The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro

Abstract

Bone marrow-derived mesenchymal stem cells (BM-MSC) are currently being investigated in preclinical and clinical settings because of their self-renewal and multipotent differentiative capacity or their immunosuppressive function. However, BM may be detrimental because of the highly invasive donation procedure and BM-MSC decline with age. Therefore, MSC derived from other sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. Human umbilical cord blood (UCB) cells are good substitutes for BM-MSC because of the immaturity of newborn cells. In this study, we successfully isolated MSC from UCB. The morphological phenotypes, cell cycle status, surface markers and differentiation potential of these clonally expanded cells are consistent with BM-MSC. Furthermore, UCB-MSC expanded in vitro retain low immunogenicity and an immunomodulatory effect. Flow cytometry analysis showed that UCB-MSC did not express CD40, CD40 ligand, CD80, CD86 and major histocompatibility complex class II molecules. We have demonstrated that UCB-MSC are incapable of inducing allogeneic peripheral blood mononuclear cell (PBMC) proliferation and have a dose-dependent inhibition of PBMC immune responses in mixed lymphocyte reactions (MLR) and phytohaemagglutinin activation assays, even after interferon-gamma treatment. Additionally, we have found that UCB-MSC can suppress the function of mature dendritic cells. Using transwell systems, we have demonstrated an inhibition mechanism that depends on both cell contact and soluble factors. Based on the findings we conclude that banked UCB could serve as a potential alternative source of MSC for allogeneic application in the future.

Figure 1

The morphology, population doubling time and cell cycle status of the fibroblastoid cells from umbilical cord blood (UCB). (a) After the initiating plate, the fibroblastoid adherent cells developed the onset of colony formation within 3 weeks (scale bars = 300 μm). (b) Single cell-derived, clonally expanded adherent cells derived from UCB displayed a fibroblastic morphology (scale bars = 200 μm). (c) Single cell-derived clones were culture expanded and gradually reached confluency with a whirlpool-like array (scale bars = 200 μm). (d) Population doubling time of UCB-derived cells (n= 6, P1–P15) was determined. The UCB-derived cells displayed high doubling numbers in all passages analysed and the mean population doubling time remained constant until approximately P15. (e) Representative cell cycle plots from UCB-derived cells. The majority of the UCB-derived cells were retained in the G0–G1 phase, whereas small populations of cells were engaged in the G2–M phase and S phase (Dip G1: 95·15%, Dip G2: 0·62%, Dip S: 4·23%, Apoptosis: 0·04%, Debris: 2·9%, Aggregates: 1·3%).

Figure 2

Phenotype and multilineage differentiation capacity of the fibroblastoid cells from umbilical cord blood (UCB). (a) Cells were cultured for three to five passages, harvested and labelled with antibodies against human antigens CD14, CD29, CD31, CD34, CD40, CD40 ligand, CD45, CD73, CD80, CD86, CD105, CD166, major histocompatibility complex (MHC) I and MHC II, as indicated and analysed by fluorescence-activated cell sorting. Only representative flow cytometric plots are shown. Shaded histogram indicates background signal; open histogram, positive reactivity with the indicated antibody. (b–f) Osteogenic, chondrogenic and adipogenic differentiation. Osteogenic differentiation evidenced by the formation of mineralized matrix shown by Alizarin red staining at original magnification × 40 (b). Osteoblastic differentiation was further confirmed by alkaline phosphatase staining after 21 days of induction (c). Chondrogenic differentiation was shown by Safranin-O staining (d) and by immunohistochemical staining for type II collagen (e). Adipocytic differentiation was detected by the formation of neutral lipid vacuoles stainable with Oil Red O staining (f). Original magnification, × 100 (c–f). (g) Reverse transcription–polymerase chain reaction showed that the fibroblastoid cells from UCB expressed the messenger RNA of stem cell factor.

Figure 3

Immunomodulatory properties of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC). (a) After UCB-MSC were treated with interferon-γ (IFN-γ) for 48 hr; the expression of major histocompatibility complex (MHC) II molecules was significantly up-regulated on UCB-MSC from undetectable to more than 80% positive expression. Shaded histogram indicates background signal; open histogram, positive reactivity with the indicated antibody. (b) UCB-MSC failed to stimulate allogeneic peripheral blood mononuclear cells (PBMC). The proliferation of PBMC self-stimulated (self), by allogeneic PBMC (allogeneic), by unstimulated allogeneic bone marrow-derived MSC (BM-MSC; grey bar), by unstimulated allogeneic UCB-MSC (hatched bar), and by stimulated allogeneic UCB-MSC (white bar) was measured using [³H]thymidine incorporation. (c) UCB-MSC and UCB-MSC + IFN-γ inhibited mixed lymphocyte reaction activated peripheral blood mononuclear cells (PBMC) in a cell dose-dependent manner. PBMC were cocultured with equal amounts of allogeneic PBMC and varying amounts of third-party UCB-MSC (hatched bar), BM-MSC (grey bar), and UCB-MSC + IFN-γ (white bar). (d) UCB-MSC and UCB-MSC + IFN-γ inhibit phytohaemagglutinin (PHA)-activated lymphocyte proliferation in a cell dose-dependent manner. The PBMC were cocultured with UCB-MSC (hatched bar), BM-MSC (grey bar) and UCB-MSC + IFN-γ (white bar), which inhibited PHA-activated PBMC proliferation at equal levels. Both UCB-MSC and UCB-MSC + IFN-γ can inhibit the stimulatory effect of PHA more efficiently at the dose of 1 × 10⁵ than the dose of 1 × 10⁴, which is consistent with BM-MSC. The results are representative of three independent experiments. The data are mean ± SD of triplicate cultures. *P<0·05, **P<0·01.

Figure 4

Suppression of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) on the function of mature dendritic cells (DC). (a) Monocytes cultured in the presence of granulocyte–macrophage colony-stimulating factor and interleukin-4 for 7 days and then tumour necrosis factor-α for another 2 days showed clustered and protruding veils resembling mature DC. Cell morphology was evaluated by phase contrast microscopy (scale bars = 120 μm). (b) Flow cytometry was employed to analyse the surface molecules expressed by immature DC (top row) and mature DC (bottom row). Results shown are representative flow cytometric plots. Shaded histogram indicates background signal; open histogram, positive reactivity with the indicated antibody. (c,d) The UCB-MSC inhibit the ability to stimulate T-lymphocyte proliferation of monocyte-derived DC. Graded doses of mature DC were cocultured with 1 × 10⁵ allogeneic responders in the presence of absence of UCB-MSC for 5 days (c). After UCB-MSC treatment, mature DC alone were used in graded doses to stimulate allogeneic responders (d). The proliferative activity was measured by adding [³H]thymidine to each well 18 hr before the cultures were terminated. The results are representative of three independent experiments. The data are mean ± SD of triplicate cultures. *P<0·05, **P<0·01.

Figure 5

Inhibitory effects of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) are mediated by cell contact and soluble factors. UCB-MSC were plated into the lower chamber of transwell plates and mixed lymphocyte reactions were set up in the upper chambers. After 5 days of culture, cells from the upper chamber were transferred to microtitre wells in triplicate, pulsed with [³H]thymidine and harvested 18 hr later. The results are representative of three independent experiments. The data are mean ± SD of triplicate cultures. *P<0·05, **P<0·01.