The Mechanism Of Human Umbilical Cord Mesenchymal Stem Cells In Immunomodulation

Part I:Interactions between human umbilical cord mesenchymal stem cells and peripheral blood mononuclear cells:respective roles of IL-1β, TNF-a, IFN-y and cell deathBackground:Mesenchymal stromal cells (MSCs) are attractive cells due to their capacity of long-term ex vivo proliferation, multilineage differentiation potential and immunomodulatory properties. These cells have emerged as powerful tools in tissue engineering and regeneration. Human umbilical cord mesenchymal stem cells were isolated from umbilical cord with higher accessibility and fewer ethical constraints than Bone Marrow holding great promise as an alternative. hUC-MSCs are believed to be more primitive than MSCs derived from more mature tissue sources and to have intermediate properties between embryonic and adult stem cells. Moreover, hUC-MSCs are available in potentially large quantities, have a fast proliferation rate, a great expansion capability, do not induce teratomas and harbor strong immunomooulatory capacities. In addition, recent findings strongly suggest that the immunomodulatory capacity of MSCs is not constitutive, which depends on the (pre-)activation of these cells and on their surrounding milieu. The potential immune-regulation of MSC under these condition is not clear.Objective:Previous works have shown that hUC-MSCs possess profound immunomodulatory capacities through IL-1 stimulation produced by peripheral blood mononuclear cells (PBMCs), their main cellular partner in most pathophysiological and therapeutic situations. The present study was designed to explore the role of TNF-a in these interactions.Methods:(1)hUC-MSCs were isolated from from umbilical cord obtained from donors with written informed consent, and its phenotype was analyzed by flow cytometry. (2) hUC-MSCs were cultured under inducing media, and then cells were stained with Oil red for lipid and alizarin red for calcium deposition, which use for hUC-MSCs identification.(3) Establish the best process for collecting the condition media from PBMCs for further study. PBMC CM were used to stimulate the BM-MSCs and hUC-MSCs, the supernatant were collected for ELISArray. In the meantime, in order to find the key cytokines in hUC-MSCs regulation, test the related cytokine from different supernatant, including PBMC CM, hUC-MSCs basal and treatment. (4)To investigate the different effect of IL-1β and TNF-α on hUC-MSCs, recombinant Il-1β, TNF-α and relating inhibitor of signal pathway (including LY2940002, JNK inhibitor II, BAY 11-7082, SC-514, U0126, SB203580) were used to stimulate the hUC-MSCs, the supernatants were collected for IL-6 and MCP-1 ELISA.(5)To test the effect TNF-a on cell death, recombinant TNF-a, IFN-y and death receptors ligands-FasL and TRAIL were used to simulate the hUC-MSCs. And in some experiment, adding pan-caspase inhibitor zVAD-fmk or specific inhibitor of RIPK1 kinase activity Necrostatin 1 in the culture system. Cell death were measured via CellTiter Glo(?) Luminescent Cell Viability Assay or PI&FITC Annexin V stain. In the same time, the supernatant from different groups were collect and IL-6 and MCP-1 were tested by ELISA.(6) hUC-MSCs were pretreated with small-molecule antagonist of IAP, GDC-0152 for 2 hours and then added TNF-a. NF-kb translocation were measured by immunofluorescence staining under laser scanning confocal microscopy or western blot.Results:(1) The isolated hUC-MSCs expressed CD44, CD73, CD90 and CD105, and negative for CD34, CD 19, CD31, CD45 and HLA-DR. Under inducing media, they could differential to osteoblast and adipocyte. All fulfilled the minimal phenotypic and functional criteria proposed by International Society for Cellular Therapy. (2) The conditional media collected from 4×104/ml PBMC for 24 hours was the best conditional media(CM). And in the following stimulating experiment, all CM were collected by this method. (3)After stimulated with PBMC CM, the cytokines secretion from hUC-MSCs were similar as BM-MSCs except that hUC-MSCs expressed high level of IL-1β and GM-CSF. (4) II-1β and TNF-α are two major cytokines involving in this process. IL-1β appears to be the master of these interactions, was able to generate TNF-a production from PBMCs. The TNF-α produced was then able to clearly contribute to IL-6 and MCP-1.(5) High concentration of TNF-αcould induce hUC-MSCs death. Death receptor FasL itself didn’t induce cell death and when combine with TNF-α,it didn’t enhance TNF-αeffect. In contrast, another death receptor-TRAIL, which could induce hUC-MSCs death. In addition, when combined with TNF-α, it enhanced cell death. Also, IFN-γ enhanced TNF-αinducing cell death, but it mechanism were different from TRAIL. When combined TNF-αand TRAIL, the cell death was caused by apoptosis, but with IFN-γ, this process was major caused by necrosis. (6) TNF-αcould stimulate hUC-MSCs producing MCP-1 and IL-6. This process was indirectly linked to cell death and involved inhibition of apoptosis proteins dependent NF-kB activity.Conclusions:we defined an important role of soluble cytokines in the interactions between hUC-MSCs and PBMCs. I1-1β and TNF-αare two major cytokines involving in this process. IL-1β appears to be the master of these interactions, was able to generate TNF-αproduction from PBMCs. The TNF-αproduced was then able to clearly contribute to IL-6 and MCP-1 production at low concentrations. However, at high concentrations and in association with TRAIL and IFN-y, TNF-a was also able to induce hUC-MSC death, again associated with IL-6 but mainly with MCP-1 production. The effect of TNF-a on hUC-MSCs involved inhibition of apoptosis proteins dependent NF-kB activity. The characterization of the local interactions of these crucial factors strongly contributes to the understanding the patho-physiological role of MSCs in the maintenance of tissue homeostasis.Part Ⅱ:CD4+type 1 T regulatory involved in the immunosuppression of human umbilical cord mesenchymal stem cellsBackground:Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. Human umbilical cord mesenchymal stem cells were isolated from umbilical cord with higher accessibility and fewer ethical constraints and more proliferation than Bone Marrow holding great promise as an alternative. Different mechanisms have been proposed as mediating the immunomodulation of MSCs that involve direct cell-cell contact, soluble factors and regulatory T-Cell generation. In other hand, it is recently discovered that Trl cells show strong immunosuppressive activity, with high level of IL-10 and independent from FoxP3 expression. It is not clear the effect of hUC-MSCs on this type of cells.Objective:Establish the co-culture system for hUC-MSCs and PBMCs, investigating the effects of hUC-MSCs on Tr1 generation.Methods:(1)hUC-MSCs were isolated from umbilical cord obtained from donors with written informed consent, and its phenotype was analyzed by flow cytometry. In addition, hUC-MSCs were cultured under inducing media, and then cells were stained with Oil red for lipid and alizarin red for calcium deposition, which use for hUC-MSCs identification.(2) Establish co-culture and transwell culture system. Plated 105-2＝105/well hUC-MSCs in 6-well plate. After adhering the plate, MSCs were irradiated (54Gy, X-ray) immediately before starting the coculture with PBMCs, which inhibit the proliferation of MSCs. Next, added fresh isolating PBMCs from health donor, and the ratio of PBMC and hUC-MSCs was 10:1. In some groups, added 5μg/ml phytohemagglutinin for stimulating the PBMCs. (3) At the end of culture, collect the cells and stain them with PE-antiCD4 (Sk3,1:100), FITC-antiCD49b(AK7,4:100), APC-antiLAG3 (FAB2319A,4:100) and preCP-Cy5.5-antiCD45RA (MI100,1: 100)for observing the effect of hUC-MSCs on Trl generation. (4) After culture, collected the cells and sorting the CD49b-/LAG3-and CD49b+/LAG3+cells via Flow cytometer. After sorting, plated 105/well sorting cells in 96-well plate, continued to culture for 24 hours, and then collected the suspernatant for IL-10 ELISA. (5) After culture, some groups were used for cytokine stain by flow cytometer to determine which type of cell secreting the IL-10.Results:(1)The isolated hUC-MSCs expressed CD44, CD73, CD90 and CD105, and negative for CD34, CD 19, CD31, CD45 and HLA-DR. Under inducing media, they could differential to osteoblast and adipocyte. All fulfilled the minimal phenotypic and functional criteria proposed by International Society for Cellular Therapy.(2)hUC-MSC induced the production of Trl. In co-culture system, no matter with or with PHA, hUC-MSCs significant increased the Trl proportion in lymphocytes(without PHA, single culture:co-culture=2.8±0.41 vs 4.59 ±0.53; and with PHA, single culture:co-culture=3.107±0.19 vs.11.07±0.85, p<0.05).(3) Through transwell system, we found that soluble cytokines were involved in the effect of hUC-MSC induing Trl. There is no significant different between the normal co-culture group and transwell group(11.07±0.85 vs.10.60±0.47, P>0.05) (4) The IL-10 producing of LAG3+/CD49+ cells were determined by ELISA after sorting by flow cytometer. The double positive cells indeed produced more IL-10 than double negative cells. These two selective makers could be used for Trl identification and purification. (5) Cytokine stain were used for determining the source of IL-10. In the co-culture system, the IL-10 was major produced by CD 14+positive cells. Since IL-10 is critical for Trl cell induction, we believed that CD 14+cells participated in the process of hUC-MSCs inducing Trl.Conclusions:The induction of Trl is an mechanism through which MSCs are able to promote immunosuppression. CD 14+monocyte were involved in this process. In addition, the use of LAG3 and CD49b makes it feasible to identify Tr1 cells and purify Tr1 cells for further study.