Umbilical cord and cord blood were obtained with the approval of the Medical Ethics Committee of Shanxi Medical University and the consent of the donors, and experiments were performed in accordance with the Declaration of Helsinki.

Umbilical cord tissue was provided by one hospital, and the donors gave informed consent. After obtaining the umbilical cords, two veins and one artery were removed, and Wharton’s jelly was cut into pieces and placed into a 10 cm² petri dish (Nice) with 5 mL of DMEM/F12 culture medium (Thermo Fisher Scientific, Waltham, MA, United States) containing 2.5% serum substitute (Shanxi Yinshi Cell Technology, Xian, China). Approximately 14 d later, the UC-MSCs were passaged, and P4 generation cells were used for experiments. UC-MSCs were cultured in a carbon dioxide incubator (Thermo Fisher Scientific) at standard oxygen tension with 5% carbon dioxide, 95% air, and 37 °C (i.e. normoxia). When UC-MSCs were 70%-80% confluent, a mixture of IFN-γ (R&D), TNF-α (R&D Systems, Minneapolis, MN, United States) and IL-1β (PeproTech, Cranbury, NJ, United States) was added to the medium. The cells were then immediately placed into a three-gas incubator (Panasonic, Osaka, Japan) with 2% O₂, 5% CO₂, and 93% N₂ at 37 °C (i.e. hypoxia). After 24 h, primed UC-MSCs (PUC-MSCs) were obtained. Therefore, our experiments were divided into two groups: Control (UC-MSCs) with no treatment and an experimental group (PUC-MSCs) in which cells were exposed to hypoxia and inflammatory factor supplementation.

Umbilical cord blood was obtained and centrifuged at 700 × g for 10 min and the plasma was extracted and placed into a water bath at 56 °C for 30 min for heat inactivation. The plasma was removed from the water bath, centrifuged at 850 × g for 10 min, and placed in a refrigerator at 4 °C for later use. The blood cells were resuspended in phosphate buffered saline (PBS) and mixed. The cell suspension was slowly added into a centrifuge tube containing Ficoll human peripheral blood lymphocyte isolation medium (Tianjin Haoyang Biological Products Technology Co., Ltd., Tianjin, China). After centrifugation at 400 × g for 30 min, white cells were extracted and washed twice with PBS. The cells [(1-2) × 10⁶ cells/mL] were inoculated in peripheral blood mononuclear cell (PBMC) medium containing 10% of the heat-inactivated autologous plasma (Shanxi Yin Cell Technology Co., Ltd., Hebei, China) in the presence or absence of UC-MSCs or PUC-MSCs. The PBMCs were treated with 100 U/mL IL-2 (PeproTech). For coculture experiments, PBMCs were inoculated with UC-MSCs or PUC-MSCs at a PBMC/MSC ratio of 3:1 so that the cells were in direct contact, which is the appropriate proportion of cells to obtain MSC-mediated inhibition of PBMC proliferation.

Umbilical cord blood was obtained and centrifuged at 700 × g for 10 min, and the plasma was placed into a water bath at 56 °C for 30 min for heat inactivation. The plasma was removed, centrifuged at 850 × g for 10 min, and placed into a refrigerator at 4 °C for later use. The blood cells were resuspended in PBS, mixed, and incubated at room temperature for 20 min with RosetteSepT NK Enrichment Cocktail. The cell suspension was slowly added into a centrifuge tube containing Ficoll human peripheral blood lymphocyte isolation medium (Tianjin Haoyang Biological Products Technology Co., Ltd.). After centrifugation at 400 × g for 30 min, white cells were extracted and washed twice with PBS. The cells [(1-2) × 10⁶ cells/mL] were inoculated into NK medium containing 10% heat-inactivated autologous plasma (Shanxi Yin Cell Technology Co., Ltd.). NK cells were cultured with or without UC-MSCs or PUC-MSCs and treated with 100 U/mL IL-2 (PeproTech).

Double fluorescence acridine orange/propidium iodide (AO/PI) cell viability counting was performed. The AO/PI reagent consists of the DNA-binding dye AO, which fluoresces green, and the DNA-binding dye PI, which fluoresces red. AO can pass through a complete cell membrane and enter the nuclei of all cells (living and dead cells), emitting green fluorescence. PI can only pass through an incomplete cell membrane and enter the nuclei of dead cells, resulting in red fluorescence. After mixing 10 μL of cell suspension with 10 μL of AOPI reagent, the viability and size of the cells in suspension were measured by a Countstar Rigel S2 (Shanghai Rui Yu Biotech, Shanghai, China).

Flow cytometry analysis of UC-MSCs and PUC-MSCs was performed. The cells were resuspended in staining buffer after centrifugation, and the corresponding antibodies were added to the buffer. Cells were incubated for 30 min, neutralized and washed 1-2 times. The UC-MSC or PUC-MSC antigen markers (BD Biosciences, San Jose, CA, United States) were CD105-APC, CD90-FITC, CD73-PE, CD142-PC5.5, CD29-PE, CD34-PE, CD14-PE, CD45-APC, human leukocyte antigen (HLA)-ABC-APC, CD47-APC, CD166-PE, CD44-PE, and CD31-APC.

Mitochondrial reactive oxygen species (ROS) production was measured by flow cytometry (Beckman Coulter Life Sciences, Brea, CA, United States) using a ROS detection kit (Biyuntian, Shanghai, China). 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted in serum-free medium at 1:1000 to a final concentration of 10 μmol/L. After collection, 1 million to 20 million/mL cells were suspended in diluted DCFH-DA and incubated in an infrared carbon dioxide incubator (Thermo Fisher Scientific) at 37 °C for 20 min. The cells were turned over and mixed every 3-5 min to ensure that the probe was in full contact with the cells. The cells were washed with serum-free cell culture solution three times to fully remove excess DCFH-DA. Finally, the results were detected by flow cytometry.

Mitochondrial membrane potential (MMP) was detected using MMP detection reagent JC-1 (Beyotime, Jiangsu, China). The culture medium was removed and the cells were washed twice with PBS. JC-1 staining solution (1 mL) was added, mixed thoroughly, and incubated with cells in an infrared carbon dioxide incubator (Thermo Fisher Scientific) at 37 °C for 20 min. During the incubation period, JC-1 staining buffer (1×) was prepared by adding approximately 4 mL distilled water to approximately 1 mL JC-1 staining buffer (5×), and the buffer was placed in an ice bath. After incubation at 37 °C, the supernatant was removed, and the cells were washed twice with JC-1 staining buffer (1×). Finally, 2 mL of the cell culture solution was added. Images were obtained with an inverted fluorescence microscope (CKX53; Olympus, Tokyo, Japan).

TRIzol (Invitrogen, Waltham, MA, United States) was used to lyse the cells, total RNA was extracted, and an ultratrace nucleic acid protein detector (Shanghai Jiapeng Materials, Shanghai, China) was used to determine RNA concentration. mRNA expression levels were quantitatively analyzed on a real-time polymerase chain reaction (RT-PCR) instrument (Bio-Rad, Hercules, CA, United States) using a One Step TB Green PrimeScript PLUS RT‒PCR Kit (Takara Bio Inc., Shiga, Japan) to measure the levels of the following transcripts: Catalase, Istanniocalcin-1 (STC1), hemeoxygenase-1 (HOMX1), B-cell lymphoma 2-associated protein (Bax), B-cell lymphoma 2 (Bcl2), silencing information regulator 2-related enzyme 1 (SIRT1), P53, P16, P21, PGE2, kynurenine (KYN), IDO, IL-1 receptor antagonist (IL-1ra), cyclooxygenaese-2 (COX2), IL-10, TGF-β1, HLA-G5, TSG-6, ligands for programmed cell death 1 (PD-L1). All primer sequences for qRT-PCR are listed in Table 1.

UC-MSCs and PUC-MSCs supernatants were collected and centrifuged at 210 × g for 5 min, and the suspended cells were removed and stored at -80 °C. The protein levels of PGE2, TSG-6, IDO, IL-10 and TGF-β1 secreted by UC-MSCs and PUC-MSCs were measured by enzyme-linked immunosorbent assay (ELISA) kits (Wuhan Eliret Biotechnology Co, Ltd., Wuhan, China). Finally, the optical density (OD) value of each well was measured on the basis of enzyme label (Thermo Fisher Scientific) absorption at 450 nm.

UC-MSC and PUC-MSC suspensions were obtained and washed twice with PBS. Annexin V-FITC apoptosis reagent (Biyuntian) was added and incubated with the cells at room temperature for 10-20 min in the dark, and flow cytometry was performed. Annexin V-FITC emits green fluorescence, and PI emits red fluorescence.

The cell culture medium was removed, the cells were washed once with PBS, and 1 mL/well β-galactosidase (SA-β-gal) staining fixation solution (Solarbio, Beijing, China) was added and incubated at room temperature for 15 min. Fixative was removed, and the cells were washed 3 times with PBS for 3 min each. The PBS was removed, and 1 mL of staining solution was added to each well. The plate was incubated at 37°C overnight and sealed with plastic wrap to prevent evaporation. The positive expression of blue particles was observed under an inverted microscope (CKX53; Olympus), and the number of positive cells per 100 cells was quantified. Each experiment was repeated three times.

NK cells were cultured in the presence or absence of UC-MSCs or PUC-MSCs for 3 d at a 3:1 ratio. The cells were prelabelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (BioLegend, San Diego, CA, United States) and the fluorescence intensity of CFSE was measured by flow cytometry.

UC-MSCs and PUC-MSCs were collected for DNA extraction and DNA concentration was determined by an ultramicro nucleic acid protein detector (Shanghai Jiapeng Materials) to evaluate the cell proliferation rate. PBMCs were cultured in the presence or absence of UC-MSCs or PUC-MSCs at a ratio of 3:1. The number of cells (Countstar Rigel S2; Shanghai Rui Yu Biotech) was counted by a cell imaging analyzer for 5 consecutive days and the growth curve was plotted.

NK cells were cultured alone or in direct contact with MSCs (NK: UC-MSCs or PUC-MSCs = 3:1 ratio) for 72 h. NK cells were then collected and cocultured with CFSE-stained (Biolegend) K562 target cells for 4 h at the effective target ratio of 1:5. All cells were collected and washed twice with PBS. K562 cell apoptosis was analyzed by PI staining (Biyuntian), and the fluorescence intensity was measured by flow cytometry.

SPSS 19.0.0 and GraphPad Prism 7 (GraphPad Prism, La Jolla, CA, United States) statistical software were used for data processing. All experimental data are expressed as the mean ± SD. A t test was performed to compare two datasets, and P < 0.05 was considered statistically significant.

We evaluated the effects of hypoxia and inflammatory factor pretreatment on UC-MSC morphology, vitality and size. The morphological differences between UC-MSCs and PUC-MSCs were compared under a microscope when cell confluency reached 80% to 90%. Untreated UC-MSCs appeared as either short rods or long spindles in an adherent state and were arranged in a spiral pattern. After 24 h of exposure to hypoxia and inflammatory factors, cell morphology changed from short and rod-like to thin and elongated (Figure 1A), but the proliferation rate did not change (Figure 1B). We further evaluated the effects of pretreatment on UC-MSC viability and size using AOPI staining and a cell imaging analyzer. UC-MSC viability (Figure 1C) in suspension was 93.59% ± 3.87% (n = 5) and the mean diameter (Figure 1D) was 18.216 ± 0.78 μm (n = 5), while PUC-MSC viability (Figure 1C) was 92.89% ± 4.13% (n = 5) and the mean diameter (Figure 1D) was 18.628 ± 0.76 μm (n = 5). These results showed that pretreated MSCs became elongated, the measured viability slightly decreased (P = 0.84), and the cells were slightly enlarged (P = 0.47), but these differences were not statistically significant.

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Figure 1 Morphology, viability, proliferation, and size of mesenchymal stem cells. A: Representative micrographs showing umbilical cord mesenchymal stem cells (UC-MSCs) and primed umbilical cord mesenchymal stem cells (PUC-MSCs). Microscopy observation of cell morphology revealed that UC-MSCs were elongated after hypoxia and inflammatory factor pretreatment (Bar = 200 μm); B: The DNA concentration was measured by an ultramicro nucleic acid protein detector. The DNA concentration of PUC-MSCs was not significantly different from that of UC-MSCs, and pretreatment had no effect on their proliferation; C: Cell viability was determined by an acridine orange (AO) propidium iodide (PI) staining cell image analyzer, and no significant difference between UC-MSCs and PUC-MSCs was observed; D: Cell size was determined with a cell imaging analyzer, and the sizes of the UC-MSCs and PUC-MSCs were comparable. ^dP > 0.05.

We further evaluated whether preconditioning altered the cellular phenotype by analyzing MSC surface marker expression by flow cytometry. UC-MSCs (Figure 2A) and PUC-MSCs (Figure 2B) from three donors were positive for CD105, CD90, CD73, CD29, CD166, CD47 and HLA-ABC surface expression and negative for CD31, CD45, CD14 and CD34. The results showed that surface marker expression was consistent between PUC-MSCs and UC-MSCs. Interestingly, CD142 expression (59.3%) (Figure 2B) was significantly lower in PUC-MSCs compared to UC-MSCs (99.6%) (Figure 2A).

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Figure 2 Phenotypes of mesenchymal stem cells were detected by flow cytometry. A: Cell surface markers of umbilical cord mesenchymal stem cells (UC-MSCs); B: Cell surface markers of primed umbilical cord mesenchymal stem cells (PUC-MSCs). The signals from the unstained control cells are shown in blue in the histogram, and the signals from stained cells are shown in red in the histogram. HLA: Human leukocyte antigen.

ROS play key roles in the proapoptotic signaling cascade, and excess ROS attacks the mitochondrial membrane and loss of MMP, leading to MSC apoptosis[44]. Therefore, we used flow cytometry to examine the effect of MSC pretreatment on ROS production. Intracellular ROS levels showed a three-fold increase in DCFH-DA fluorescence intensity in PUC-MSCs compared to untreated UC-MSCs (Figure 3A), but all the effects were within the range of values obtained in positive controls. STC1, catalase and HOMX1 expression levels increased (Figure 3B), indicating that the antioxidant capacity of the cells was increased and that mitochondrial ROS removal was increased, enhancing antioxidant defense effects and preventing ROS-induced DNA damage. Maintaining a stable MMP (Ψm) is essential to ensure efficient ROS clearance and prevent apoptosis or other stress-related events caused by excessive ROS[45]. Therefore, we further determined whether hypoxia and inflammatory factor preconditioning induced MMP dysfunction in UC-MSCs. We used the sensitive fluorescent probe JC-1 to evaluate the effect of pretreatment. We determined whether the membrane potential changed in UC-MSCs by measuring a change in JC-1 red fluorescence to green fluorescence. Microscopically, untreated UC-MSCs showed a normal MMP, as shown by red fluorescence staining with JC-1 (Figure 3C). Neither the red and green fluorescence nor the MMP was significantly different between PUC-MSCs and UC-MSCs. We further measured the JC-1 red/green fluorescence ratio by flow cytometry and confirmed our microscopy results (Figure 3D). These results showed that mitochondrial function in UC-MSCs was not damaged by hypoxia and inflammatory factor pretreatment.

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Figure 3 Mitochondrial function. A: Reactive oxygen species (ROS) levels in umbilical cord mesenchymal stem cells (UC-MSCs) and primed UC-MSCs (PUC-MSCs) were analyzed by flow cytometry. The fluorescence intensity of 2,7-dichlorodihydrofluorescein (DCFH-DA) in PUC-MSCs increased by 3-fold compared to UC-MSCs; B: The expression of the antioxidant-related genes catalase, stanniocalcin-1 (STC1), and hemeoxygenase-1 (HOMX1) was increased; C: Representative image showing the mitochondrial membrane potential (MMP) of mesenchymal stem cells stained with JC-1. The red fluorescence of potential-dependent JC-1 aggregation in each group and the green fluorescence of the JC-1 monomer in the cytoplasm after depolarization of the mitochondrial membrane were observed. Fluorescence microscopy showed no significant difference in red and green fluorescence between UC-MSCs and PU-MSCs; Bar = 200 μm; D: Flow cytometry was used to detect the red and green fluorescence of the JC-1 probe in UC-MSCs and PUC-MSCs. There was no difference in the ratios of red and green fluorescence. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP > 0.05.

Bax expression decreased and Bcl2 and SIRT1 expression increased after pretreatment, suggesting that the anti-apoptotic capacity increased after pretreatment (Figure 4A). The expression of the senescence-related genes P53, P16 and P21 was upregulated by pretreatment (Figure 4B). We analyzed apoptosis by Annexin V-PI staining and flow cytometry. We compared the proportion of cells that were positive for both Annexin V-FITC and PI (necrotic cells), and the apoptotic index of PUC-MSCs (15.08% ± 4.11%) significantly increased (Figure 4C) compared to UC-MSCs (6.22% ± 3.03%). Next, we investigated the effect of preconditioning on cell senescence. SA-β-gal has been the most widely accepted senescence marker since Dimri first published its use in 1995[46], so we performed SA-β-gal staining to evaluate cell senescence ratios. SA-β-gal levels increased in PUC-MSCs (14.83% ± 1.57%) compared to UC-MSCs (4.83% ± 1.34%) (Figure 4D). These results suggest that hypoxia and inflammatory factor preconditioning not only induces UC-MSC apoptosis and senescence but also increases anti-apoptotic factor levels.

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Figure 4 Effects of pretreatment on apoptosis and senescence in mesenchymal stem cells. A: Apoptosis-related gene analysis showed that the expression of B-cell lymphoma 2 (BCL-2)-associated protein (Bax) was decreased and the expression of Bcl2 and silencing information regulator 2-related enzyme 1 (SIRT1) was increased after hypoxia and inflammatory factor pretreatment; B: Expression of P53, P16 and P21 was upregulated after hypoxia and inflammatory factor pretreatment; C: Umbilical cord mesenchymal stem cells (UC-MSCs) and primed UC-MSCs (PUC-MSCs) were stained with Annexin V-FITC apoptosis assay kit reagents and apoptotic cells were detected by flow cytometry. The results showed that the apoptosis index of PUC-MSCs increased; D: Representative images of β-galactosidase (SA-β-gal) staining and quantitative analysis of positive SA-β-gal staining. Compared with that of the UC-MSCs, the number of SA-β-gal-positive PUC-MSCs was significantly increased; Bar = 100 μm. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

MSCs are pluripotent stem cells that have been shown to hold promise in tissue regeneration due to their ability to self-renew and differentiate and their broad immunomodulatory properties[1-5]. We evaluated whether UC-MSC immunomodulatory activities increased after pretreatment. qRT-PCR analysis revealed that the expression of PGE2, IDO, KYN, COX2, IL-10, TGF-β1, TSG-6, HLA-G5, and PD-L1 increased in PUC-MSCs compared to untreated UC-MSCs. IL-1ra expression was slightly elevated, but the difference was not statistically significant (Figure 5A). Because paracrine activity is a key mechanism underlying MSC effects, we compared the bioactive factor secretion levels in PUC-MSCs and untreated UC-MSCs. We assessed MSC paracrine function by measuring the protein levels of immunosuppression-related soluble factors released into the culture supernatant, including IDO, PGE2, TGF-β1, TSG-6, and IL-10 by ELISAs. IDO, PGE2, TGF-β1 and TSG-6 levels significantly increased in the PUC-MSC supernatant, while IL-10 levels slightly increased but did not reach statistical significance (Figure 5B). In summary, these data strongly suggest that UC-MSC preconditioning with hypoxia and inflammatory factors upregulates the expression of soluble immunomodulatory factors and enhances their immunomodulatory activity.

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Figure 5 Relative expression of immunomodulatory genes and proteins in umbilical cord mesenchymal stem cells after pretreatment. A: Gene expression levels of umbilical cord mesenchymal stem cells (UC-MSCs) and primed UC-MSCs (PUC-MSCs) were analyzed by quantitative real-time polymerase chain reaction. Expression levels of prostaglandin E2 (PGE2), kynurenine, idoleamine-2,3-dioxygenase (IDO), cyclooxygenaese-2 (COX2), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-β1), human leukocyte antigen-G5 (HLA-G5), tumor necrosis factor-α-induced protein-6 (TSG-6) and ligands for programmed cell death 1 (PD-L1) were all increased after hypoxia and inflammatory factor preconditioning. IL-1 receptor antagonist (IL-1ra) was not significantly different between the two groups; B: Levels of immunoregulatory proteins in UC-MSCs and PUC-MSCs were detected by enzyme-linked immunosorbent assay. After hypoxia and inflammatory factor pretreatment, the protein levels of IDO, PGE2, TGF-β1 and TSG-6 were increased, while the expression of IL-10 was not significantly different, as determined via three independent experiments. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP > 0.05.

Considering the high expression of immunomodulatory molecules in PUC-MSCs, we evaluated their immunosuppressive capacity. We compared UC-MSC and PUC-MSC immunosuppressive capacity using PBMC and NK cell proliferation assays by analyzing growth dynamics of PBMCs cultured alone, with UC-MSCs or with PUC-MSCs (Figure 6A). Both UC-MSCs and PUC-MSCs significantly inhibited PBMC proliferation compared to PBMCs cultured alone. However, PUC-MSCs had a stronger inhibitory effect on PBMC proliferation, further indicating that pretreatment enhanced UC-MSC immunosuppressive abilities. We further investigated MSC-mediated inhibition of NK cell proliferation in the presence of the two MSC populations. We cultured CFSE-stained NK cells in the presence of IL-2 alone or with UC-MSCs or PUC-MSCs and analyzed CFSE fluorescence intensity by flow cytometry after 3 d. The proliferation rate of NK cells was reduced in cocultures with UC-MSCs or PUC-MSCs compared to NK cells cultured alone, but the PUC-MSC group exerted stronger inhibitory effects on NK cells compared to UC-MSCs (Figure 6B).

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Figure 6 Detection of the immunomodulatory function of umbilical cord mesenchymal stem cells after preconditioning. A: Peripheral blood mononuclear cells (PBMCs) alone or directly exposed to umbilical cord mesenchymal stem cells (UC-MSCs/primed UC-MSCs) for 5 d in a 3:1 ratio. The number of PBMCs in three groups was measured daily and the growth curve was plotted; B: Carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained natural killer (NK) cells were cultured alone or cocultured with UC-MSCs/PUC-MSCs at a 3:1 ratio for 72 h. NK cells were harvested, and the fluorescence intensity of CFSE was measured by flow cytometry to measure the proliferation rate of NK cells; C: NK cells were collected and incubated with CFSE-stained K562 target cells for 4 h at an effector ratio (E:T = 1:5). Effector cell (NK cell)-mediated cytotoxicity of K562 cells was analyzed by flow cytometry. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP > 0.05.

Finally, we evaluated the effect of UC-MSC pretreatment with hypoxia exposure and inflammatory factor treatment on NK cell cytotoxic activity. NK cells were cultured alone or with UC-MSCs/PUC-MSCs at a 3:1 ratio for 72 h, then collected and incubated with CFSE-stained K562 target cells at an effective target ratio (E:T = 1:5) for 4 h. The cells were collected, and K562 cell apoptosis was analyzed by PI staining. The results showed that NK cells cocultured with UC-MSCs or PUC-MSCs had lower cytolytic activity, and the inhibition of NK cell-mediated cytotoxic activity was higher with PUC-MSCs than UC-MSCs (Figure 6C).

Mesenchymal stem cells (MSCs) have great potential in the treatment of a variety of immune-related diseases due to their unique immunomodulatory and anti-inflammatory abilities. However, after intravenous transplantation, MSCs cannot effectively exert their biological effects when they encounter a harsh environment in vivo, which reduces the efficacy of cell therapy. To increase transplantation efficacy, appropriate pretreatment methods are particularly important.

Although a variety of pretreatment methods are used to increase MSC transplantation efficacy, suitable and effective in vitro pretreatment methods are still worth studying.

To evaluate whether umbilical cord MSCs (UC-MSCs) pretreated with hypoxia exposure and inflammatory factors show enhanced immunosuppressive effects without affecting cell biological characteristics.

In this study, we used a combination of hypoxia (2% O₂) and inflammatory factors (interleukin-1β, tumor necrosis factor-α, interferon-γ) to pretreat UC-MSCs for 24 h to simulate the in vivo injury environment. We then comprehensively evaluated the biological properties of pretreated UC-MSCs and investigated their immunosuppressive properties.

Our results showed that compared to UC-MSCs, pretreated UC-MSCs were morphologically elongated, but their viability, proliferation and size were not affected, the expression of coagulation-related tissue factors was significantly reduced, and mitochondria maintained their function and integrity. Although some cells underwent apoptosis or senescence, polymerase chain reactions and enzyme-linked immunosorbent assays revealed a significant increase in the levels of immunomodulation-related factors. Coculture with peripheral blood mononuclear cell and natural killer cells exerted a stronger immunosuppressive effect.

The combined pretreatment of hypoxia exposure and inflammatory factors enhanced the immunosuppressive ability of MSCs but did not affect the biological characteristics of these cells.

Our study provides new strategies for the preconditioning of UC-MSCs.