| Background:Mesenchymal stem cells (MSCs), also termed multipotent mesenchymal stromal cells, are a non-hematopoetic adult stem cell with capacity for self-renewal and differentiation with a broad issue distribution. The immunomodulatory properties of MSCs, together with their multilineage differentation potential, easy accessibility, relatively rapid in vitro expansion, minimal loss of potency after biopreservation, ability to point-of-care delivery, low imunogenicity, no significant ethical issues and adverse reactions, has led to their wide clinical application in treating a variety of diseases. Although adult bone marrow-derived MSCs (BM-MSCs) have earliestly been studied and been investigated more deeply than MSCs from other tissues, clinical application of BM-MSCs is limited due to the invasive procedure during obtaining a BM sample, the high degree of viral exposure, the significant decreases in cell number and proliferation/differentiation capacity with age. A large number of studies have proved that non-bone marrow tissues also have MSCs, such as umbilical cord (UC), adipose and so on. Therefore, we intend to compare the biological characteristics of UC-MSCs and BM-MSCs in order to provide a better MSCs source. In addition, many studies have shown that cytokines secreted by MSCs play a vital role in MSCs therapy, but no high-through analysis of UC-MSCs has yet been reported. Therefore, the high-throughput cytokine array (including174cytokines) was used for screening of cytokines secreted by MSCs for the purpose of determinating the better MSCs passage and laying a good theoretical basis for subsequent mechanism study.Objective:The four characteristics (such as morphological features, proliferation ability, surface markers and differentiation capacity) of UC-and BM-MSCs were compared in order to provide experimental evidence for better MSCs source. The biological characteristics and activity change of long-term ex vivo cultured UC-MSCs was studied in order to provide experimental evidence for the stability of above UC-MSCs. The positive cytokines secreted by UC-MSCs were screened and these cytokine levels among different UC-MSCs passages were compared. Methods:Enzyme digestion and density gradient centrifugation were used to isolate UC-and BM-MSCs, respectively, and the UC-and BM-MSCs were further purified by adherent culture. The morphological features of UC-and BM-MSCs were observed and compared using microscopy technology; the proliferation ability of UC-and BM-MSCs was detected by cell counting; the ability to continuous ex vivo passage of UC-and BM-MSCs was monitored; phenotypes of UC-and BM-MSCs were analyzed by flow cytometry; adipogenic, osteogenic and chondrogenic differentiation potential of UC-and BM-MSCs were examined ex vivo, the cells were stained after induction. Subsequently, we focused on the continuous ex vivo passage of UC-MSCs and cytokines secreted by UC-MSCs, the cell features, biological activities and cytokine levels during the culturing process were analyzed. The high-throughput cytokine array (including174cytokines) was used to analyze the supernatant obtained from different MSCs passages, the positive cytokines were screened according to fluorescence signal intensity, and the data were analyzed with SAS9.2software and comparisons among groups were made with analysis of variance. And LSD method (homogeneity of variance) or Dunnett’s (uneven, variance) were used in multiple comparisons.Results:The results of morphological experiment showed that UC-and BM-MSCs shared a similar spindle-shaped morphology and adhered to the culture flask. The results of long-term ex vivo culture indicated that BM-MSCs had a poor ability to proliferate than UC-MSCs and BM-MSCs lost the proliferation ability after passage9-10, while UC-MSCs also kept the proliferation ability until passage15. Phenotypes of UC-and BM-MSCs showed that they had a consistent immunophenotype:negative for CD34, CD45, CD11b, CD31, CD80, CD86and HLA-DR, and positive for CD44, CD105, CD166and CD73. The expression of CD80on BM-MSCs was significantly higher than that of UC-MSCs (P=0.0137), it suggests that the imunogenicity of UC-MSCs is lower than that of BM-MSCs. The results of cell differentiation experiments indicated that both UC-and BM-MSCs can differentiate into three lineages:adipogenic, osteogenic and chondrogenic. The results of cytokine array analysis showed that UC-MSCs at passage3,5and9secreted102postive cytokines (strongly positive:34, positive:68), mainly involved in cell migration (24.5%), receptor (20.6%), cell growth and differentiation (20.6%) and immunodulatory (12.7%). The results of analysis of variance indicated that there were significant difference among UC-MSCs at passage3,5and9about11cytokine secretion, including TNF-beta (P=0.0147), IL-12p70(P=0.0265), MSP-alpha (P=0.0289), HCC-4/CCL16(P=0.027), IP-10/CXCL10(P=0.0202), GITR(P=0.024), PDGF R alpha (P=0.0277), VEGF R2(P=0.038), IL-9(P=0.0291), MIP-1beta/CCL4(P=0.0497) and CNTF (P=0.0073)Conclusions:UC-MSCs could be substituted for BM-MSCs for clinical application. There are no significant difference among most cytokine secretions of UC-MSCs at different passages. Cytokine array is an ideal method to high-throughputly screen the cytokines in UC-MSCs with little sample volume. |
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