Influence of Umbilical Cord Blood Biochemical Parameters and Disease Condition on the Expression of the TSG-6 Gene in Umbilical Mesenchymal Stem Cells

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.

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.