Investigation of conditioned medium properties obtained from human umbilical cord mesenchymal stem/stromal cells preconditioned with dimethyloxalylglycine in a correlation with ultrastructural changes

Mesenchymal stem/stromal cells (MSCs) hold significant therapeutic value due to their regeneration abilities, migration capacity, and immunosuppressive and immunomodulatory properties. These cells secrete soluble and insoluble factors, and this complex secretome contributes to their therapeutic effect. Furthermore, stimulation of cells by various external stimuli lead to secretome modifications that can increase the therapeutic efficacy. So, this study examined the effect of dimethyloxalylglycine (DMOG), a hypoxia‐mimetic agent, on secretome profiles and exosome secretions of MSCs by evaluating conditioned medium (CM) and ultrastructural morphologies of the cells in comparison with unpreconditioned MSCs. The appropriate dose and duration of the use of DMOG were determined as 1000 μM and 24 h by evaluating the HIF‐1α expression. DMOG‐CM and N‐CM were collected from MSCs incubated in serum‐free medium with/without DMOG for 24 h, respectively. The content analysis of conditioned mediums (CMs) revealed that VEGF, NGF, and IL‐4 levels were increased in DMOG‐CM. Subsequently, exosomes were isolated from the CMs and were shown by transmission electron microscopy and Western blot analysis in both groups. The effects of CMs on proliferation and migration were determined by in vitro wound healing tests; both CMs increased the fibroblast's migratory and proliferative capacities. According to the ultrastructural evaluation, autophagosome, autolysosome, myelin figure, and microvesicular body structures were abundant in DMOG‐preconditioned MSCs. Consistent with the high number of autophagic vacuoles, Beclin‐1 expression was increased in those cells. These findings suggested that DMOG could alter MSCs' secretion profile, modify their ultrastructural morphology accordingly, and make the CM a more potent therapeutic tool.


Research Highlights
• Preconditioning mesenchymal stem/stromal cells with dimethyloxalylglycine, a hypoxia-mimetic agent, could modify cellular metabolism.
• Hypoxic mechanisms lead to alterations in the ultrastructural characteristics of mesenchymal stromal/stem cells.
• Preconditioning with dimethyloxalylglycine leads to ultrastructural and metabolic changes of mesenchymal stromal/stem cells along with modifications in their secretome profiles.
• Preconditioning of mesenchymal stromal/stem cells could render them a more potent therapeutic tool.

| INTRODUCTION
Mesenchymal stem/stromal cells (MSCs), with their higher proliferation capacities, multipotencies, immune modulator properties, and paracrine effects, have been considered one of the important therapeutic cell types in regenerative medicine (González-González et al., 2020).MSCs can be isolated from different sources, such as bone marrow (BM), adipose tissue, and umbilical cord (UC).Recent studies showed that UC is more advantageous than other sources, including BM which has been considered as gold standard due to not requiring invasive procedures, less ethical concern and higher quality of the isolated cells like lower risk of immunological reaction and higher differentiation and proliferation capacities (Bharti et al., 2018;El Omar et al., 2014).MSCs get involved in various biological processes such as cellular survival, immunomodulation, neurogenesis, angiogenesis, wound healing, and tissue repair (Praveen Kumar et al., 2019).However, the direct link between the differentiation of those cells to target injured cells and their beneficial effect has not been proved (El Omar et al., 2014).Eggenhofer et al. showed that MSCs were mainly localized in the lungs rather than livers with the ischemia-reperfusion injury, which indicates that MSCs could not survive in vivo for a long time and their therapeutic effects are mainly conducted through paracrine interactions (Calcat-i-Cervera et al., 2023;Eggenhofer et al., 2012).Direct application of MSCs and conditioned medium (CM) derived from those cells have similar therapeutic effects in different injury models (Ebrahim et al., 2018;Nagaishi et al., 2016).Furthermore, conditioned mediums (CMs) are more advantageous than the direct application of MSCs regarding their more accessible storage and transport and no risk of tumorigenicity (Barreca et al., 2020;Pawitan, 2014).
The content of CMs is called the secretome and consists of soluble factors such as growth factors, cytokines and chemokines, and insoluble extracellular vesicles (ECVs) (González-González et al., 2020).In this respect, it was shown that growth factors secreted by MSCs like pro-angiogenic vascular endothelial growth factor (VEGF) and neuroprotective glial cell-derived neurotrophic factor (GDNF), neuron growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have significant therapeutic effects by improving wound healing, tissue repair and survival of neuronal cells respectively (Bai et al., 2016;Guo et al., 2015).Moreover, MSCs-derived CMs could be effective in treating autoimmune diseases thanks to their immune modulatory contents, including anti-inflammatory cytokines like IL-4, IL-10 (Ahmadi et al., 2023;Doorn et al., 2012;Kay et al., 2017;Konala et al., 2020;Zhang et al., 2019) Extracellular vesicles secreted from MSCs have sizes changing from 30 to 2000 nm and investigated under three subgroups as exosomes, microvesicles, and apoptotic bodies (Kurian et al., 2021).Exosomes, the smallest ECVs with 30-120 μm, contain lipids, proteins, and RNAs.Exosomes are essential lipid bilayer vesicles as a natural delivery vehicle for therapeutic contents, which can be modified or strategically engineered.
In addition, exosomes are able to penetrate the blood-brain barrier, which makes them suitable therapeutic agents for the treatment of a wide range of diseases, including neurodegenerative ones (Sarko & McKinney, 2017).
The therapeutic potential of MSCs can be ameliorated by various strategies, including genetic modification or preconditioning (Noronha Nc et al., 2019).Hypoxia, hypoxia mimetic agents, or biomaterials with appropriate topography are some of the strategies used for preconditioning MSCs (Ferreira et al., 2018;Noronha Nc et al., 2019).In this respect, preconditioning with hypoxia has improved the stemness and survival of MSCs and their therapeutic potency by increasing the angiogenic and immune modulator capacities (Ferreira et al., 2018).
Dimethyloxalylglycine (DMOG), which is one of the hypoxia mimetic agents, is also able to increase hypoxia-inducible factor-1 alpha (HIF-1α) (Duscher et al., 2017).Therefore, the aim of this study is comparative analysis of secretome profiles and exosome contents of two types of CMs obtained from hUC-derived MSCs, which are incubated with/ without preconditioning DMOG and evaluation of ultrastructural changes in both groups of MSCs.

| Isolation and characterization of MSCs
The required ethical approval for the use of umbilical cords (UCs) was acquired from Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Clinical Research Ethics Committee, and the study was carried out in accordance with the ethical principles.UC obtained from the patient who delivered by cesarean section was transferred to the laboratory under appropriate conditions.MSCs were isolated from the umbilical cord by the tissue explant method, as described in our previous studies (Isildar et al., 2019;Ozkan et al., 2018).
Immunophenotypic characterization of MSCs was performed by flow cytometry.For this purpose, the following antibodies were used: CD44-PE and CD90-APC as positive markers, endothelial CD34-FITC and hematopoietic CD45-APC-Cy (BD the Biosciences, USA) as negative markers, APC mouse IgG1, κ isotype control, APC/Cyanine7 mouse IgG1, κ isotype control, PE mouse IgG1, κ isotype control, FITC mouse IgG1, κ (Biolegend, San Diego).Briefly, MSCs at the third passage were harvested and filtered through the 70 μm filter and transferred to FACS tubes (approximately 10 6 cells).The tubes were centrifugated at 3000 RPM for 5 min, and the pellet was resuspended with a staining solution and incubated with the antibodies for 30 min at dark.After the incubation, non-conjugated antibodies were removed by centrifugation, the pellet was resuspended with the staining solution, and the cells were analyzed with BD FACS Callibur.The FACS analyzes were performed in three replicates.The multipotency of the MSCs was assessed by following the instruction of adipogenic and osteogenic differentiation-inducing medium (Sigma 811D-250 and Sigma 417D-250).Briefly; at 3rd passage, the isolated cells were collected using trypsinization.Subsequently, 1.6 Â 10 6 cells were seeded in 12 culture dishes for adipogenic differentiation, and 8 Â 10 5 cells were seeded for osteogenic differentiation.Following 48 h of incubation in the normal culture medium, the culture medium was replaced with the respective differentiation mediums for adipogenic and osteogenic differentiation.Throughout the differentiation period, the differentiation medium was refreshed every 3 days.
Upon completion of the 21-day differentiation period, the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min.
After fixation, the cells stained with Oil Red O for adipogenic differentiation and Alizarin Red for osteogenic differentiation.

| Immunocytochemical analysis of HIF-1α and Beclin-1 expressions
The MSCs at the 3rd passage were cultured on coverslip placed in 24-well plates (3 Â 10 4 cells in each well), and then the growth medium was changed with serum-free culture medium, including different doses of DMOG.In this respect, the time and dose for preconditioning with DMOG were determined by evaluating HIF-1α expression levels with immunocytochemical staining after incubation of the cell at different doses of DMOG (250, 500, 750, and 1000 μM) for 24 or 48 h.Following incubation, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT).Briefly, the cells were permeabilized with 0.1% Triton X-100 first.Following blocking for 5 min (ScyTek Laboratories SensiTek HRP kit), they were incubated with 5 μg/mL HIF-1α antibody at +4 C overnight.Then, biotinylated secondary antibodies were applied for 20 min.After the washing step, streptavidin peroxidase solution incubation was performed for 20 min.In the last step, the coverslips were treated with 3-amino-9-ethyl carbazole (AEC) chromogen.H-score method was used for the quantification of HIF-1α expressions out of 50 cells.
Beclin-1 expression was evaluated in cells preconditioned with 1000 μL and 24 h DMOG.The procedure was performed as described above, except for 5% BSA blocking.Beclin-1 (1/200) was applied at +4 C overnight.Immunocytochemical analysis were performed in at least 3 replicates.A semi-quantitative evaluation was performed out of 10 random regions at 200Â magnification, scoring from 0 to +3 by the absence or highest expression level, respectively.All sections were examined with the Olympus BX61 microscope and photographed with an Olympus DP72 camera.

| Collection and analysis of conditioned mediums
MSCs at the 3rd passage were cultured on T75 flasks, and when 70%-80% confluency was reached, the mediums were changed to serum-free medium with/out 1000 μM DMOG.At the end of the 24-hour-long incubation period, two types of conditioned mediums (N-CM and DMOG-CM) were collected and centrifugated at 3000 RPM for 5 min to remove cellular debris.The CMs were stored at À80 C for further analysis.

| Quantitative analysis of protein and paracrine contents of CMs
The total protein contents of CMs were analyzed with calorimetric bicinchoninic acid assay (BCA).The concentrations of growth factors including VEGF, GDNF, NGF, and BDNF, and cytokines, including IL-4, IL-10, IL-17, and IFN-γ were determined by enzyme-linked immunosorbent assay (ELISA) following the instruction of the manufacturer.The ELISA test was performed in three repetitions.

| Isolation and characterization of exosomes
Exosome isolation from CMs was performed using the commercial kit (ExoEasy Maxi Kit) and following the manufacturer's instructions.For the characterization, an exosome-specific marker CD63 was analyzed by Western blot.Briefly, exosome samples were subjected to lysis with RIPA buffer, vortexed for 30 s, and incubated for 30 min.Exosome lysate was centrifugated at 12000 rpm for 15 min.Supernatants were mixed with Laemmli buffer and denatured by incubating in a 100 C water bath for 5 min.Equal volumes of samples were loaded in each well of polyacrylamide gel.After PAGE and semi-dry blotting, the nitrocellulose membrane was blocked with a 5% fat-free milk powder solution for 1 h.Subsequently, the membrane was incubated overnight with anti-human CD63 primary antibody at +4 C on the shaker.The next day, after washing the membrane with TBS-T, it was incubated with HRP-conjugated secondary antibody for 2 h at RT and washed again.The membrane was treated with a chemiluminescence kit to visualize protein bands.Band intensities were quantified with the Fiji ImageJ software program (Sheller-Miller & Menon, 2020).
Western blot analysis was repeated three times.
In addition, the morphological characterization of isolated exosomes was performed with transmission electron microscopy (TEM).
In this respect, an equal volume of exosome solution was mixed with 2% glutaraldehyde and incubated for 30 min.Following fixation, 20 μL of the sample was placed on carbon/formvar coated grids and incubated for 10 min.Subsequently, grids were washed with distilled water two times and then contrasted with 1.5% uranyl acetate for 1 min.The grids were examined under the Jeol Jem-1011 model TEM and photographed with the Olympus Soft Imaging camera system (Rikkert et al., 2019).

| Assessment of effects of CMs on wound healing
The effects of the CMs on wound healing were assessed by in vitro scratch assay in which a straight line was scratched in the middle of the 24-h cultured 3 T3 fibroblast cells was scratched.Then each well was separately treated with DMEM, N-CM, and DMOG-CM for 24 h and investigated with an inverted microscope.The level of wound healing or the distance of each scratch closure was calculated by the formula ΔX (μm) = X1 À X2 (X1: the distance between cells border at time 0 of generating the scratch, X2: the distance between cells border at the end of 24th h).The wound healing model of each group was repeated three times.

| Ultrastructural evaluation of UC-MSCs
MSCs were prepared for ultrastructural analysis as described previously (Ozkan et al., 2018).Briefly, after the collection of CMs, the cells were harvested with trypsin and pelleted, fixed first with glutaraldehyde and then with osmium tetroxide.The cells were embedded in 2% liquid agar following fixation, and when the agar solidified, it was minced into 1-2 mm 3 pieces.The pieces were dehydrated by passing through graded alcohol series and embedded in araldite.0.5 μm thick semi-thin sections were cut to decide the interested location, and then 40-50 nm thin sections were placed on copper grids.The grids were contrasted with uranyl acetate and lead citrate.The cells were examined under the Jeol Jem-1011 model TEM and photographed with the Olympus Soft Imaging camera system (Ozkan et al., 2018).

| Isolation and characterization of UC-MSCs
On the 10th day of incubation of UC fragments on the plastic Petri dish, spindle-shaped cells were observed around the tissue fragments (Figure 1a,b).Expression levels of MSC-specific CD44, CD90, and non-MSC specific CD34, CD45 cell surface markers were determined as 97.95%, 99.24%, and 0.32%, 5.27%, respectively (Figure 1c-f).
The isolated cells' adipogenic and osteogenic differentiation potentials were determined by staining lipid droplets and extracellular calcium deposits with Oil Red O and Alizarin Red S, respectively (Figure 1g,h).

| Immunocytochemical analysis of HIF-1α and Beclin-1 expressions
Immunocytochemical analysis revealed that the expression level of HIF-1α has a positive correlation with increasing doses of DMOG.
The highest HIF-1α was determined for the MSCs preconditioned with 1000 μM DMOG for 24 h (Figure 2).In the following, the expression levels of Beclin-1 in the MSCs incubated with/out 1000 μM DMOG were evaluated.Semi-quantitative immunocytochemical evaluations showed that cytoplasmic Beclin-1 expression was significantly increased in the MSCs preconditioned with DMOG (Figure 3).

| Content analysis of CMs
Total protein concentration of N-CM and DMOG-CM were determined as 93.91 μg/mL and 56.71 μg/mL by BCA analysis (Figure 4a).
On the other hand, the concentrations of growth factors, including VEGF (1394.4pg/mL vs. 36.3pg/mL) and NGF (167.1 pg/mL vs. 79.1 pg/mL), and anti-inflammatory IL-4 (9.9 vs. 5.8 pg/mL) cytokine in the content of DMOG-CM were increased compared to ones of N-CM.In both types of CMs, GDNF, BDNF, IL-10, IL-17, and IFNγ were not detected (Figure 4b).

| Characterization of exosomes
Exosomes isolated from both types of CMs have CD63 expressions and band intensity of N-CM was higher than the ones of DMOG-CM but the difference was not significant (Figure 5a).Exosomes from both types of CM were demonstrated by TEM.It was observed that they were similar in size but their morphologies had slight variations.The exosomes obtained from DMOG-CM had uniform round shape while the ones of N-CM had multiform morphologies including cup, rhombus and round shapes (Figure 5b,c).

| Assessment of effects of conditioned mediums on wound healing
Both CMs' effects on wound healing were evaluated with in vitro scratch assay.Levels of closures of scratched areas were increased in both types of CM applications compared to one of only DMEM applied control group.However, the increases were not statistically significant for both types of CMs (Figure 6).

| Ultrastructural evaluation of UC-MSCs
According to the ultrastructural evaluations, both groups of MSCs have euchromatic nuclei with the distinctive nucleolus and heterochromatin material noted just beneath the nuclear membrane.MSCs without preconditioning have endocytic vesicles, enlarged rough endoplasmic reticulum (RER), abundant mitochondria and autolysosomal structures and a few multivesicular bodies (MVBs) (Figure 7a).At the same time, it was observed that there was a higher amount of MVB, autophagosomes (AP), autolysosomes (AL), and myeline figures (MF) than the ones noted for MSCs, which were not preconditioned (Figure 7b).This observation was supported by counting the number of structures out of 10 cells (as control MSC vs. DMOG-MSC; 0.2 vs. 0.5 MVB per cell; 2.5 vs. 3.9 AL per cell; 0.2 vs. 2.0 AP per cell; 0.3 vs. 0.7 MF per cell).In addition, the sizes of intraluminal vesicles in MVBs were compatible with the sizes of exosomes noted in the literature (Figure 7a4,b7).Double membraned phagophore structures (Figure 7b3) observed during the vesicle nucleation stage of autophagosome formation and characterized by cup-shaped appearance were also noted (Figure 7b).

| DISCUSSION
Here, we demonstrated the changes in the secretion profiles and ultrastructural properties of UC-MSCs preconditioned with the hypoxia-inducing agent DMOG.Although MSCs have been prominent with their differentiation abilities since they were discovered, it has been recently revealed that they derive their therapeutic features from the factors they secrete (Kay et al., 2017;Pawitan, 2014).For MSCs to be used as effective therapeutics, it is necessary to explain their secretome properties and regulate these secretome.This regulation, which will increase the antiapoptotic, cytoprotective, immunomodulatory, and immunosuppressor properties of MSCs, could be done by preconditioning MSCs.Therefore, this study evaluated the changes in MSCs and secretory profiles using the hypoxia-inducing agent DMOG.
Preconditioning MSCs through hypoxia, chemical agents, trophic factors, cytokines, and physical factors is crucial to improving MSC function in vitro and in vivo (Ferreira et al., 2018;Isildar et al., 2022).
It is known that the improvement in the biological properties of MSCs preconditioning with hypoxia is mainly induced by the HIF complex, which is the principal transcription factor of the hypoxia response in the organism (Liang et al., 2019).The HIF-1α subunit, a functional subunit regulated by hypoxia, translocates into the nucleus under hypoxic conditions, where HIF complexes with other components initiate the transcription of HIF target genes (Zhou et al., 2020).HIF-1α is rapidly observed that 1000 μM DMOG for 24 h resulted in the highest expression of HIF-1α.Furthermore, there are studies indicating that 1000 μM of DMOG protects bone marrow-derived MSCs from apoptotic cell death in a dose-dependent manner (Liu et al., 2014).Similarly, another study showed that 1000 μM of DMOG enhanced the expression of survival and angiogenic factors in bone marrow-derived MSCs following 24 h of preconditioning (Liu et al., 2014).In light of these studies and our findings regarding HIF-1α expressions and morphological observation, 1000 μM of DMOG was decided as the optimal dose for preconditioning.Total protein analysis showed that DMOG-preconditioned CM (DMOG-CM) has a lower protein concentration than unpreconditioned CM (N-CM).However, in DMOG-CM, VEGF, NGF, and IL-4 were in higher amounts.In particular, the increase in VEGF, which is known for its angiogenic and antiapoptotic effects (Zhou et al., 2020), shows that DMOG can be used as an agent to increase the therapeutic power of MSCs by acting on HIF-1α.On the other hand, GDNF and BDNF growth factors and IL-10, IL-17, and IFN-γ cytokines could not be detected in the CM.Since IL-17 and IFN-γ were evaluated as proinflammatory cytokines according to the general cytokine paradigm, their absence in the CM was evaluated positively.Due to the current limited understanding of the secretome content of MSCs and the factors that regulate their secretion, it is crucial to reveal that other factors cannot be measurably detected in current experimental settings.Research investigating the secretome contents of MSCs has produced diverse and sometimes contradictory findings regarding the cytokine-secreting abilities of these cells.For instance, while there are studies in the literature showing that these cells secrete IL4 and IL10 (Blaber et al., 2012;Zagoura et al., 2012), there are also studies in which the secretion of these cytokines remains undetectable in the absence of specific stimuli or triggers (Kiselevskii et al., 2021;Kyurkchiev, 2014) Although it is thought that the immunomodulatory properties of MSCs are intrinsic, it has been shown in recent studies that these properties emerge in response to the presence of inflammation, hypoxia, or extracellular matrix (Su et al., 2023).As a result of these recent findings, it is now understood that MSCs do not actively secrete cytokines, chemokines, and other immunomodulatory molecules in their secretion content under normal circumstances, or if they do, it is at very low levels.However, when exposed to certain stimuli, the levels of these factors increase significantly, leading to the emergence of their immunomodulatory effects (Gorgun et al., 2021).As mentioned above, BDNF and GDNF could not be detected in the CMs obtained in this study.However, our previous study demonstrated presence of BDNF and GDNF factors in CMs collected from normal and deferoxamine-preconditioned MSCs.
The probable reason for this occurrence could be attributed to the concentration of CM before conducting the analysis (Ozkan et al., 2022).Considering all these, it becomes clear that a better understanding of the secretome properties of MSCs requires careful evaluation of various parameters.These parameters include investigating the specific stimuli that influence the secretion, acknowledging differences arising from the source of MSCs used, and thoroughly assessing the concentration of the secretome.New studies should address these aspects and further enhance the knowledge.
The wound-healing process is a multifaceted cellular reaction to injury, coordinated by many growth factors and cytokines.These bioactive molecules are released by various cell types under physiological conditions, including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets (Brem & Tomic-Canic, 2007).It is known that these factors are also present in the secretome secreted by MSCs and that MSCs thus increase proliferation and migration and inhibit apoptosis (Vizoso et al., 2017).Since fibroblasts play a crucial role in wound healing (Saheli et al., 2020), an in vitro wound healing model was established using the 3 T3 cell line to test the effect of DMOG-CM and N-CM.After incubation of the 3 T3 cell line with CMs for 24 h, it was observed that DMOG-CM and N-CM accelerated the closure of the stretch in 3T3s.Accordingly, in our study, both MSC-CMs successfully increased the rate of stretch closure by increasing the proliferation and migration of cells in vitro, and no statistical difference was detected between them.Previous studies show that MSC-CM accelerates wound healing both in vitro (Li et al., 2017;Walter et al., 2010) and in vivo (Fridoni et al., 2019;Yuan et al., 2016).In this context, it would be helpful to check with more specific tests whether the preconditioning of MSCs with DMOG is superior to normal MSCs in terms of wound healing.
Exosomes were isolated from DMOG-CM and N-CM, and CD63 was evaluated by Western blot analysis to compare the quantities of exosome secreted by the preconditioned and unpreconditioned MSCs.The results showed the presence of exosome in both CMs and no significant difference between the groups.TEM analysis also showed that both CMs contain cup-shaped exosomes and measured 40-50 nm in diameter.It was determined that both groups' exosomes were similar in size and morphology and compatible with the literature findings (Cizmar & Yuana, 2017;Rikkert et al., 2019).As a result of the investigations, it was decided that the induction of MSCs with DMOG did not cause any difference in exosome amount, morphology, and size.At this stage, it is necessary to emphasize the importance of performing content analysis in exosomes to see whether exosome contents are affected by this induction.
Ultrastructural examination of DMOG-preconditioned (DMOG-MSCs) and unpreconditioned normal MSCs (N-MSCs) revealed that the cells from both groups have a euchromatic nucleus, prominent nucleolar structure, enlarged GER, and abundant mitochondria, consistent with active cell morphology (Isildar et al., 2019;Ozkan et al., 2018).It was noted that DMOG-MSCs were more vacuolized than N-MSCs.Consistent with this result, in a study in which UC-MSCs have been preconditioned with hypoxia mimetic agents deferoxamine and cobalt chloride, it was stated that intracellular vacuole-like structures increased in MSC morphologies compared to the control group, which was associated with the pre-apoptotic morphology of the cells in the related study (Zeng et al., 2011).Additionally, it was observed that autophagosome and autolysosome structures increased in these cells.Autophagy is a dynamic and catabolic process that recycles damaged intracellular structures in lysosomes and supports cellular homeostasis, regeneration, and survival.
Before these intracellular structures are transported to lysosomes, they are surrounded by double-membrane autophagosomes and fuse with lysosomes to form autolysosomes. Autolysosomes are structures surrounded by a single membrane.The outer membrane of autophagosomes fuses with lysosomes, while the inner membrane is destroyed.Lysosomal enzymes break down the cytoplasmic structures exposed by the inner membrane's destruction.Thus, nutrients and energy are provided to the cells (Jakovljevic et al., 2018;Miller & Zachary, 2017).The autophagy mechanism can be affected by hypoxia, nutrient deprivation, and radiation, which are known to be induced by stress factors (Jakovljevic et al., 2018).released into the extracellular space by exocytosis as exosomes (Gruenberg & Van Der Goot, 2006;Xu et al., 2022).As an alternative pathway, MVBs could be transferred to fuse with lysosomes for degradation (Huber & Teis, 2016).Therefore, the increased MVBs observed in DMOG-MSCs indicate that exosome release is triggered in these cells.However, these ultrastructural changes associated with the exosome secretion mechanism have not been revealed as an increase in exosome quantity in the CM.Consistent with observation of increased autolysosomal structures and expression of Beclin-1 in the preconditioned cells, it could be conjectured that significant amount of MVBs are degraded with this activated autophagic pathway.

| CONCLUSION
In conclusion, we demonstrated for the first time that hUC-MSCs could potentiate their therapeutic secretory properties after preconditioning with DMOG, a hypoxia mimetic agent.In addition, new data that have been gained on the secretome profile of MSCs is essential considering the contradictory data in this area.Finally, findings revealing the ultrastructural changes of MSCs preconditioned with DMOG, will contribute to understanding the response mechanisms of the cells to hypoxic stress.These findings suggested that the preconditioning of MSCs with DMOG, a hypoxia mimetic agent, could modify their secretory profile, adapt their ultrastructural morphology accordingly and render their conditioned medium a more potent therapeutic tool.

F
I G U R E 1 Isolation and characterization of mesenchymal stem/stromal cells (MSCs): morphologies of 1st generation of MSCs (a,b).Flow cytometric analysis of CD34, CD45, CD44, and CD90 cell surface markers (c-f).Detection of adipogenic and osteogenic differentiation potential of MSCs with Oil Red O and Alizarin Red S stains respectively (g,h).F I G U R E 2 Immunocytochemical analysis of HIF-1α expression of mesenchymal stem/stromal cells (MSCs) preconditioned with different doses of dimethyloxalylglycine (DMOG) for 24 and 48 h (a-e).Graphical demonstration of HIF-1α expression (f).F I G U R E 3 Immunocytochemical analysis of Beclin-1 expression of mesenchymal stem/stromal cells (MSCs): Unpreconditioned control (a) and preconditioned with dimethyloxalylglycine (DMOG) (b) and semi-quantitative evaluation of Beclin-1 expression (c).F I G U R E 4 Quantification of total protein (a) and growth and cytokine factors (b) concentrations.F I G U R E 5 Analysis of expression levels of CD63 protein in N-conditioned medium (CM) and dimethyloxalylglycine (DMOG)-CM by Western blot (a).Transmission electron microscopic demonstration of morphologies of exosomes isolated from N-CM and dimethyloxalylglycine (DMOG)-CM.Round (arrow), rhombus (diamond), and cup (arrowhead) shaped exosomes.MSCs preconditioned with DMOG have active RER but decreased number of mitochondria (3.1 in DMOG-MSC vs. 10.1 in control MSC).
degraded under normal oxygen conditions by hydroxylation of prolyl hydroxylase (PHD).Proline hydroxylase inhibitors (PHIs) inhibit PHD activity and reduce the degradation of HIF-1α under constant oxygen conditions.PHI stabilizes HIF-1α levels and upregulates the HIF-1 signaling pathway by inhibiting the hydroxylation of HIF-1α(Zhou et al., 2020).DMOG is a PHIs that inhibits prolyl hydroxylase to regulate the stability of HIF-1α under normal oxygen levels by mimicking hypoxia in cells and was used in this study for hypoxic preconditioning of MSCs(Liang et al., 2019).Studies examining the properties of MSCs after preconditioning with DMOG are very limited in the literature.Two studies that analyzed HIF-1α expressions due to preconditioning bone marrow MSCs with DMOG stated that the optimal DMOG dose was 750 and 1000 μM, respectively(Esmaeilzade et al., 2019;Liang et al., 2019).However, another study showed that 500 μM DMOG could attenuate cell viability of adipose tissuederived MSCs following 24 h of treatment, even reducing cell viability significantly following 3 days long treatment(Abu-Shahba et al., 2020).At this point, MSCs were preconditioned with different doses of DMOG to determine at which dose DMOG would be most effective.Based on the dose trial experiments in our study, it was F I G U R E 6 Assessment of effects of conditioned mediums on wound healing (T 0 -T 24 h).Control (a), N-conditioned medium (CM) (b), and dimethyloxalylglycine (DMOG)-CM (c) groups.Graphical representation of covered distances (d).
The ultrastructural findings in this study indicate that the increased number of autophagosomes and autolysosomes observed in MSCs preconditioned with DMOG activate their autophagic mechanisms in response to the stress to which the cells are exposed.These ultrastructural findings were also supported by immunocytochemical analysis of autophagyrelated Beclin-1 protein, which increased cytoplasmic expression in the MSCs treated with DMOG.Beclin-1 protein is one of the important proteins of the autophagy signaling pathway and is needed for the processes including the nucleation of the phagophore and maturation of the autolysosome(Liu et al., 2013).Another important finding in the ultrastructural analyses is the myelin figures seen in the cells preconditioned with DMOG.Myelin figures are the lamellar structures formed by damaged membranes in the cell(Miller & Zachary, 2017) and no study has been found in the literature on myelin figure formation in UC-MSCs after hypoxic exposure.Finally, increased number of microvesicular bodies (MVB) were observed in the cells preconditioned with DMOG.These vesicles were also present in N-MSCs as they are active cells.It was noted that intraluminal vesicles inside the MVBs were compatible with exosomes.Exosomes are a class of secreted membrane vesicles originating from endosomes.To produce endosomes, first, the cell membrane is internalized.Then many small vesicles are formed inside the endosome by invaginating parts of the endosome membranes, called MVBs.Finally, MVBs fuse with the cell membrane, and their intraluminal endosomal vesicles are