Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions

N. S. Shuvalova1, V. A. Kordium1, 2 1 State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 2 Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 riyena@yandex.ua Aim. To optimize the cultivation of Wharton jelly-derived mesenchymal stem cells (WJ-MSCs) using physiological oxygen concentrations, and to compare the effect of “hypoxic” gas mixtures, based on nitrogen and argon, on their proliferative activity. Methods. From the fi rst passage, WJ-MSCs were cultivated during fi ve passages in the nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen) and argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon), 7 days before replating. At each passage the fi nal cell number was estimated and the number of population doublings was calculated. Results. The proliferation level of WJ-MSCs, cultured in both gas mixtures with 3 % of O2, was signifi cantly higher compared to that under the regular CO2-incubator conditions. In argon-based mixture, the WJ-MSCs proliferation was higher than in the control but lower than in nitrogen-based mixture. Conclusion. Cultivation of human WJ-MSCs under 3 % O2 had a stimulating effect on the cell proliferation potential. The highest intensity of the cell multiplication was observed in the nitrogen-based mixtures.


Introduction
Numerous experimental works and theoretical analysis, focused on mesenchymal stem cells (MSCs), allow considering them not only as an interesting object of studies on fundamental processes of fetal and adult life, but also as a key element in various methods of regenerative therapy.A promising position of MSCs in the cell-based therapeutic strategies results from their high proliferative and differential potential, unique paracrine effects and immune properties [1,2,3].
MSCs were fi rst identifi ed in bone marrow and described as a population of non-hematopoietic mul tipotent cells [4,5].Further studies showed that the cells with similar properties can be found in both tissues of adult organism and birth-associated tis-sues: amnion, placenta and umbilical cord [6].Accor ding to the current literature, the latter are often defi ned as the «perinatal» stem cells, possessing the properties of both adult and embryonic stem cells [7].Among them, MSCs from umbilical cord matrix -Wharton jelly (WJ-MSCs), are thought to be especially attractive.The formation of WJ-MSCs population at the early stages of embryogenesis [8], permits them to preserve the features of the embryonic stem cells [7,9,10], prominent differentiation [11], immune [12] and paracrine properties [13].At the same time, they possess the characteristics of adult somatic mesenchymal multipotent stromal cells [14], determined by the International Society for Cellular Therapy [15].
One of the most important characteristics of MSCs, particularly for the clinical usage, is their prolifera-tive activity.For instance, according to the literature, during the treatment of acute «graft-versus-host» disease, patients received 2 × 10 6 -8×10 6 MSCs/kg body weight [16].The percent of MSCs in their classical source -bone marrow for the newborns is 0.01 %, and decreases to 0.001-0.005% with aging [17].Therefore, it is vitally important to develop the cultivation technologies, which would allow maximal cell multiplication with preservation of the MSCs therapeutically relevant properties.The complexity of this task is related to the process of cultivation itself.Some works report that a long-term cultivation increases the risks of genetic abnormalities [18], thus a reasonable approach to provide the most effective MSCs cultivation should grant obtaining the maximal number of cells while minimizing the duration of culturing.
In the organism, a crucial role in the regulation of MSCs behavior and preservation of their properties belongs to their natural site of localization -«stem cell niche».Its components include the extracellular matrix, surrounding cells and signal molecules, produced by them.One of the key factors of niche regulation is the oxygen concentration, which is generally lower comparing not only to ambient atmospheric concentration , but also to that in other regions of the tissue.For instance, the oxygen concentration in bone marrow ranges from 2 % to 7 %, depending on the distance from capillary, and MSCs locate in the areas remote from vessels , which are, the most «hypoxic» zones [19].Thus, the generally accepted environmental conditions of CO 2 -incubator, where the oxygen concentration is similar to atmospheric, are actually «hyperoxic» for MSCs, which inevitably leads to the oxidative damages [20,21].Taking this into account, the MSCs cultivation at physiological oxygen concentrations, often referred to as «hypoxic», is considered to be a perspective approach [22][23][24].
The studies focused on the infl uence of hypoxic conditions on the MSCs cultivation reported their benefi cial effect on multiplication, reducing oxidative stress, and engraftment in the transplantation [25][26][27][28].However, it is hard to compare the results obtained in different works.MSCs, used in various studies, originate from different sources, the design of experiments varies from short-term preconditioning to a long-term cultivation, and the O 2 concentrations used range from 1.5 % to 8 %.The works on the infl uence of hypoxic conditions on WJ-MSCs, still remain rare cases.
Generally, the gas mixtures used for cultivation include nitrogen as a major «fi lling» component [29][30][31].However, recent works have shown the cytoprotective effect of noble (or inert) gases (argon and xenon) on the cell cultures [32].Taking this into account, we hypothesized, that using the «hypoxic» gas mixture based on the noble gas would enhance the benefi cial effect of physiological oxygen concentration on the MSCs culture.
Thus, the aim of the present work was to optimize the MSCs cultivation using physiological oxygen concentrations, and to compare the effects of «hypoxic» nitrogen-and argon-based gas mixtures on the human WJ-MSCs proliferation.

Materials and Methods
MSCs were obtained from WJ of umbilical cord (UC) from three healthy donors (39-40 weeks of gestation, normal delivery), after obtaining the informed written consent, in Kyiv maternity clinic N 5.The cells were isolated using the explant method [33].The UC fragment (5-10 cm) was washed with PBS, the vessels were mechanically removed.WJ was mechanically sliced, the fragments were placed in the cultural fl acks, 75 cm 2 , containing complete growth medium (DMEM with low glucose (PAA Austria) supplemented with 10 % fetal bovine serum (PAA, Austria), glutamine 2 mM (PAA, Austria), penicillin 100 U/ml (Arterium, Ukraine), streptomycin 100 μg/ml (Arterium, Ukraine).The fi rst adherent cells were visible on 7-10 day.After 14 days the clones reached 70-80 % confl uence, and the cells were passed using trypsin-EDTA (0.1 % trypsin and 0.02 % EDTA) solution.At the fi rst passage the cells were characterized for the surface marker proteins CD90, CD73, CD105 expression (over 85 % positive), using fl ow cytometry ( BD FACS Aria) with fl uorescein-and rhodamine-conjugated antibodies (UsBiological, USA).For microscopy, inverted microscope Leica DMIL was used.
From the fi rst passage, MSCs were seeded on plastic fl acks (25 cm 2 ) at a density of 75,000 per fl ack and cultivated during 5 passages in the nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen) and argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon), 7 days before replating.The control group was maintained under standard CO 2 incubator conditions.For creating the hypoxic conditions, the cultural fl acks with cells were placed in polyethylene bags with hermetical clasp «ZipLock».The bags were washed twice with the oxygen-free gas mixture, containing 4 % CO 2 and 96 % nitrogen or argon (depending on the group), and after that were fi lled with the cultivation gas mixture (see above).The bags were placed in the vacuum containers (Scarlet).The percentage «liquid media/gas» for normal gas exchange must be 1:100 [34], the volume of gas mixture must be no less than 0.7 l.The volume of bags used was 1.5 l.
The data on cell numbers are represented as mean ± standard deviation for 3 samples.Statistical signifi cance was determined using Mann-Whitney U-test at P < 0.05.

Results and Discussion
WJ-MSCs were expanded for fi ve consecutive passages under 3 % O 2 , in the gas mixtures based on nitrogen, argon, and ambient O 2 concentration (near-ly 20 %) in CO 2 -incubator.In order to assess the proliferation activity, the cells were counted at each passage.The results, summarized in Table 1 an Figure 1, show a fi nal number of cells after 7 days of cultivation.
The proliferation level of WJ-MSCs, cultured at 3 % O 2 , in both gas mixtures was signifi cantly higher compared to that of WJ-MSCs under the CO 2 -incubator conditions.This was observed at each passage.
Interestingly, a comparison of the culture growth rates at different passages within each group allows distinguishing two separate «phases» during the cultivation.The fi rst «phase» -at the fi rst and second passages the cultures had practically similar proliferation levels.The second «phase» -beginning from the third passage, a progressive decrease in proliferation can be observed with each new passing.For WJ-  MSCs, cultured in the gas mixtures, this tendency was less pronounced.At the fi rst passage of culture under 3 % O 2 in the nitrogen-based and argon-based gas mixtures, the final number of expanded WJ-MSCs was respectively 6.8 and 6.4 times higher, was higher comparing to cells from control group (6.1-fold increase).The results for the second passage were 6.9, 6.4 and 6 respectively.The data for the entire culture duration are summarized in Table 2.
The PD estimation showed that, despite a general decrease in the number of PD up to the 5th passage, the MSCs cultured under the physiological oxygen tension, had generally higher intensity of division (Table 3).
The proliferative activity is an important criterion for an estimation of the state of culture, and a suffi -cient activity is necessary for practical application.It strongly depends on the conditions of cultivation, each factor of environment having its own mechanisms of infl uence.In this context, proliferation itself can be considered a subject of research.In the present work, the proliferation rates of MSCs cultures, expanded under physiological oxygen tensions -3 %, in the mixtures based on nitrogen and argon, were validated.The estimation of cells number at 1-5 passages showed signifi cantly higher numbers in the gas mixtures at each passage, comparing to the groups from control CO 2 -incubator.The results also revealed a higher level of PD under hypoxic conditions up to the 5th passage.The effect of mild hypoxia appeared to be stimulating in both mixtures, at the same time, the differences between these conditions were detected.
Although a high variability in the object and methods of studies on hypoxia complicates the comparison of results, we can assume that the tendencies, observed in the present work, are generally in line with those described in literature.Nevertheless, it is important to point out the differences.The work by Basciano et al. revealed the slowing of culture growth at early passages under hypoxic conditions [22].We have not observed any similar effect.Ren et al detected the morphological signs of accelerated cellular senescence in hypoxic conditions, probably associated with a higher population doublings number [35].In the present study there was no evidence of degenerative changes of morphology in the cultures expanded under hypoxic conditions (data not shown).
The current literature distinguishes several potential mechanisms of physiological oxygen concentrations impact.First, the reactive oxygen species are one of the major sources of DNA damage [36].Hypoxic conditions were shown to prevent the accumulation of damages of genetic apparatus, which in control groups can be detected since the second passage [17].This can play an important role during long-term cultivation.Next, the researches show that hypoxic conditions increase the level of cytokine receptors expression, as well as the production of growth factors.In this case, hypoxia « sensibilizes « the cells to serum growth factor, and to that produced by the cells themselves [27].Next, there are data showing that hypoxia activates the signal cascades of cell survival.The cultures in hypoxic conditions are shown to have lower levels of necrosis comparing to those cultured under the ambient oxygen concentration [24].Each of these mechanisms can contribute to the general effect of physiological oxygen concentration, and determination of their roles is still considered the question of interest.
To date, the data about the effect of noble gases on MSCs culture are still lacking.There are only few works conducted to determine the infl uence of inert gases (xenon and argon), which demonstrated a cytoprotective effect on the cell cultures of neural origin.For example, the modeling of ischemic and traumatic damages of hippocampal slice culture in gas mixtures, containing various concentrations of argon (25 %, 50 % and 74 %) and atmospheric oxygen concentration (21 %), showed a decreased level of cellular death compared to the control samples.The underlying mechanisms are unknown [32].Though, Fahlenkamp et al. demonstrated the activation of ERK 1/2, kinase that plays an important role in the processes of cells proliferation and survival, in the primary cultures of mouse embryonic astrocytes and neurons, cultured under 20 % O 2 and 50 % argon [37].It is possible that MSCs possess the similar mechanism.
The present work showed that the level of proliferation of WJ-MSCs, cultured in argon-based gas mixtures was higher comparing to WJ-MSCs from CO 2 -incubator conditions, but lower than that for the cultures from nitrogen-based mixtures.To explain these results, further research is required.Besides, taking into account the variety of existing cultivation protocols, changing the cultural strategy could probably lead to a different effect.

Conclusions
Cultivation of human WJ-MSCs under 3 % O 2 in gas mixtures, based on nitrogen and argon, had a beneficial effect on the cells proliferative activity and preservation of multiplication potential.The hypoxic argon-and nitrogen-based gas mixtures had different effects.The highest intensity of cell proliferation was observed in the nitrogen-based mixtures.