Effect of human umbilical cord versus placenta mesenchymal stem cells in prevention of mouse acute graft versus host disease

doi:10.3969/j.issn.2095-4344.2017.05.007

Abstract

Abstract:

BACKGROUND: Recently, the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) and placenta-derived mesenchymal stem cells (PDMSCs) on treatment of acute graft versus host disease (aGVHD) have been confirmed in some in vitro studies or animal models. But there are still no reports comparing the therapeutic effects of these two cell types.
OBJECTIVE: To compare the immunosuppressive function of hUCMSCs and PDMSCs in vitro or in a mouse aGVHD model.
METHODS: (1) In vitro experiment. Human peripheral blood mononuclear cells (PBMCs) were isolated and divided into four groups: PBMCs cultured alone, PBMCs stimulated with phytohaemagglutinin (PHA), PHA stimulated-PBMCs cocultured with hUCMSCs, PHA stimulated-PBMCs cocultured with PDMSCs. After 5 days, PBMCs proliferation and interferon-γ level in cell supernatant were measured. (2) In vivo experiment. Fifty-seven BABL/C(H-2d) mice exposed to 8.5 Gy irradiation were randomly divided into five groups: only saline injection group, syngeneic bone marrow transplantation group, allogeneic bone marrow transplantation group, aGVHD group, hUCMSCs treatment group, PDMSCs treatment group. The clinical aGVHD score, histopathology of skin, liver, and small intestine, and survival time were analyzed at days 11, 14, 21 after transplantation.
RESULTS AND CONCLUSION: (1) In vitro test: compared with the hUCMSCs, PDMSCs had stronger anti-inflammatory function. (2) In vivo test: The clinical scores on acute graft versus host disease were significantly lower in the hUCMSCs and PDMSCs treatment groups than that in the aGVHD group (P < 0.05). The survival rates of mice were significantly increased in the hUCMSCs and PDMSCs treatment groups compared to the aGVHD group (P < 0.05). Evident skin lesions were not found in all groups. Although small intestine mucosal lesions were found in all groups, the damage level seemed similar. Notably, significant difference was found in the liver that multifocal necrosis and a large number of inflammatory cells were seen in the aGVHD group, but less necrosis and inflammatory cells in the hUCMSC and PDMSC treatment groups. In conclusion, hUCMSC and PDMSC are comparably effective in the treatment of aGVHD in mice.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Placenta, Mesenchymal Stem Cells, Graft vs Host Disease, Mice, Tissue Engineering

CLC Number:

R394.2

Li Juan-juan, Wang You-wei, Ma Feng-xia, Du Wen-jing, Song Bao-quan, Wang Xin, Feng Ying, Tian Jian-jian,Han Zhong-chao. Effect of human umbilical cord versus placenta mesenchymal stem cells in prevention of mouse acute graft versus host disease[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 693-700.

Figures/Tables 2

2.1 脐带和胎盘间充质干细胞的提取鉴定从形态观察脐带和胎盘组织来源间充质干细胞呈细小梭形(图1A)；均表达CD73、CD105、CD90，不表达造血细胞表面分子CD34、CD45、CD11b及CD19，也不表达HLA-DR(图1B)；两者均可向成脂成骨细胞诱导分化(图1 C)，符合间充质干细胞的基本特征。脐带和胎盘间充质干细胞成长较快，初始接种密度低，生长速度稍慢，当细胞达到一定密度时生长速度加快，两者之间未见明显差异(图1D)。 2.2 两种干细胞免疫调节相关基因的表达情况实时定量PCR结果显示，脐带和胎盘间充质干细胞均表达转化生长因子β、吲哚胺2，3-二氧化酶、环氧酶2、肝细胞生长因子、白细胞介素6基因转录本(图2)。两种干细胞转化生长因子β和白细胞介素6表达水平比较差异无显著性意义(P > 0.05)；脐带间充质干细胞中吲哚胺2，3-二氧化酶、肝细胞生长因子的表达水平高于胎盘间充质干细胞(P < 0.05)，中环氧酶2表达水平低于胎盘间充质干细胞(P < 0.05)。 2.3 两种干细胞抑制外周血单个核细胞增殖和干扰素γ分泌的作用单个核细胞增殖：流式检测CFSE标记外周血单个核细胞增殖情况，见图3A，B。单独培养组、植物凝集素组、植物凝集素联合脐带间充质干细胞组、植物凝集素联合胎盘间充质干细胞组增殖细胞比例分别为(1.000±0.173)% 、(44.770±0.914)%、(28.030±2.451)%、(27.000±1.595)%。与植物凝集素组比较，脐带和胎盘间充质干细胞均明显抑制了植物凝集素刺激的单个核细胞增殖(P < 0.05)，但不能完全抑制单个核细胞的增殖；两干细胞干预组相比较差异无显著性意义(P > 0.05)。培养上清干扰素γ分泌水平：采用ELISA检测各组培养上清干扰素γ分泌水平，结果见图3C。单独培养组、植物凝集素组、植物凝集素联合脐带间充质干细胞组、植物凝集素联合胎盘间充质干细胞组干扰素γ分泌水平分别为(491.0±31.2)，(1 657.0±128.1)，(926.4±36.6)，(770.9±7.5) μg/L。与植物凝集素组比较，两干细胞干预组干扰素γ量均显著减低(P < 0.01)；植物凝集素联合脐带间充质干细胞组干扰素γ量高于植物凝集素联合胎盘间充质干细胞组(P < 0.05)，结果表明，胎盘间充质干细胞可能比脐带间充质干细胞具有更强的抗炎功能。 2.4 两种干细胞对急性GVHD小鼠临床症状及生存时间的影响临床症状：单纯照射组小鼠从第7天开始出现死亡，到第13天全部死亡，体质量减轻，活动减少，但未出现GVHD症状。同基因及异基因骨髓移植组在21 d的观察期内均未出现GVHD症状。急性GVHD组在移植后第6天开始出现弓背(图4A)、体质量明显减轻(图5A)、活动减少、颤抖、腹泻症状(图4B)，而脐带和胎盘干细胞组也发生了GVHD症状，但症状明显减轻。移植后11 d临床症状评分结果显示(图5B)，急性GVHD组为7.40±0.75(n=5)，脐带和胎盘干细胞组分别为4.43±0.43(n=7)、4.00±0.26(n=6)，脐带和胎盘干细胞组临床症状评分低于急性GVHD组(P < 0.01)。小鼠生存情况：通过kaplan-meier 的方法绘制生存曲线，Log-rank 分析方法比较各组小鼠生存率之间的差异，结果显示见图5C：单纯照射组小鼠在移植后13 d全部死亡；同基因骨髓移植组、异基因骨髓移植组、脐带干细胞组、胎盘干细胞组小鼠存活率分别为71.43%、62.50%、0、54.55%及60%。与急性GVHD组相比较，脐带和胎盘间充质干细胞均可延长小鼠的生存时间(P < 0.05)，但两干细胞组间比较差异无显著性意义(P > 0.05)。 2.5 皮肤、肝脏、小肠组织的病理变化移植后2周，取急性GVHD组、脐带干细胞组和胎盘干细胞组发生GVHD症状的小鼠皮肤、肝脏和小肠组织，进行苏木精-伊红染色，观察组织病理变化，结果见图6，各组发病小鼠皮肤组织变薄，均未见明显炎症细胞浸润；肝脏病理切片显示，急性GVHD组小鼠肝脏发生了多发性坏死病灶，大量炎症细胞浸润，而脐带和胎盘干细胞组小鼠肝脏仅有小的坏死病灶，可见少量炎症细胞浸润或在肝汇管区可见炎症因子浸润，较急性GVHD 组明显减少；各组小肠组织均可见小肠黏膜绒毛变性、脱落，隐窝受损，未见明显差异。"

References

[1] Baron F,Storb R.Mesenchymal stromal cells: a new tool against graft-versus-host disease? Biol Blood Marrow Transplant. 2012;18(6):822-840.

[2] Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement. Cytotherapy.2006;8:315-317.

[3] Lu LL,Liu YJ,Yang SG, et al.Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica.2006;91:1017-1026.

[4] Yen BL,Huang HI,Chien CC,et al.Isolation of multipotent cells from human term placenta.Stem Cells.2005;23:3-9.

[5] Tondreau T,Lagneaux L,Dejeneffe M,et al.Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential.Cytotherapy.2004;6:372-379.

[6] Le Blanc K,Rasmusson I,Sundberg B,et al.Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet.2004; 363: 1439-1441.

[7] Muroi K,Miyamura K,Okada M,et al. Bone marrow-derived mesenchymal stem cells (JR-031) for steroid-refractory grade III or IV acute graft-versus-host disease: a phase II/III study. Int J Hematol.2016;103:243-250.

[8] Galipeau J.The mesenchymal stromal cells dilemma--does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy. 2013;15:2-8.

[9] Wu Y,Wang Z,Cao Y,et al.Cotransplantation of haploidentical hematopoietic and umbilical cord mesenchymal stem cells with a myeloablative regimen for refractory/relapsed hematologic malignancy.Ann Hematol.2013;92:1675-1684.

[10] Jang MJ,Kim HS,Lee HG,et al.Placenta-derived mesenchymal stem cells have an immunomodulatory effect that can control acute graft-versus-host disease in mice.Acta Haematol.2013;129(4):197-206.

[11] Cooke KR,Kobzik L,Martin TR,et al.An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood.1996;88:3230-3239.

[12] Yang ZX,Han ZB,Ji YR,et al.CD106 identifies a subpopulation of mesenchymal stem cells with unique immunomodulatory properties.PloS One.2013;8:e59354.

[13] Manochantr S,U-pratya Y,Kheolamai P,et al. Immunosuppressive properties of mesenchymal stromal cells derived from amnion, placenta, Wharton's jelly and umbilical cord.Intern Med J.2013;43(4):430-439.

[14] Ferrara JL,Levine JE,Reddy P,et al.Graft-versus-host disease. Lancet. 2009;373:1550-1561.

[15] Ferrara JL,Reddy P.Pathophysiology of graft-versus-host disease.Semin Hematol.2006;43:3-10.

[16] Teshima T,Ferrara JL.Understanding the alloresponse: New approaches to graft-versus-host disease prevention.Semin Hematol.2002;39(1):15-22.

[17] Murphy WJ,Welniak LA,Taub DD,et al.Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.J Clin Invest.1998;102:1742-1748.

[18] Soleymaninejadian E,Pramanik K,Samadian E.Immunomodulatory properties of mesenchymal stem cells: cytokines and factors.Am J Reprod Immunol.2012;67:1-8.

[19] Yagi H,Soto-Gutierrez A,Parekkadan B,et al.Mesenchymal stem cells: Mechanisms of immunomodulation and homing. Cell Transplant.2010;19:667-679.

[20] Arnberg F,Lundberg J,Olsson A,et al.Intra-arterial Administration of Placenta-Derived Decidual Stromal Cells to the Superior Mesenteric Artery in the Rabbit: Distribution of Cells, Feasibility, and Safety.Cell Transplant.2016;25(2): 401-410.

[21] Kean TJ,Lin P,Caplan AI,et al.MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation.Stem Cells Int.2013;2013:732742.

[22] Aggarwal S,Pittenger MF.Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood.2005; 105:1815-1822.

[23] Waterman RS,Tomchuck SL,Henkle SL,et al.A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype. PloS One.2010;5:e10088.