The Immunosuppressive Activity of Amniotic Membrane Mesenchymal Stem Cells on T Lymphocytes

Fatemeh Alikarami, Fatemeh Yari, Naser Amirizadeh, Mahin Nikougoftar, Mohammad Ali Jalili, Fatemeh Alikarami, Fatemeh Yari, Naser Amirizadeh, Mahin Nikougoftar, Mohammad Ali Jalili

Abstract

Background: Mesenchymal Stem Cells (MSCs) are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane (AM) are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells (hAM-MSCs). The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro.

Methods: Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin (PHA). Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-γ was examined by ELISA method. Additionally, the expression of activation markers (CD38, HLA-DR) was studied on T lymphocytes by flow cytometry technique.

Results: It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes (p≤0.01) and significantly decrease the production of IFN-γ by T cells (p<0.05). hAM-MSCs also down regulated the expression of activation markers on the surface of T lymphocytes, CD38 and HLA-DR. The difference was significant between the case and control samples (p<0.05). All the comparisons were carried out between the case (Tcell+PHA+hAM-MSCs) and control (Tcell+PHA) groups.

Conclusion: In conclusion, hAM-MSCs could inhibit the (mitogen-activated) T cells even in the absence of blood monocytes. Besides, hAM-MSCs-mediated inhibition of T lymphocytes was combined with down regulation of activation markers.

Keywords: Amnion; Mesenchymal stem cell; T-lymphocyte.

Figures

Figure 1.
Figure 1.
Flow cytometry plot. hAM-MSCs were prepared and their surface markers were assessed by flow cytometry technique. A) hAM-MSC were gated, B-C) isotype controls, D-E) hAM-MSCs were positive for CD29 and CD166, respectively. F-I) hAM-MSCs were also positive for CD105, CD73, CD44 and CD90, respectively. J-K) hAM-MSCs were negative for CD45 and CD34, respectively.
Figure 2.
Figure 2.
The in vitro osteogenic differentiation of hAM-MSC. A) The osteogenic differentiation of hAM-MSC was followed by Alizarin red S staining. B) Red calcium deposits could not be seen in negative control that was cultured in the absence of the differentiation medium.
Figure 3.
Figure 3.
The inhibitory effects of hAM-MSCs on the proliferation of T cells. T cells (1×10 6 ) were co-cultured with different densities of irradiated hAM-MSCs in the presence or absence of PHA in the final volume of 250 μl for 72 hr. Each bar was compared with the T cell+PHA (control T cells) group. Data were presented as the mean ±SD of four independent experiments **p≤0.01.
Figure 4.
Figure 4.
Cytokine levels in the supernatant of the co-culture medium of hAM-MSCs and Tcells in the presence or absence of PHA. A) Production of IL-4 and B) IFN-γ was detected using ELISA method. Each bar was compared with T cell+PHA (control T cells) group. Data were presented as the mean±SD of four independent experiments *p

Figure 5.

T cells were co-cultured with…

Figure 5.

T cells were co-cultured with hAM-MSCs for 72 hr in the presence or…

Figure 5.
T cells were co-cultured with hAM-MSCs for 72 hr in the presence or absence of PHA. The inhibitory effects of hAM-MSCs on the expression of A) CD38 and B) HLA-DR on the surface of T cells were shown. Each bar was compared with T cell+PHA (control T cells) group. Data were presented as the mean±SD of four independent experiments *p<0.05, ** p≤ 0.01.
Figure 5.
Figure 5.
T cells were co-cultured with hAM-MSCs for 72 hr in the presence or absence of PHA. The inhibitory effects of hAM-MSCs on the expression of A) CD38 and B) HLA-DR on the surface of T cells were shown. Each bar was compared with T cell+PHA (control T cells) group. Data were presented as the mean±SD of four independent experiments *p<0.05, ** p≤ 0.01.

References

    1. Yoo KH, Jang IK, Lee MW, Kim HE, Yang MS, Eom Y, et al. Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol 2009; 259 (2): 150– 156.
    1. Sensebé L, Krampera M, Schrezenmeier H, Bourin P, Giordano R. Mesenchymal stem cells for clinical application. Vox Sang 2010; 98 (2): 93– 107.
    1. Gieseke F, Böhringer J, Bussolari R, Dominici M, Hand-gretinger R, Müller I. Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells. Blood 2010; 116 (19): 3770– 3779.
    1. Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal 2011; 9: 12.
    1. Vija L, Farge D, Gautier JF, Vexiau P, Dumitrache C, Bourgarit A, et al. Mesenchymal stem cells: Stem cell therapy perspectives for type 1 diabetes. Diabetes Metab 2009; 35 (2): 85– 93.
    1. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringdén O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003; 57 (1): 11– 20.
    1. Bacigalupo A, Valle M, Podestà M, Pitto A, Zocchi E, De Flora A, et al. T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia. Exp Hematol 2005; 33 (7): 819– 827.
    1. Groh ME, Maitra B, Szekely E, Koç ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells. Exp Hematol 2005; 33 (8): 928– 934.
    1. Asari S, Itakura S, Ferreri K, Liu CP, Kuroda Y, Kandeel F, et al. Mesenchymal stem cells suppress B-cell terminal differentiation. Exp Hematol 2009; 37 (5): 604– 615.
    1. Spaggiari GM, Capobianco A, Abdelrazik H, Becchetti F, Mingari MC, Moretta L. Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood 2008; 111 (3): 1327– 1333.
    1. Veryasov VN, Savilova AM, Buyanovskaya OA, Chulkina MM, Pavlovich SV, Sukhikh GT. Isolation of mesenchymal stromal cells from extraembryonic tissues and their characteristics. Bull Exp Biol Med 2014; 157 (1): 119– 124.
    1. Fibbe WE, Noort WA. Mesenchymal stem cells and hematopoietic stem cell transplantation. Ann NY Acad Sci 2003; 996: 235– 244.
    1. Bensidhoum M, Chapel A, Francois S, Demarquay C, Mazurier C, Fouillard L, et al. Homing of in vitro expanded Stro-1− or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment. Blood 2004; 103 (9): 3313– 3319.
    1. Bartholomew A, Patil S, Mackay A, Nelson M, Buyaner D, Hardy W, et al. Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo. Hum Gene Ther 2001; 12 (12): 1527– 1541.
    1. Díaz-Prado S, Muiños-López E, Hermida-Gómez T, Rendal-Vázquez ME, Fuentes-Boquete I, de Toro FJ, et al. Isolation and characterization of mesenchymal stem cells from human amniotic membrane. Tissue Eng Part C Methods 2011; 17 (1): 49– 59.
    1. Mihu CM, Rus Ciucă D, Soritău O, Suşman S, Mihu D. Isolation and characterization of mesenchymal stem cells from the amniotic membrane. Rom J Morphol Embryol 2009; 50 (1): 73– 77.
    1. Parolini O, Caruso M. Review: Preclinical studies on placenta-derived cells and amniotic membrane: an update. Placenta 2011. ; 32 Suppl 2 : S186 – 195 .
    1. Kang JW, Koo HC, Hwang SY, Kang SK, Ra JC, Lee MH, et al. Immunomodulatory effects of human amniotic membrane-derived mesenchymal stem cells. J Vet Sci 2012; 13 (1): 23– 31.
    1. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105 (4): 1815– 1822.
    1. Le Blanc K, Rasmusson I, Sundberg B, Götherström C, Hassan M, Uzunel M, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363 (9419): 1439– 1441.
    1. Ghannam S, Bouffi C, Djouad F, Jorgensen C, Noël D. Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications. Stem Cell Res Ther 2010; 1 (1): 2.
    1. Uccelli A, Moretta L, Pistoia V. Immunoregulatory function of mesenchymal stem cells. Eur J Immunol 2006; 36 (10): 2566– 2573.
    1. Sato K, Ozaki K, Oh I, Meguro A, Hatanaka K, Nagai T, et al. Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood 2007; 109 (1): 228– 234.
    1. Si ZG, Bai H, Wang CB, Xue ZW, Wang Q, Wu T, et al. [Effect of human bone marrow mesenchymal stem cells on T lymphocyte killing K562 cells]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2007; 15 (6): 1216– 1219. Chinese.
    1. Ueta M, Kweon MN, Sano Y, Sotozono C, Yamada J, Koizumi N, et al. Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction. Clin Exp Immunol 2002; 129 (3): 464– 470.
    1. Dazzi F, Krampera M. Mesenchymal stem cells and autoimmune diseases. Best Pract Res Clin Haematol 2011; 24 (1): 49– 57.
    1. Klyushnenkova E, Mosca JD, Zernetkina V, Majumdar MK, Beggs KJ, Simonetti DW, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci 2005; 12 (1): 47– 57.

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