Human mesenchymal stem cells derived from umbilical cord and bone marrow exert immunomodulatory effects in different mechanisms

Figure 1 Characterization of umbilical cord-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells. A: Surface protein expression was analyzed using flow cytometry. Mesenchymal stem cells (MSCs) were assessed at the fourth passage; B: Chemokine receptor expression was analyzed at passages 3–7 by flow cytometry; C: Representative images of umbilical cord-derived MSCs and bone marrow-derived MSCs differentiation into adipocytes (bone marrow-derived MSCs: day 7; umbilical cord-derived MSCs: day 21), chondrocytes (day 21) and osteocytes (day 14). Scale bars: 200 μm. Adipocytes were detected by goat anti-mouse FABP4 polyclonal antibody under a fluorescence microscope. Osteoblasts and chondrocytes were stained with Alizarin Red S and Alcian Blue, respectively and observed under a light microscope; D: Growth rate of MSCs at different passages; and E: Pluripotency transcription factor expression levels of cultured MSCs measured by quantitative real-time polymerase chain reaction (target, GAPDH), D and E: Bone marrow-derived MSCs. Statistical analysis was performed by Student’s t-tests, ^aP < 0.05 vs indicated group. iso: Isotype; Klf4: Kruppel‐like factor 4; Nanog: Nanog homeobox; NS: No significance; OCT4: Octamer-binding transcription factor 4; UC-MSC: Umbilical cord-derived mesenchymal stem cells; BM-MSCs: Bone marrow-derived mesenchymal stem cells.

Figure 2 Immunomodulatory effects of interferon-gamma-stimulated mesenchymal stem cells. Umbilical cord-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells were stimulated with 0, 1, 5 or 10 ng/mL of interferon-gamma in culture for 72 h. A and C: Expression of indoleamine 2,3-dioxygenase (A) and cyclooxygenase-2 (C) of interferon-gamma-stimulated mesenchymal stem cells measured by western blotting; B, D and E: Expression of indoleamine 2,3-dioxygenase (B), prostaglandin E2 (D), and interleukin-6 (E) in cultured medium measured by ELISA; F and G: Expression of PD-L1 (F) and PD-L2 (G) after interferon-gamma stimulation analyzed by flow cytometry. Statistical analysis was performed by Student’s t-tests, ^aP < 0.05; ^bP < 0.01; ^cP < 0.0001 vs indicated group. B, D and E: ^aP < 0.05; ^bP < 0.01; ^cP < 0.0001 vs first bar of each group (F and G). BM-MSC: Bone marrow-derived mesenchymal stem cells; IDO: Indoleamine 2,3-dioxygenase; IFN-γ: Interferon-gamma; IL-6: Interleukin 6; iNOS: Inducible nitric oxide synthase; MFI: Mean fluorescence intensity; ND: Not detected; PGE2: Prostaglandin E2; UC-MSC: Umbilical cord-derived mesenchymal stem cells.

Figure 3 Immunomodulatory effects of mesenchymal stem cells treated with combinations of cytokines. Umbilical cord-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells were stimulated with different combinations of interferon-gamma (5 ng/mL), tumor necrosis factor alpha (5 ng/mL), and/or IL-1β (5 ng/mL) for 72 h. A: Expression levels of indoleamine 2,3-dioxygenase and cyclooxygenase-2 of cytokine-stimulated mesenchymal stem cells measured by western blotting; B and C: Expression levels of prostaglandin E2 (B) and interleukin-6 (C) in culture medium measured by ELISA. Statistical analysis was performed by Student’s t-tests, ^aP < 0.05; ^bP < 0.01; ^cP < 0.0001 vs first bar of each group. (B and C) BM-MSC: Bone marrow-derived mesenchymal stem cells; IDO: Indoleamine 2,3-dioxygenase; IFN-γ: Interferon-gamma; IL-1β: Interleukin 1 beta; IL-6: Interleukin 6; ND: Not detected; PGE2: Prostaglandin E2; TNF-α: Tumor necrosis factor alpha; UC-MSC: Umbilical cord-derived mesenchymal stem cells.

Figure 4 T cell inhibition by mesenchymal stem cells. T cell proliferation was inhibited by either umbilical cord-derived mesenchymal stem cells (MSCs) or bone marrow-derived MSCs in coculture. A: Human peripheral blood mononuclear cells (1 × 10⁵/well) from heathy adult donors were stimulated with phytohemagglutinin (5 μg/mL) with or without MSCs (5 × 10³, 1 × 10⁴, 5 × 10⁴, 1 × 10⁵) for 72 h. In the final 18 h, ³H-thymidine was added to the cultures, ^aP < 0.05; ^bP < 0.01; ^cP <0.0001 vs control of each group, ^eP < 0.01; ^fP < 0.0001 vs control; and B: Human peripheral blood mononuclear cells were treated with L-NAME (iNOS inhibitor), indomethacin (cyclooxygenase-2 inhibitor), anti-IL-10, anti-transforming growth factor-β, hemin (heme oxygenase inducer), alloxazine (selective A_2B adenosine receptor antagonist), or adenosine 5'-(α,β-methylene)diphosphate (CD73 inhibitor) with or without MSCs for 72 h. In the final 18 h, ³H-thymidine was added to the cultures. Statistical analysis was performed by Student’s t-tests ^aP < 0.05; ^bP < 0.01; ^cP < 0.0001 vs control. Anti-IL-10: Anti-interleukin 10 antibody; Anti-TGF-β: Anti-transforming growth factor β antibody; APCP: Adenosine 5'-(α,β-methylene)diphosphate; BM-MSC: Bone marrow-derived mesenchymal stem cells; cpm: Count per minute; L-NAME: N-nitro-L-arginine methyl ester; UC-MSC: Umbilical cord-derived mesenchymal stem cells.

Figure 5 Roles of prostaglandin E2 and interleukin-10 in the immunomodulatory functions of mesenchymal stem cells. A: Human peripheral blood mononuclear cells were stimulated with phytohemagglutinin (10 μg/mL) and cocultured with mesenchymal stem cells treated with indomethacin (20 μM) and/or anti- interleukin (IL)-10 (5 μg/mL). Then, interferon-gamma-producing CD4⁺ T cells were analyzed by flow cytometry, ^aP < 0.05; ^bP < 0.01 vs first bar; B: Human peripheral blood mononuclear cells were treated with anti-interferon-gamma (10 μg/mL), anti-IL-4 (10 μg/mL), IL-6 (10 ng/mL), IL-23 (5 ng/mL), IL-1β (10 ng/mL), tumor necrosis factor alpha-α (5 ng/mL) and transforming growth factor-β (2 ng/mL) to induce T helper 17 cells. Induced T helper 17 cells were cocultured with mesenchymal stem cells treated with indomethacin (20 μM) and/or anti-IL-10 (5 μg/mL). Populations of CD4⁺Foxp3⁺ T cells and CD4⁺IL-17⁺ T cells were analyzed by flow cytometry. Statistical analysis was performed by Student’s t-tests, ^aP < 0.05; ^bP < 0.01; ^cP < 0.0001 vs indicated group, ^dP < 0.05; ^fP < 0.0001 vs phytohemagglutinin control. Anti-IL-10: Anti-interleukin 10 antibody; BM-MSC: Bone marrow-derived mesenchymal stem cells; IFN-γ: Interferon-gamma; IL-17: Interleukin 17; MSC: Mesenchymal stem cells; PHA: Phytohemagglutinin; Th17: T helper 17; UC-MSC: Umbilical cord-derived mesenchymal stem cells.

Figure 6 Inhibitory effects of mesenchymal stem cells therapy on graft-versus-host disease severity. A-C: NOG mice were administered 200 cGy of total body irradiation before transplantation of human peripheral blood mononuclear cells (2 × 10⁷). At days 0, 7 and 14 after transplantation, mice were administered with umbilical cord-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells (1 × 10⁵). All NOG mice were monitored for survival (A), clinical signs of graft-versus-host disease (B) and weight (C). BM-MSC: Bone marrow-derived mesenchymal stem cells; GVHD: Graft-versus-host disease; UC-MSC: Umbilical cord-derived mesenchymal stem cells.