Therapeutic effects of human adipose tissue-derived stem cell (hADSC) transplantation on experimental autoimmune encephalomyelitis (EAE) mice

Abstract

This study is to investigate the therapeutic effects of human adipose tissue-derived stem cell (hADSC) transplantation on experimental autoimmune encephalomyelitis (EAE) in mice. EAE mouse model was established by MOG35-55 immunization. Body weight and neurological function were assessed. H&E and LFB staining was performed to evaluate histopathological changes. Flow cytometry was used to detect Th17 and Treg cells. ELISA and real-time PCR were performed to determine transcription factor and pro-inflammatory cytokine levels. Transplantation of hADSCs significantly alleviated the body weight loss and neurological function impairment of EAE mice. Inflammatory cell infiltration and demyelination were significantly increased, which were relieved by hADSC transplantation. Moreover, the Th17 cells and the ROR-γt mRNA level were significantly elevated, while the Treg cells and the Foxp3 mRNA level were significantly declined, resulting in significantly increased Th17/Treg ratio. This was reversed by the transplantation of hADSCs. Furthermore, serum levels of IL-17A, IL-6, IL-23, and TGF-β, were significantly increased, which could be influenced by the hADSC transplantation. Transplantation of hADSCs alleviates the neurological function impairment and histological changes, and reduces the inflammatory cell infiltration and demyelination in EAE mice, which might be associated with the regulation of Th17/Treg balance.

Figure 1. Morphological observation of cultured hADSCs.

(A) The 2^nd passage cells (100×). (B) The 3^rd passage cells (400×).

Figure 2. Effects of hADSC transplantation on the body weight and neurological function of the EAE mice.

EAE mouse model was established by the immunization with the MOG35-55/CFA emulsion, and 14 days later, these mice were transplanted with hADSCs via tail vein. Animal body weight (A) and neurological function (B) of the mice in the control, EAE model, and hADSC transplantation groups were recorded and assessed since modeling. Compared with the EAE model group, ^##P < 0.01.

Figure 3. Effect of hADSC transplantation on inflammatory cell infiltration in the spinal cord of the EAE mice.

(A) Inflammatory cell infiltration in the spinal cord of the mice in the control, EAE model (EAE + PBS), and hADSC transplantation (EAE + hADSCs) groups was detected with H&E staining (40×, 100×, and 200×, respectively). (B) Statistical analysis of inflammatory cell infiltration. Compared with the control group, **P < 0.01; compared with the EAE model group, ^#P < 0.05.

Figure 4. Effect of hADSC transplantation on demyelination in the EAE mice.

(A) Demyelination in the mice of the control, EAE model (EAE + PBS), and hADSC transplantation (EAE + hADSCs) groups was detected with LFB staining (40×, 100×, and 200×, respectively). (B) Statistical analysis of relative demyelination area. Compared with the control group, **P < 0.01; compared with the EAE model group, ^#P < 0.05.

Figure 5. Effect of hADSC transplantation on the Th17 and Treg cells in the spleen of EAE mice.

(A) Statistical analysis of the percentage of CD4⁺ IL-17A⁺ Th17 cells out of the total CD4⁺ T cells. (B) Statistical analysis of the percentage of CD4⁺ CD25⁺ Foxp3⁺ Treg cells out of the total CD4⁺ T cells. (C) Statistical analysis of the Th17/Treg ratio. (D) The mRNA expression levels of ROR-γt (Th17-specific marker) and Foxp3 (Treg-specific marker) were detected with real-time PCR. Compared with the control group, *P < 0.05, **P < 0.01; compared with the EAE model group, ^#P < 0.05, ^##P < 0.01.

Figure 6. Effect of hADSC transplantation on the levels of pro-inflammatory cytokines in the EAE mice.

(A–D) The serum levels of IL-17A (A), IL-6 (B), IL-23 (C), and TGF-β (D) in the control, EAE model, and hADSC transplantation groups were detected with flow cytometry. (E) The mRNA expression level of IL-17A in the spleen was detected with real-time PCR. Compared with the control group, **P < 0.01; compared with the EAE model group, ^#P < 0.05, ^##P < 0.01.

Figure 7. Homing of PKH67-labeled hADSCs in EAE mice.

The hADSCs were labeled with PKH67 on day 14 of immunization, and tracked in the spleen, brain, and liver at day 8 after injection (400×).