Immune-modulation Effect Of Cord Blood-derived Multipotent Stem Cells On Lymphocytes Of Patients With Alzheimer’s Disease

Background:Alzheimer’s disease (AD) is a degenerative disease, one of the main pathogenesis is Aβ deposition, in which inflammation is an important component to promote the progress of AD pathology physiology. Studies have shown that immune-inhibit functional regulatory T (Treg) cells decreased in the peripheral blood of AD, and there is excessive lymphocyte immune responses and inflammatory responses. Another study showed that the pro-inflammatory cytokine IL-1 in peripheral blood is an important risk factor for AD, anti-inflammatory cytokines IL-4, IL-10 can reduce Aβ-induced inflammation, while AD displayed increased concentration of IL-1, and decreased concentration of IL-4 and IL-10 in peripheral blood. Thus immunotherapy is required. Immunomodulatory functional bone marrow-derived mesenchymal stem cells (MSCs) and embryonic stem cells(ESCs) have obvious technical and ethical limitations. The cord blood derived-multipotent stem cells(CB-SCs) have a good immunomodulatory effects, have a wide source, easy to be extracted and cultured, and there is no limit ethics. The experiments is to co-culture the CB-SCs and the peripheral blood lymphocytes of patients with AD in vitro and to explore the therapeutic potential of CB-SCs for AD.Method:1. Stem cell preparationsUmbilical cord blood samples were collected from healthy maternal infant at Ji’nan Centre Hospital. Mononuclear cells were isolated with Ficoll-Hypapue, then red blood cells were removed using red blood cell lysis buffer. The remaining Cells were cultured in X-VIVO15 serum-free culture medium.2. Identification of CB-SCsThe morphology of CB-SCs were observed using inverted microscope. The CB-SCs were recovered by using 0.25% trypsin-EDTA, digestion was terminated by X-VIVO15 containing 10% FBS. After washing 3 times by PBS, the surface antigens of CB-SCs were detected by immunofluorescence labeling. The selected surface markers were CD34 and CD45.3. Preparation of lymphocytes of AD patient10 mL fasting venous blood was collected form AD patients, the same method of separation of the peripheral blood mononuclear cells in patients with AD was inoculated in the culture medium of RPMI1640 containing 20% FBS in 37℃,5% CO2 full wet conditions and incubated 24 h, collecting the suspended lymphocytes. The ratio of co-cultured CB-SCs and lymphocytes is 1:3, the experimental group and the control group were set lymphocyte stimulation respectively, the concentration of lymphocyte stimulator PHA-P is 4 mg/L. To co-cultured for 3 days in X-VIVO15 serum-free medium.4. Counting the lymphocytes within cell suspensionCollect the cells within cell suspension and to count the number of the collected lymphocytes.5. ELISA testThe levels of anti-inflammation and pro-inflammatory factors in the supernatant were detected with the ELISA test. The concentration of IL-1、 IL-4 and IL-10 be detected according to the instructions from the company Blue Gene.6. Flow cytometryCollect the cells within cell suspension and to be detected by flow cytometry. The surface antigens CD4 and CD8 of CB-SCs were detected by immunofluorescence labeling. Surface antigens CD4、CD25 and the intracellular antigens Foxp3、IL-10 and TGF-betal were detected according to the instructions of human regulatory T cell staining kit from the company of eBioscience.Results:1. Morphology and the markers of CB-SCsPrimary CB-SCs displayed round or oval, and adhering to the bottom of Petri dish tightly. The immunofluorescence staining by flow cytometry showed positive rate of CD45 100% and CD34 2.5%.2. CB-SCs showed an inhibitory effect on lymphocyte proliferationCB-SCs can inhibit the number of lymphocytes, and can inhibit the gathers with the existed of lymphocyte stimulator PHA-P.3. CB-SCs regulate the concentration of cytokines in the supernatant.CB-SCs can decrease the pro-inflammatory IL-1 of supernatant obviously, while increase the anti-inflammatory IL-4 and IL-10 apparently.4. CB-SCs can regulate the subsets of co-cultured T lymphocytes.By flow cytometry data analysis, CB-SCs can significantly reduce the ratio of CD4+ /CD8+T lymphocyte which co-cultured with them. CB-SCs can increase the positive rate of CD4+CD25+Foxp3+Tregs in CD4+T cells, and increase the positive rate of Foxp3+cells in CD4+CD25+T cells. While with the exist of PHA-P, CB-SCs increase the positive rate of Foxp3、IL-10 and TGF-βin CD4+CD25+T cells. CB-SCs can also down regulate the CD4+CD25+Foxp3" cells, which means that decrease the active CD4+T cells.Conclusions:1. It has been established that CB-SCs make an apparent expression of CD45 and show negative expression of CD34.2. CB-SCs can inhibit immune response of co-cultured AD patients’ lymphocytes. And mitigate the inflammatory response of lymphocytes.3. CB-SCs regulate the proportion of lymphocyte subsets, and up-regulate the rate of Tregs and enhance their immune suppression.