Moreover, the reduced proportion of Treg cells in EAU rats was restored upon hAEC treatment during disease development (Fig. therapeutic strategy is urgently required. The present study investigated the therapeutic efficacy of human amniotic epithelial cells (hAECs) on autoimmune uveitis in a rat model. Herein, experimental autoimmune uveitis (EAU) was induced in rats via a subcutaneous injection of interphotoreceptor retinoid-binding protein. EAU rats were treated with hAECs or the vehicle solution via a subretinal injection on day 0 and day 6 after immunization, and rats were sacrificed on day 12 and day 18 for further analysis. The pathological development of EAU was evaluated by slit lamp microscopy. Immune cell infiltration and retinal structure damage were examined by histological examination of hematoxylin TGR-1202 and eosin (H&E) and immunofluorescence staining. T-cell subsets were detected by flow cytometry, and the levels of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). hAEC treatment ameliorated the pathological progression of EAU and preserved the retinal structure organization and thickness, especially in the preventive group that received a subretinal injection on day 0. Moreover, hAECs RBM45 inhibited the retinal infiltration of macrophages and T-cells. Mechanistically, hAECs modulated the balance of T-cell subsets by downregulating T helper (Th)17 cells and upregulating T regulatory (Treg) cells, as confirmed by decreased interleukin (IL)-17 and increased IL-10 levels in the spleens and lymph nodes of EAU rats. Furthermore, hAECs improved the local cytokine environment in EAU rats by suppressing the monocyte chemoattractant protein (MCP)-1, IL-17 and interferon (IFN)- levels and enhancing the IL-10 in the aqueous humor. Therefore, subretinal transplantation of hAECs in EAU rats ameliorated ocular inflammation, preserved the retinal structure and coordinated the immune balance. The current study provides a novel therapeutic strategy for autoimmune uveitis and related ocular inflammatory diseases in the clinic. H37RA (Sigma-Aldrich). To evaluate the therapeutic effect of hAECs on EAU, EAU rats were injected with hAECs on day 0 and day 6 after immunization (termed as preventive group and therapeutic group, respectively). EAU rats injected with a vehicle solution of balanced salt solution (BSS) at the same time points were set as control groups. 3105 hAECs in 2 l BSS or equal volume of BSS were injected into EAU rats by subretinal injection. Rats were sacrificed on day 12 and day 18 after immunization in different groups for further analysis. hAEC Isolation and Culture Human amniotic membranes were obtained with written and informed consent from healthy mothers undergoing Cesarean section. Human placentas were obtained from healthy mothers who provided TGR-1202 written informed consent after uncomplicated elective Cesarean section. The procedure was approved by the Institutional Patients and Ethics Committee of the International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University School of Medicine. All donors were negative for hepatitis A, B, C, and D as well as human TGR-1202 immunodeficiency virus (HIV)-I and antibody (TPAB). hAECs were isolated from the collected placenta. In brief, the amniotic membrane was peeled from the placental chorion and washed in Hanks balanced salt solution (HBSS, Thermo Scientific, MA, USA) to discard blood cells. TGR-1202 The amniotic membrane was digested with 0.25% trypsin (ethylenediaminetetraacetic acid) for 30 min at 37C in a water bath. Two volumes of complete culture medium (F12/Dulbeccos modified Eagles medium containing 10% KnockOut Serum Replacement (KSR), 2 mM L-glutamine, 1% nonessential amino acid, 55 M 2-mercaptoethanol, 1 mM sodium pyruvate, 1% antibiotic-antimycotic (all from Thermo Scientific, Waltham, MA, USA) and 10 ng/ml epidermal growth factor (Peprotech, Rocky Hill, NJ, USA)) were added to the trypsin digestion medium, and the cell suspension was centrifuged for 10 min at 300test or two-way analysis of variance (ANOVA) followed by Tukeys multiple comparison test. = 6 in each group. *< 0.05; **< 0.01; ***< 0.001. Statistical analysis was performed using an unpaired Students test (B) as well as a two-way ANOVA and Tukeys multiple comparison test (C, D). A representative slit lamp image of a normal control is shown in (E). Scale bar=1 mm. ANOVA: analysis of variance; BSS: balanced salt solution; EAU: experimental autoimmune uveitis; hAEC: human amniotic epithelial cells; SEM: standard error of the mean. hAECs Ameliorate Retinal Structure Damage To investigate the effect of hAEC treatment on tissue injuries, retinal structure changes were analyzed by histological examination. Consistent with the pathological observation, the retinal structure in the control groups was severely damaged and disorganized with massive inflammatory cell infiltration into the vitreous cavity and retina around day 12, which corresponded to the peak of ocular inflammation of EAU (Fig. 3A with quantification in 3B). The progressive retinal injury further led to decreased retinal thickness around day 18 in control groups (Fig. 3C). In contrast, less severe retinal damage was observed in the therapeutic group, and even better improvement in the preventive group on day.
Moreover, the reduced proportion of Treg cells in EAU rats was restored upon hAEC treatment during disease development (Fig
