Comparison of biological properties of umbilical cord-derived mesenchymal stem cells from early and late passages: immunomodulatory ability is enhanced in aged cells

Abstract

Mesenchymal stem cells (MSCs) are a potential source of adult stem cells for cell-based therapeutics due to their substantial multilineage differentiation capacity and secretory functions. No information is presently available regarding the maintenance of immunosuppressive properties of this cell type with repeated passages. It was therefore the aim of the present study to analyze the biological properties, particularly the immunoregulatory effect, of MSCs from late passages. The differences between young and old MSCs in morphology, cell surface antigen phenotype, proliferation, gene expression and immunomodulatory ability were investigated. The results of the current study demonstrated that with the passage of cells, senescent MSCs displayed a characteristically enlarged and flattened morphology, different gene expression profiles and stronger immunosuppressive activities. Increased interleukin-6 production may be a possible underlying mechanism for this enhanced immunomodulatory ability of MSCs. These findings suggest that aged MSCs may provide a treatment option for patients with graft versus host disease and other diseases associated with dysregulation of the immune system.

Figure 1

Morphology and immunophenotype of umbilical cord-derived cells. (A) Hematoxylin and eosin staining of umbilical cord-derived cells. Cells exhibited a bipolar spindle-shaped morphology and grew as a monolayer. Scale bar, 200 μm. Surface markers of (B) hUC-MSC-p3 and (C) hUC-MSC-p15 were detected by flow cytometric analysis. hUC-MSC-p3 and hUC-MSC-p15 expressed CD29, CD44, CD73, CD90, CD105 and CD106, but not CD34 and CD45, and no significant difference was identified between the expression levels (P>0.05). CD29 and CD106 are not shown in the figure.

Figure 2

Morphologic changes in hUC-MSCs during long-term culture. (A and B) Giemsa staining of umbilical cord-derived cells. With the passage of cells, MSCs became flat, displayed (A) increased cell size, and (B) possessed reduced nucleocytoplasmic ratio. Scale bar, 100 μm. (C) Morphologic changes identified by SEM. hUC-MSC-p3 were plump and round in size and had equally distributed microvilli on the cell surface, whereas hUC-MSC-p15 exhibited more podia and less microvilli, spread more widely and contained more actin stress fibers. Scale bar: Left, 10 μm; right, 60 μm. MSC, mesenchymal stem cell; SEM, scanning electron microscopy; P, passage.

Figure 3

SA-β-gal activity and cell proliferation of hUC-MSCs. (A) Senescence of hUC-MSCs was detected by SA-β-gal staining. hUC-MSC-p15 exhibited high levels of SA-β-gal activity, as determined by the number of flattened and blue-stained cells. Few SA-β-gal-positive cells were observed in hUC-MSC-p3. Scale bar, 200 μm. (B) The number of blue-stained hUC-MSC-p15 cells were significantly higher than those of hUC-MSC-p3 (^**P<0.01). (C) Cell proliferation was measured by MTT analysis. hUC-MSC-p15 exhibited a lower growth activity level than hUC-MSC-p3.

Figure 4

Senescence affects the capacity of cells to suppress allogeneic lymphocyte proliferation (^*P<0.05 and ^**P<0.01). hUC-MSC-p3 and hUC-MSC-p15 significantly inhibited PHA-stimulated peripheral blood mononuclear cell proliferation following 72-h co-culture (P<0.01), and the inhibitory activity of hUC-MSC-p15 was higher than that of hUC-MSC-p3 (P<0.05). Quantitative data are expressed as the mean ± standard error. PHA, phytohemagglutinin.

Figure 5

Changes in immunoregulatory-related cytokines. (A) RT-qPCR of immunoregulation-related genes (^*P<0.05). The hUC-MSC-p3 group values were set at fold change=1. Four immunosuppressive genes, HMOX-1, IL-10, IL-6, and iNOS, were upregulated in hUC-MSC-p15 (P<0.05), whereas no significant difference was observed in IDO-1 and TGF-β1 (P>0.05). For the anti-inflammatory genes, IL-1α, IL-1β, and IFN-γ were downregulated (P<0.05). Quantitative data are expressed as the mean ± standard error. (B) ELISA results indicated that IL-6 production was greatly increased during senescence (P<0.05). HMOX-1, heme-oxygenase 1; IL, interleukin; iNOS, inducible nitric oxide synthase; IDO-1, indoleamine 2,3-dioxygenase-1; TGF-β1, transforming growth factor β1; IFN-γ, interferon γ.