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. 2016 Apr 1:18:77.
doi: 10.1186/s13075-016-0979-0.

Human embryonic stem cell-derived mesenchymal stromal cells ameliorate collagen-induced arthritis by inducing host-derived indoleamine 2,3 dioxygenase

Elena Gonzalo-Gil  1 , María J Pérez-Lorenzo  1 , María Galindo  1   2 , Rafael Díaz de la Guardia  3 , Belén López-Millán  3 , Clara Bueno  3 , Pablo Menéndez  3   4 , José L Pablos  1   2 , Gabriel Criado  5   6
Affiliations

Human embryonic stem cell-derived mesenchymal stromal cells ameliorate collagen-induced arthritis by inducing host-derived indoleamine 2,3 dioxygenase

Elena Gonzalo-Gil et al. Arthritis Res Ther. .

Abstract

Background: The immunosuppressive and anti-inflammatory properties of mesenchymal stromal cells (MSC) have prompted their therapeutic application in several autoimmune diseases, including rheumatoid arthritis. Adult MSC are finite and their clinical use is restricted by the need for long-term expansion protocols that can lead to genomic instability. Inhibition of Smad2/3 signaling in human pluripotent stem cells (hPSC) provides an infinite source of MSC that match the phenotype and functional properties of adult MSC. Here, we test the therapeutic potential of hPSC-MSC of embryonic origin (embryonic stem cell-derived mesenchymal stromal cells, hESC-MSC) in the experimental model of collagen-induced arthritis (CIA).

Methods: CIA was induced in DBA/1 mice by immunization with type II collagen (CII) in Complete Freund's Adjuvant (CFA). Mice were treated with either a single dose (10(6) cells/mouse) of hESC-MSC on the day of immunization (prophylaxis) or with three doses of hESC-MSC every other day starting on the day of arthritis onset (therapy). Arthritis severity was evaluated daily for six weeks and ten days, respectively. Frequency of Treg (FoxP3(+)), Th1 (IFNγ(+)) and Th17 (IL17(+)) CD4(+) T cells in inguinal lymph nodes (ILN) was quantified by flow cytometry. Serum levels of anti-CII antibodies were determined by ELISA. Detection of hESC-MSC and quantification of murine and human indoleamine 2,3 dioxygenase (IDO1) expression was performed by quantitative real-time PCR. Statistical differences were analyzed by ANOVA and the Mann-Whitney U test.

Results: Administration of hESC-MSC to mice with established arthritis reduced disease severity compared to control-treated mice. Analysis of CD4 T cell populations in treated mice showed an increase in FoxP3(+) Treg and IFNγ(+) Th1 cells but not in Th17 cells in the ILN. Anti-CII antibody levels were not affected by treatment. Migration of hESC-MSC to the ILN in treated mice was associated with the induction of murine IDO1.

Conclusion: Treatment with hESC-MSC ameliorates CIA by inducing IFNγ(+) Th1 cells and IDO1 in the host. Thus, hESC-MSC can provide an infinite cellular source for treatment of rheumatoid arthritis.

Keywords: Arthritis; Cytokines; Human embryonic stem cell-derived mesenchymal stromal cells; Indoleamine 2,3 dioxygenase; T cells.

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Figures

Fig. 1
Fig. 1
Administration of embryonic stem cell-derived mesenchymal stromal cells (hESC-MSC) ameliorates established collagen-induced arthritis (CIA). a Progression of arthritis after prophylactic administration of hESC-MSC in one experiment representative of two performed. Incidence (left panel) and arthritis score (right panel) were comparable to the control group when hESC-MSC were administered on the day of type II collagen (CII) immunization. b Dose-response effect of the therapeutic administration of hESC-MSC on arthritis score. Statistical differences between control and single-dose groups are indicated. c Amelioration of CIA induced by administration of three doses (arrowheads) of hESC-MSC is sustained in mice with established disease. Data pooled from three independent experiments with four to ten mice per experimental group. d Representative hematoxylin-eosin staining of joints after treatment with hESC-MSC in mice with established disease. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 2
Fig. 2
Embryonic stem cell-derived mesenchymal stromal cells (hESC-MSC) administration alters the regulatory T cell (Treg)/effector T cell (Teff) balance in mice with collagen-induced arthritis. The frequency of Treg and Teff cells was analyzed by flow cytometry in the inguinal lymph nodes (ILN) 10 days after arthritis onset in mice with established disease. a Representative flow cytometry plots and quantification of CD4 Treg and Teff cell frequency in hESC-MSC-treated and control mice. b Treg/T helper (Th)17 ratio is significantly increased in hESC-MSC-treated mice compared to the control group. Data shown are values from individual mice and the mean of experimental groups. Statistical differences were analyzed by the Mann-Whitney U test. *P < 0.05; **P < 0.01. PBS phosphate-buffered saline, IFN interferon
Fig. 3
Fig. 3
Type II collagen (CII)-specific T cell responses in vitro and anti-CII antibody levels in embryonic stem cell-derived mesenchymal stromal cells (hESC-MSC)-treated mice. a Proliferation and cytokine production in response to CII by inguinal lymph node cells from hESC-MSC and phosphate-buffered saline (PBS)-treated mice with established collagen-induced arthritis. Data represent the mean ± the standard error of the mean of the experimental groups. b Comparable levels of anti-CII IgG1 and IgG2a were measured in sera from control and hESC-MSC-treated mice. Data shown are values from individual mice and the mean of experimental groups. Statistical differences were analyzed by the Mann-Whitney U test and no significant differences were found. NS non-stimulated
Fig. 4
Fig. 4
Embryonic stem cell-derived mesenchymal stromal cells (hESC-MSC) migrate to the inguinal lymph nodes (ILN) and induce expression of murine indoleamine 2,3 dioxygenase (IDO1). a Expression of HLA-C transcripts in the ILN of mice with collagen-induced arthritis (CIA) treated with hESC-MSC compared to the phosphate-buffered saline (PBS)-treated group. b Expression of murine IDO1 (mIDO1) in the ILN of mice with CIA treated with vehicle (PBS) or with hESC-MSC and showing evidence of migration (HLA+) or not (HLA–). Data show values from individual mice and the mean of the experimental groups. Statistical differences were analyzed by Kruskall-Wallis analysis of variance and Dunn’s multiple comparisons test. *P < 0.05; **P < 0.01
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