14 June 2023: Clinical Research
Influence of Umbilical Cord Blood Biochemical Parameters and Disease Condition on the Expression of the Gene in Umbilical Mesenchymal Stem Cells
Aleksandra Ozygała1ABDEFG, Gabriela Wojciechowska1BDEF, Kinga Traczyk1BDEF, Dominika Przywara23ABDEF, Alicja Petniak 2BDEF, Rafał Szymanowski2BE, Agnieszka Wilińska2BE, Arkadiusz Krzyżanowski4BF, Anna Kwaśniewska4BF, Bartosz J. Płachno5EF, Paulina Gil-Kulik 6ABCDEF*, Janusz Kocki2ADEGDOI: 10.12659/MSM.939716
Med Sci Monit 2023; 29:e939716
Abstract
BACKGROUND: Mesenchymal stem cells (MSCs) are capable of secreting different substances, including the anti-inflammatory protein TSG-6, which can be useful in the treatment of diseases with inflammatory reactions. The main aim of this study was to evaluate the expression of the TSG-6 gene in MSCs derived from the umbilical cord. For better understanding of the anti-inflammatory properties of MSCs, we additionally assessed the expression of some interleukins (ILs).
MATERIAL AND METHODS: The study group included 45 patients after delivery, aged from 21 to 46 years; the average patient age was 33 years. MSCs were isolated enzymatically from umbilical cord Wharton’s jelly, in vitro cultured, and characterized using flow cytometry; qPCR was performed to assess expression of the studied genes. The expression of genes of a number of pro-inflammatory ILs in MSCs was investigated in relation to the health of patients (coexistence of hypertension), the level of leukocytes, pCO2, and hemoglobin in the blood.
RESULTS: Our research showed that the expression of the TSG-6 gene in MSCs depends on coexisting diseases in the patient and the biochemical parameters of umbilical cord blood, including the important role of cord blood pH. We found that the levels of IL2 and IL6 expression were correlated with pCO2, and IL6 expression were correlated with pO2.
CONCLUSIONS: Our study suggests that maternal health status and cord blood biochemical parame-ters could affect the anti-inflammatory properties of MSCs; however, this needs to be confirmed in a future study.
Keywords: Gene Expression, Mesenchymal Stem Cells, MSC 127, TNFAIP6 Protein, Human, Humans, Adult, Fetal Blood, Interleukin-6, Umbilical Cord, Biological Transport, Family
Background
Mesenchymal stem cells (MSCs) are multipotential cells. According to the International Society for Cell and Gene Therapy, the correct name is “multipotent mesenchymal stromal cells”. The term “mesenchymal cells” is synonymous with embryonic connective tissue, which, as the name suggests, only occur in the dividing embryo [1]. MSCs are derived from the third germ layer, the mesoderm, which makes the production of the cells that ultimately make up the connective tissue in the body possible [1].
Clinically, human MSCs are derived from adult bone marrow, adipose tissue, dental pulp, and peripheral blood. The MSCs extracted from perinatal tissues (such as the placenta, umbilical cord, or amnion) in newborns have characteristics that make them superior to cells obtained from adults. These characteristics include proliferative capacity, capability for differentiation, cell longevity (and growth), and a reduced capacity to provoke an immune response (lower immunogenicity) [2,3].
The features that are particularly related to MSCs are a high differentiation potential, especially toward bone tissue, cartilage tissue, and adipose tissue, and the ability of the MSCs to renew themselves. Visually, they are fusiform in shape and resemble fibroblasts. In vitro they show adhesion to plastic, and in regards to human phenotype, they also possess human surface antigens, which are also MSC markers: CD73, CD90, and CD105. Negative MSC markers, which do not show expression in these cells, are CD11b, CD14, CD19, CD34, CD45, and CD79a. It is significant that there are no class II histocompatibility antigens on the MSC surface [1,4,5].
Apart from differentiation potential, MSCs have many other properties. MSCs are capable of secreting substances such as growth factors, angiogenesis stimulation factors, and anti-apoptotic factors [6]. Moreover, MSCs show anti-inflammatory effects, which are mainly caused by the tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) protein secreted by these cells [7].
TSG-6 is a multifunctional protein with anti-inflammatory and tissue protective properties that mediate the beneficial effects of MSCs. In recent years, the potential of TSG-6 as a therapeutic agent has been emphasized in a wide range of indications [8].
Expression of the
It was observed that TSG-6 can be used in the therapy of inflammatory diseases. It was shown that TSG-6 has a positive effect on neural system inflammation [13], reduces swelling in arthritis [14], and also limits the size of a myocardial infarction [7]. Moreover, the latest studies report the possible use of TSG-6 in the diagnosis of acute ischemic stroke [15] and rheumatoid arthritis [16]. It has been shown that the use of MSCs to induce neuroprotection and the lasting alleviation of neuropathic pain is dependent on the secretion of TSG-6 by these cells [13]. Additionally, Li et al indicated the potential use of MSCs in clinical recurrent miscarriages is possible due to the TSG-6-dependent mechanisms [17].
Because numerous studies have shown that the immunomodulatory properties of MSCs are dependent on TSG-6, we speculated that the level of
The anti-inflammatory activity of MSCs mediated by the TSG-6 protein has already been proven, but no studies indicate which sources of MSCs are the most optimal in this regard. There is no information of laboratory parameters affecting expression of that gene. To the best of our knowledge, the present study is the first to evaluate the level of
Our study will provide new information about TSG-6, and therefore about the anti-inflammatory properties of MSCs, what may be crucial for further use of MSCs and TSG-6 in therapy.
Material and Methods
SAMPLE COLLECTION AND ETHICS APPROVAL:
The material used for the research was the umbilical cord, which was collected shortly after delivery from 45 patients hospitalized at the Department of Obstetrics and Pathology of Pregnancy of the Independent Public Clinical Hospital No. 1 in Lublin. The research was conducted with the consent of the Bioethics Committee at the Medical University of Lublin (KE-0254/128/2014).
The age of the patients ranged from 21 to 46 years; the average patient age was 33 years. A total of 6 patients (13%) had a vaginal delivery, while 37 patients had a cesarean section. Ten patients (22%) out of 45 patients were treated with oxytocin during labor.
Eleven patients gave birth before 37 weeks of pregnancy, and 22 patients gave birth for the first time (18 of them were pregnant for the first time). The women gave birth to 25 boys and 20 girls. Gestational diabetes was diagnosed in 13 patients, hypertension in 8 patients, and hypothyroidism in 14 patients; 3 patients had diabetes and hypertension at the same time, and 4 patients had diabetes and hypothyroidism at the same time. A total of 17 patients had no comorbidities and had healthy babies born full term (control group). Patient characteristics are shown in Tables 1 and 2.
ISOLATION OF MSCS FROM UMBILICAL CORD:
MSCs were isolated using an enzymatic digestion method. Immediately after delivery, the umbilical cord (10-cm piece) was placed in a culture medium containing Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine (584 mg/L) and glucose (4.5 g/L; Corning, Manassas, VA, USA) with 10% fetal bovine serum (FBS; ATCC, Teddington, UK) and 1% addition of antibiotic (penicillin-streptomycin, 10000 U and 10 mg/mL, respectively; Sigma-Aldrich, Israel). The umbilical cord was then rinsed several times in sterile phosphate buffered saline (PBS; Biomed Lublin, Poland) containing the antibiotic and cut into smaller pieces with a scalpel under aseptic conditions. Fragments of umbilical cord (2–5 mm) were digested in a sterile type I collagenase solution (1 mg/mL; Gibco by Life Technologies, Grand Island, NY, USA) for 2 to 3 h, while maintaining a temperature of 37°C at 300 rpm (Eppendorf Thermomixer comfort). After this, the sample was washed twice with warm PBS buffer (21°C, 128 g; Eppendorf Centrifuge 5810 R). The last step was to filter the sample after it was rinsed through a 100-μm sieve to clean the mixture for further testing. After the cells were filtered, they were rinsed in warm PBS with 1% antibiotic, the collagenase was stopped by adding FBS, the sample was centrifuged with warm PBS buffer (21°C, 128 g, 10 min Eppendorf Centrifuge 5810R), and the cell pellet was transferred to a culture vessel. The procedure of cell isolation and cell culture was done as described in a previous study [18].
CELL CULTURE:
The isolated umbilical cord cells were cultured in vitro for up to 14 days in adherent conditions in a tissue incubator (New Brunswick Galaxy 170 R). Incubation conditions were as follows: temperature 37°C, O2 concentration 15%, CO2 concentration 5%, and moisture 95%. The culture medium (10 mL) consisted of DMEM medium with L-glutamine and glucose, with 10% FBS and a 1% addition of antibiotic (penicillin-streptomycin). The bottle for cell culture had bottom surface area of 25 cm2 (TC Flask T 25, Cell+; Sarsted, Germany). The cultures were observed every few days, and the medium was changed every 3 days. After successful in vitro culture, the isolated MSCs were detached from the surface of the culture vessel using a sterile cell scraper (Corning). Finally, these cells were washed in PBS buffer. Cells after culture were divided and some were used for cytometric analysis, some for RNA isolation.
CYTOMETRIC ANALYSIS:
Cytometric analysis was performed using a Navios flow cytometer (Beckman Coulter) to assess cell phenotype. The analysis was performed according to protocols presented in previous studies [19–21]. In vitro cultured umbilical cord MSCs, at a concentration of about 106/mL, were taken and suspended in a volume of 100 μL and added to a DuraClone SC Mesenchymal Tube antibodies panel (Beckman Coulter, Bangalore, Karnataka, India), then shaken for 6 s. Subsequently, the cells were incubated in the dark for 15 min at room temperature. The following DuraClone MSCs containing a lyophilisate set of fluorescently labeled monoclonal antibodies against MSC surface antigens were used: CD45-APC, CD73-PE, CD90-FITC, CD105-PC7, CD146-PC 5.5, CD31-PBE, CD14/CD19-Krome Orange, dedicated to the determination of characteristic MSC antigens. After incubation, 2 mL of PBS without calcium and magnesium ions (Biomed, Lublin, Poland) was added. Then, they were centrifuged at 200 g for 5 min. After centrifugation, the cell pellet was dissolved in 400 μL PBS without calcium and magnesium ions. The labeled cells were placed on a Navios flow cytometer carousel and subjected to cytometric analysis. Typically, an analysis of 10 000 events was recorded for the assays. Cells without any staining were used as a negative control, and an isotype control was performed. Single-stained samples were used for compensation. Fluorescence minus one (FMO) samples were used to determine the gating.
RNA ISOLATION:
Total cellular RNA of the obtained umbilical cord MSCs was isolated by the modified method of Chomczyński and Sacchi [22], using TRIzol (Thermo Fisher Scientific, USA), chloroform (Sigma), isopropanol (Sigma), and 75% ethanol (Poch).
After culture, the cell sample was homogenized in 0.5 mL of TRIzol, then incubated for 5 min at room temperature; 0.1 mL of chloroform was successively added, and the sample was shaken for 15 s, incubated for 15 min at room temperature, then centrifuged for 15 min at 4°C, at a speed of 12000 g (Eppendorf Centrifuge 5415 R). After centrifugation, the aqueous phase was withdrawn into a new tube. An amount of 0.25 mL of isopropanol was added to the aqueous phase, incubated for 20 min at room temperature, and then centrifuged for 20 min at 4°C, at 12 000 g (Eppendorf Centrifuge 5415 R). After centrifugation, the supernatant was discarded and the RNA pellet was purified in 75% ethanol, then dissolved in ultrapure water, free of RNase and DNase. Before the next stages of research with the use of isolated RNA, qualitative and quantitative analysis of these isolates was performed using the spectrophotometric method (Nanodrop 2000 c) at wavelengths 260 nm and 280 nm.
REVERSE TRANSCRIPTION:
The cDNA synthesis was performed using the High-Capacity cDNA Transcription Kit from Applied Biosystems (Thermo Fisher Scientific, Lithuania) and 1 μg of test RNA isolated in the previous step, according to the manufacturer’s protocol.
The reverse transcription reaction was carried out in a volume of 20 μL, consisting of: 1 μL (RNase 40 U/μL); 1 μL (reverse transcriptase 50 U/μL); 2 μL (10xRT Buffer); 3.2 μL (Ultrapure water); 0.8 μL (10 xdNTPs (100 mM); 2 μL (10 xRT Random Primer); and 10 μL (1 μg RNA dissolved in 10 μL ultrapure water). The reaction was conducted in a Verit Thermal Cycler (Life Technologies); the reaction mixture was incubated at 25°C for 10 min, then at 37°C for 2 h, and then at 95°C for 5 min. The obtained cDNA was used for real-time PCR.
EVALUATION OF THE EXPRESSION LEVEL OF THE STUDIED GENES:
To assess the expression level of the studied genes, IL1A, IL1B, IL1R, IL2, IL6, IL6R, and TSG-6, the real-time PCR method was used. Real-time PCR reactions were performed in 0.1-mL 96-well plates (Applied Biosystems, USA) using cDNA reverse transcription as a template, in a volume of 10 μL/well consisting of 0.5 μL gene-specific primers and probe, 4.5 μL cDNA synthesized by reverse transcription with ultrapure RNAse- and DNAse-free water, and 5 μL TaqMan Gene Expression Master Mix (Applied Biosystems, USA). The reaction, after an initial 10-min denaturation at 95°C, was conducted for 40 cycles according to the following scheme: 15 s at 95°C, then 60 s at 60°C. mRNA expression was detected using the StepOnePlus System (Applied Biosystems, USA). The level of relative expression (RQ) was assessed according to the formula proposed by Livak, where RQ=2−ddCt [23]. To normalize the expression of the tested genes, the cycle threshhold value was determined for each sample against the reference gene that showed the greatest stability in the tested material, namely, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. In the study, B2M and ACTB were also considered as endogenous controls, but GAPDH turned out to be the best control. Final gene expression was determined relative to the sample used to calibrate the entire experiment. The expression of the studied genes was analyzed using ExpressionSuite Software v.1.0.3. (Life Technologies). The following TaqMan primers and probes were used for the genes tested: (for TSG-6: NM_007115.3 and Hs00200180_m1, for IL6: NM_000600.4 and Hs00174131_m1, for ILR6: NM_000565.3 and Hs00169842_m1, for Il1A: NM_000575.4 and Hs00174092_m1, for IL1B: NM_000576.2 and Hs00174097_m1, for Il1R: NM_000877.3 and Hs00168392_m1, for Il2: NM_000586.3 and Hs00174114_m1). The reference gene for endogenous control was the GAPDH gene (NM_001289746.1 and Hs99999905_m1). Three technical repetitions were performed on each test sample. The procedure for RNA isolation and evaluation of gene expression is described in the work of Gil-Kulik et al [24,25].
STATISTICAL ANALYSIS:
The results of statistical analyzes were obtained with the Statistica v.13 program, using the Mann-Whitney U test and the Spearman’s rank correlation coefficient. Statistical significance was established at the level of
EVALUATION OF CLINICAL DATA:
In taking the medical history of patients, doctors of the Department of Obstetrics and Pathology of Pregnancy assessed the age and comorbidities of the patients, weeks of gestation, weight of the newborn, sex of the newborn, and patient’s past illnesses. The biophysical parameters of umbilical cord blood were assessed in the Department of Obstetrics just after delivery on an ABL90 FLEX gas analyzer (Radiometer, Denmark).
Results
INFLUENCE OF PH:
Analyzing the expression of genes tested from umbilical cord blood pH, we observed that the expression of the TSG-6 gene in MSCs was statistically significantly nearly 5 times higher (P=0.032) at umbilical cord blood pH greater than or equal to 7.35, compared with at lower pH values (Figure 3A). Spearman’s rank correlation analysis showed that the level of TSG-6 gene expression in MSCs was statistically significantly positively correlated with cord blood pH (r=0.402, P<0.05; Figure 3B, Table 3). Moreover, the analysis of the correlation of the expression of the studied genes with the cord blood pH in MSCs collected from healthy women showed a negative correlation with the expression of IL1A (r=−0.625, P<0.05) and with the expression of IL6 (r=us;0.638, P<0.05; Table 4). There was no significant correlation between the expression levels of IL1B, IL1R, IL2, and IL6R genes with cord blood pH.
:
The analysis of the dependence of the expression of the studied genes on the pCO2 level in the umbilical blood, including the entire study group, showed that the level of IL2 expression was significantly negatively correlated with pCO2 (r=−0.494 P<0.05), while the level of IL6 expression was significantly positively correlated with pCO2 (r= 0.470 P<0.05; Table 3). Moreover, it was observed that with a pCO2 in the umbilical blood above 45 mm Hg, the expression of IL2 was statistically significantly 22 times lower (P=0.024) than the pCO2 in the range of 35 to 45 mm Hg. On the other hand, the level of IL6 expression at pCO2 above 45 mm Hg was statistically significantly more than 2.5 times higher (P=0.016) than the range of 35 to 45 mm Hg (Figure 3C). The analysis in the group of healthy women showed a positive correlation of pCO2 with the level of IL6 (r=0.628, P<0.05) and the level of IL1A (r=0.594 P<.05; Table 4). There was no significant dependence of TSG-6, IL1B, IL1R, and IL6R gene expression on the pCO2 in the umbilical cord blood.
:
The analysis of the dependence of the expression of studied genes in MSCs on pO2 showed a statistically significant negative correlation with the level of IL6 expression (r=−0.441, P<0.05) (Table 3). In the group of healthy women, the level of IL1R was negatively correlated with pO2 (r=−0.676, P<0.05; Table 4). There was no significant relationship between the expression of TSG-6, IL1A, IL1B, IL2, and IL6R genes in the MSCs from umbilical cord blood pO2.
INFLUENCE OF THE PARAMETERS OF CORD BLOOD MORPHOLOGY:
Considering the parameters of cord blood morphology, it was noted that the expression of IL1A (P=0.044) and IL1B (P=0.04) in MSCs was more than 2 times higher when the level of WBC in cord blood was in the range of 10 to 15×109/L, compared with levels of WBC lower than 10×109/L (Figure 3D), and this result was statistically significant. There were no relationships between the other genes tested and the level of WBC in the umbilical blood.
A number of correlations between the expression of genes under study and the RBC parameters of umbilical blood have been demonstrated. Among other things, we observed that the level of IL6 gene expression depended on the hemoglobin level. When HGB values exceeded 11.5 g/dL, the expression of IL6 in the MSCs was statistically significantly 2.5 times higher (P=0.041) than that of lower hemoglobin levels (Figure 3E). Correlation analysis in the entire study group showed a positive relationship between the level of IL6 expression and the level of hemoglobin (r=0.361, P<0.05; Table 3). Correlation analysis in the group of healthy women also showed a significant positive relationship between the level of IL6 expression and HGB (r=0.599, P<0.05). Moreover, it was noted that IL6 was positively correlated with RBC (r=0.590, P<0.05) and HCT (r=0.627, P<0.05; Table 4).
The relationship between the expression of the tested genes and the number of platelets was also noted. During the correlation analysis of the whole study group, we found the level of IL2 expression (r=0.545, P<0.05) and IL6R (r=0.451, P<0.05) were positively correlated with PLT (Table 3). During the analysis, in the group of healthy women, a positive relationship between TSG-6 expression and PLT (r=0.767, P<0.05) and IL1B with PLT (r=0.603, P<0.05) and a negative relationship between the expression of IL6R and PLT (r=−0.593, P<0.05) were observed (Table 4).
INFLUENCE OF PATIENTS’ DISEASES:
The analysis of the dependence of the expression of genes in the examined patients with comorbidities showed that the expression of IL1A in the MSCs of the umbilical cord obtained from patients with hypertension was statistically significantly 4 times higher (P=0.004) than that in healthy patients. In turn, the expression of the gene encoding IL6R in MSCs obtained from healthy patients was statistically significantly 3.5 times higher (P=0.037) than in patients with hypertension (Figure 4A). The other tested genes did not differ significantly depending on the presence of hypertension.
The expression of the TSG-6 gene was statistically significantly 5 times higher (P=0.026) in the umbilical cord MSCs obtained from women with hypothyroidism (Figure 4B). In the case of other tested genes, no significant relationships with the occurrence of hypothyroidism were found.
The analysis of the correlation between the studied genes in the whole study group showed a significant positive relationship between the expression of the TSG-6 gene and IL1B (r=0.514, P<0.05) and between TSG-6 and IL1R (r=0.511, P<0.05). Also, the level of IL1A expression in MSCs was positively correlated with patient age (r=0.366, P<0.05), while the level of IL6R in MSCs was negatively correlated with patient age (r=−0.322, P<0.05; Table 3).
It was also shown that the expression of the TSG-6 gene in a group of healthy women was positively correlated with the number of deliveries (r=0.527, P<0.05; Table 4).
Analysis of the correlation between the expression of the studied genes and clinical parameters in MSCs from patients with hypertension showed the existence of several strong relationships: IL1B expression was positively correlated with IL6R expression (r=0.982, P<0.05), IL1R expression was positively correlated with TSG-6 expression level (r=0.962, P<0.05), IL6 expression was positively correlated with the number of pregnancies and deliveries (r=0.988, P<0.05), TSG-6 expression in hypertensive patients was positively correlated with the number of pregnancies and deliveries (r=0.892, P<0.05), TSG-6 expression was negatively correlated with the gestational week of delivery (r=−0.934, P<0.05), IL1B expression was negatively correlated with newborn weight (r=−0.982, P<0.05), IL6R expression was negatively correlated with newborn weight (r=−0.974, P<0.05), and a positive relationship between IL2 expression and PLT was noted (r=0.959, P<0.05) (Table 4). It should be mentioned here that the study group with hypertension consisted of only 8 patients; moreover, 3 of them also had diabetes, and therefore the obtained results need to be confirmed in a larger group of patients.
The evaluation of the correlation between the tested genes and clinical data in MSCs collected from patients with hypothyroidism showed the following: the level of IL6 expression was positively correlated with the level of IL1A expression (r=0.555, P<0.05), the level of IL6 expression was negatively correlated with IL6R expression (r=−0.587, P<0.05), IL1R1 expression was positively correlated with the level of TSG-6 expression (r=0.618, P<0.05), IL1A expression was positively correlated with the gestational week in which delivery occurred (r=0.740, P<0.05), TSG-6 expression was positively correlated with the pH of umbilical cord blood (r=0.675, P<0.05), and TSG-6 expression level was negatively correlated with pCO2 (r=−0.578, P<0.05). In addition, relationships between IL1R, IL1A, IL2, and IL6 with the parameters MCV, MCH, and MCHC were noted (Table 4).
In MSCs from patients with gestational diabetes, the following relationships were observed:
Moreover, in this study, we showed a certain tendency regarding the influence of vaginal delivery and the administration of oxytocin during labor on
Discussion
The results of the present study have shown that MSCs express the
TSG-6 is a hyaluronan-binding protein. It is one of the mechanisms responsible for the anti-inflammatory effect of this protein. It has been observed that the affinity of TSG-6 for hyaluronan is pH-dependent [27–29]. This indicates that the anti-inflammatory effect of TSG-6 will depend on the pH value, which is confirmed by the results of our research, in which we have shown that the expression of
In addition to the effect of cord blood acid-base balance parameters on the level of
Moreover, arteritis has been shown to worsen after suppression of TSH, compared with the state of hypothyroidism [30]. This is consistent with our study, in which it was observed that
We also observed that the expression of
It has been shown that in inflammation caused by trauma, the platelet lysate activates NF-κB in MSCs, promoting the release of pro-inflammatory cytokines. TSG-6, which also acts through the NF-κB pathway in an inhibitory manner, increases in this situation [31]. Regarding our study, which also showed a significant, positive correlation of
Our results showed that there was a tendency to different values of
We also observed that there is a tendency for different values of
In our study, apart from the evaluation of
Treatment with TSG-6 has already been shown to reduce IL1B and IL6 levels [37]. Similar effects were observed in another study, in which cells cultured with IL1 and IL6 were shown to have lower TSG-6 production [38]. The aim of our study was to assess whether similar relationships occur in MSCs at the level of gene expression.
Liu et al tested the effect of transplantation of MSCs isolated from the human umbilical cord on the healing of post-burn wounds. MSCs reduced the amount of infiltrating inflammatory cells, lowered the concentration of pro-inflammatory interleukins IL1 and IL6, and increased the concentration of anti-inflammatory factors, such as TSG-6 [39]. During our research, we decided to check whether
Despite the lack of satisfactory effects of the potential positive impact of MSC on the course and symptoms of toxic shock syndrome presented in the publication by Kim et al [40], our team was inspired to check the relationship between the expression level of pro-inflammatory interleukins, namely
One study has shown that MSC has a positive effect on reducing the concentration of pro-inflammatory cytokines in chronic exposure to cigarette smoke. It turns out that after endotracheal administration of MSC, the concentration of IL1B and TSG-6 are reduced. We were interested in whether there is an effect of the anti-inflammatory TSG-6 in MSCs on the production of IL1B and other pro-inflammatory interleukins [41].
Data in the literature indicate a significant relationship between concentration of IL1A and arterial hypertension. The cytokine IL1A is a predictor of high systolic and diastolic blood pressure [42]. IL6, on the other hand, has multidirectional effects, such as inter alia initiating chronic inflammation. The presence of IL6R on the MSC surface has also been demonstrated [43]. The results of the reports are consistent with the results of our study concerning significantly higher
Reduction of blood oxygen saturation is associated with an increase of IL6 level in serum [46]. Reports on this subject are consistent with the results we obtained, according to which
Moreover, IL6, apart from its role in the immune response, is responsible for the regulation of hemopoiesis in the organism [47]. Hemoglobin constitutes the majority of intracellular proteins of RBCs [48]. Our study showed an increase in the level of
This is supported by other studies which show that maternal anemia has an effect on the increase in umbilical cord pO2 [49], which is further negatively correlated with
One of MSC’s functions is inhibition of the development of an excessive inflammatory response. By negative feedback, pro-inflammatory cytokines stimulate MSCs to secrete TSG-6, which then reduces signaling of NF-κB in macrophages and thus models the pro-inflammatory cytokine cascade [50]. This is consistent with our results, in which we obtained a positive correlation between
Conclusions
Our research has shown that the expression of the
Figures
Figure 1. Mesenchymal stem cells from a 5-day culture. Bright field microscopy (BF), 200× magnification, using Xcellence RT system with an IX81 inverted microscope (Olympus). Figure 2. Cytometric evaluation of the expression of CD146, CD73, CD90, and CD105 surface antigens on the tested cells. DuraClone SC Mesenchymal Tube antibodies, Navios cytometer (Beckman Coulter). Sample immunophenotype analysis of umbilical cord-derived mesenchymal stem cells (MSCs) after in vitro culture. (A) Cytogram: cell population negative for CD45 antigen (CD45−). (B) Cytogram: percentage of CD31-negative and CD146-positive cells (CD146+CD31−). (C) Cytogram: percentage of cell population expressing CD90 antigen and CD73 antigen (CD90+CD73+). (D) Cytogram: percentage of umbilical cord MSCs positive for CD90, CD105, CD73 (CD90+CD105+CD73+). (E) MSC positive for CD90, CD105, CD73 and negative for CD14 and CD19 (CD90+CD105+CD73+CD14−CD19−). Figure 3. (A) Mean TSG-6 gene expression (relative level of expression±standard error [RQ±SE]) in tested mesenchymal stem cells (MSCs) depending on umbilical cord blood pH. * P<0.05 Mann-Whitney U test. (B) Graph of distribution TSG-6 gene expression in tested MSCs and umbilical cord blood pH. Spearman’s rank factor r=0.402, P<0.05. (C) Mean IL2 and IL6 gene expression (relative level of expression±standard error [RQ±SE]) in tested MSCs, based on the umbilical cord blood pCO2. * P<0.05 Mann-Whitney U test. (D) Mean IL1A and IL1B gene expression (RQ±SE) in tested MSCs depending on umbilical cord blood white blood cell count. * P<0.05 Mann-Whitney U test. (E) Mean IL6 gene expression (RQ±SE) in tested MSCs, based on umbilical cord blood hemoglobin level. * P<0.05 Mann-Whitney U test. Figure 4. (A) Mean IL1A and IL6R gene expression (relative level of expression±standard error [RQ±SE]) in tested mesenchymal stem cells (MSCs) depending on hypertension in patients. (B) Mean TSG-6 gene expression (RQ±SE) in tested MSCs depending on the hypothyroidism in patient. * P<0.05 Mann-Whitney U test.Tables
Table 1. Characteristics of the study group (n=45) in terms of: mother’s age, number of pregnancies, number of deliveries, weeks of gestation, newborn’s weight, and cord blood parameters. Table 2. Characteristics of the control group (n=17), patients with gestational diabetes (n=13), patients with hypertension (n=8), and patients with hypothyroidism (n=14) in terms of mother’s age, number of pregnancies, number of deliveries, weeks of gestation, newborn’s weight, and cord blood parameters. Table 3. Correlations between the parameters studied in the entire study group (n=45). Table 4. Correlations between clinical parameters and expression of genes in the group of healthy women (n=17), patients with gestational diabetes (n=13), patients with hypertension (n=8), and patients with hypothyroidism (n=14).References
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