vitro O2 level affects the cellular senescence via the fed-back loop of
mitochondria-ROS-mitochondria-lysosomes and can decelerate it .These data would be of
considerable interest for researchers and clinicians working in the modern gerontology and
regenerative medicine.
This work was supported by “Integrative Physiology” Program of Presidium of RAS and RFBR
Grant # 16-04-01244.