Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis

Abstract

Human amniotic epithelial cells (hAEC) have stem cell-like features and immunomodulatory properties. Here we show that hAEC significantly suppressed splenocyte proliferation in vitro and potently attenuated a mouse model of multiple sclerosis (MS). Central nervous system (CNS) CD3(+) T cell and F4/80(+) monocyte/macrophage infiltration and demyelination were significantly reduced with hAEC treatment. Besides the known secretion of prostaglandin E2 (PGE2), we report the novel finding that hAEC utilize transforming growth factor-β (TGF-β) for immunosuppression. Neutralization of TGF-β or PGE2 in splenocyte proliferation assays significantly reduced hAEC-induced suppression. Splenocytes from hAEC-treated mice showed a Th2 cytokine shift with significantly elevated IL-5 production. While transferred CFSE-labeled hAEC could be detected in the lung, none were identified in the CNS or in lymphoid organs. This is the first report documenting the therapeutic effect of hAEC in a MS-like model and suggest that hAEC may have potential for use as therapy for MS.

Figure 1. Dose-dependent suppression of splenocyte proliferation by hAEC.

(A) ConA (5 µg/ml) stimulated proliferation of splenocytes from naive mice was dose-dependently inhibited by hAEC in hAEC∶splenocyte ratios ranging from 1∶5 to 1∶10240 (n = 5). (B) Proliferation of splenocytes from EAE mice stimulated by 1 µg/ml and 10 µg/ml MOG peptide was inhibited by hAEC (n = 3) at hAEC∶splenocyte ratios of 1∶10 and 1∶40. Splenocyte proliferation is expressed as counts per minute (cpm) of ³H-thymidine incorporation. All data are means±SEM.

Figure 2. hAEC infusion potently ameliorated EAE and reduced spinal cord pathology.

EAE was induced by immunization with 200 µg MOG_35–55 peptide in 100 µl CFA followed by 350 ng pertussis toxin and the clinical scores were evaluated. (A) hAEC (2×10⁶) injected intravenously on day 9 after EAE induction ameliorated disease development (n = 25 in total) while control animals developed EAE (n = 26 in total). Data shown are combined results of three independent experiments each using pooled hAEC from n = 4 donors. (B) Spinal cord sections were stained by H&E and LFB for assessment of cellular infiltrates and demyelination, respectively. Representative spinal cord sections from hAEC-treated mice showed intact myelin sheath (blue color) from LFB staining and no cellular infiltrate from H&E staining. EAE control sections show regions of demyelination (arrows) and cellular infiltrate (arrow heads). (C) hAEC-treated mice (n = 9) showed significantly lower histological scores in both H&E and LFB assessments compared to EAE control (n = 5). All data are means±SEM.

Figure 3. Reduced CD3⁺ T cells and F4/80⁺ monocytes/macrophages in CNS.

Average number of CD3⁺ T cells (A) and F4/80⁺ cells (B) enumerated per spinal cord section (mean of 3 sections per mouse) showed significant reduction in hAEC-treated mice (n = 8) compared to EAE control (n = 4). Photomicrographs showing representative CD3 (black arrowheads) and F4/80 (white arrowheads) staining in hAEC-treated and EAE control spinal cords. All data are means±SEM.

Figure 4. hAEC-treated mice showed reduced splenocyte proliferation and Th2 cytokine shift following stimulation in vitro with MOG peptide but serum MOG antibody and lymphocyte populations in spleen and lymph nodes remained unaffected.

(A) Splenocytes from MOG-immunized and hAEC-treated mice (n = 8) stimulated with MOG peptide proliferated significantly lower than EAE control mice (n = 5) at 10 and 100 µg/ml MOG peptide. ConA (10 µg/ml) stimulated proliferation from both groups was similar. (B) Cytokines in supernatants from splenocyte cultures in (A) stimulated with 10 µg/ml of MOG for 72 hr were analyzed. IL-5 was significantly elevated. (C) MOG-specific antibodies in sera of hAEC-treated mice (n = 8) and EAE control (n = 5) were not significantly different. (D) FACS analysis of T cell subsets (CD4⁺, CD8⁺, CD4⁺CD25⁺ FoxP3⁺) and B cells (CD19⁺) from the spleen (upper panel) and from inguinal lymph node (lower panel) showed no significant differences between hAEC-treated mice (n = 9) and EAE control (n = 5). All data are means±SEM. NS: Not significant.

Figure 5. TGF-β blocking antibody and indomethacin reversed hAEC suppression of splenocyte proliferation stimulated by ConA and MOG peptide.

hAEC∶splenocyte at 1∶40 ratio were co-cultured with splenocytes from EAE mice and MOG (10 µg/ml), or with splenocytes from naïve mice and ConA (5 µg/ml). Data shown is from n = 3 experiments each using pooled cells from n = 4 donors. Addition of TGF-β neutralizing antibody (A) or PGE2 antagonist indomethacin (B) significantly reduced the suppression exerted by hAEC in both settings. Data presented as percentage of suppression compared with TGF-β neutralizing antibody or PGE2 antagonist treated control groups (splenocytes+MOG/ConA), respectively. All data are means±SEM.

Figure 6. Tracking of CFSE-labeled hAEC.

Lung section obtained from mice injected with CFSE-labeled hAEC after MOG immunization and organ collected 7 days later (5 mice for each group, 9–16 sections per organ were examined). (A) Frozen lung section staining with DAPI. (B) Same field as (A) showing CFSE positive cells. (C) Overlay of (A) and (B). Insert showing enlarged view of CFSE-DAPI positive cells.