Embryonic Stem Cell Derived Mesenchymal Stem Cell In The Treatment Of Inflammatory Bowl Disease And Its Immunomodulation Property

Background and IntroductionInflammatory bowel disease(IBD)is a kind of digestive disease mediated by abnormal immune responses and includes ulcerative colitis(UC)and Crohn's disease(CD).The mechanism of IBD remains unknown and is associated with multiple factors such as genetic susceptibility,gut microbiota,mucosal barrier,immune response and autophagy.A commonly known mechanims of IBD is intestinal inflammation caused by pathological immune response towards gut commensal bacteria in genetic susceptible people.Therefore,abnormal immune response is the central part and the final pathway of the occurrence of IBD.Mesenchymal stem cell(MSC)is one type of stem cell that both obtain the property of self-renewal and differentiate into multiple lineage.MSC plays an important role in the treatment of immune diseases and regenerative medicine.The source of MSC includes adult tissues,umbilical cord,umbilical cord blood and embryonic stem cells through proper inducement.MSC routinely express certain surface markers and can differentiate into osteoblast,chondroblast and adipocyte(tir-linage differentiate)in vitro under certain conditions.MSC occupies potent immunomodulatory ability and can affect various immune cells.MSC can also affect antigen presenting cells(APC)and cause change in phenotype,ability of antigen presenting,chemokines secretion etc.Due to the central role of abnormal immune response in IBD and the strong immunomodulation ability of MSC,the treatment of this kind of cell in IBD caused much attention.Among various types of MSC,embryonic derived MSC outperform other MSC in some animal models whereas its treatment in MSC still lacks report.Thus this paper learns the immunomodulatory properties and therapeutic efficiency in mouse model of IBD of embryonic derived MSCs.Methods1.The induction and phenotype of embryonic derived MSC(1)ESI-053 cell line was used in this research and it can convert to MSC by stimulation of morphogenetic protein 4(BMP-4)and ALK inhibitor.(2)The basic phenotype of MSC(CD73,CD90,CD105)were stained and tested by flow cytometry.2.Xeno-antigen and immunomodulatory properties of MSC(1)N-Glycolylneuraminic acid(Neu5Gc)is expressed in almost all mammals but not human.This study measured Neu5Gc expression of embryonic derived MSC cultured under different conditions by flow cytometry.(2)Embryonic derived MSC and K562 cells were co-cultured with human NK cells and 7-AAD was detected by flow cytometry to test the killing ability of NK cells towards MSC and K562.(3)Embryonic derived MSCs were co-cultured with pan-T cells and BrdU was stained and analyzed by flow cytometry to test the proliferation of T lymphocytes.3.Embryonic derived MSC in the treatment of mouse Dextran Sulfate sodium(DSS)colitis and its in vivo engraftment(1)C57BL/6 mice were divided to 4 groups.Control group received DPBS/heparin sodium through tail vein.Intravenous group received 1×106 embryonic derived MSC suspend in DPBS/heparin sodium through tail vein.Intraperitoneal group received 1×106 embryonic derived MSC suspend in DPBS/heparin sodium intraperitoneally.UC-MSC group received 1×106 umbilical cord MSC suspend in DPBS/heparin sodium through tail vein.All groups had 2%DSS for 5 days and water for 4 days.Body weight and disease activity index(DAI)were measured daily.Mice were sacrificed on day 9 and colon,spleen,liver,lung,kidney performed HE staining and observed by microscope.The histological severity of inflammation in colon tissue were evaluated under certain criteria and histological activity index(HAI)were recorded.Serum inflammatory cytokines(TNF-?,IFN-?,IL-4,IL-6)were tested using cytokine beads array kit.(2)Embryonic derived MSCs were labeled by CM-Dil.C57BL/6 mice were divided into three group.Control group had no intervention.Intravenous group received 1×106 CM-Dil labeled embryonic derived MSC suspend in DPBS/heparin sodium through tail vein.ntraperitoneal group received 1x106 CM-DiI labeled embryonic derived MSC suspend in DPBS/heparin sodium intraperitoneally.Mice were sacrificed 1 day after injection.Colon,intestine,lung and liver were cryosectioned and stained DAPI.Slides were observed under a fluorescence microscope to measure the engraftment.Results1.The induction and phenotype of embryonic derived MSC(1)Human embryonic stem cell can be induced to MSC through a "trophoblast"intermediate stage.(2)Embryonic stem cell derived MSC express surface markers CD73(91.2%),CD90(90.6%)and CD 105(99.4%).2.Xeno-antigen and immunomodulatory properties of MSC(1)Neu5Gc is expressed in mouse embryonic fibroblasts(MEF)and embryonic stem cell derived MSC cultured on matrigel coated plates(7.85%).Embryonic stem cell derived MSC cultured on gelatin and collagen coated plates have a low expression of Neu5Gc(0.064%,0.85%respectively).(2)In the co-culture of K562 and NK cell,7-AAD increase with the number of NK cell(from 2.35%to 31%).In the co-culture of embryonic stem cell derived MSC and NK cell,7-AAD do not increase with the number of NK cell(from 0%to 0.13%).(3)The proliferation of CD4+T lymphocytes decreases when the number of embryonic stem cell derived MSCs increased(BrdU positive rate decrease from 59.3%to 12.1%).The proliferation of CD8+T lymphocytes also decreases when the number of embryonic stem cell derived MSCs increased(BrdU positive rate decrease from 70.4%to 27.0%).3.Embryonic derived MSC in the treatment of mouse Dextran Sulfate sodium(DSS)colitis and its in vivo engraftment(1)Intravenous administration of embryonic stem cell derived MSC alleviate mouse DSS colitis.The therapeutic effect includes reduced weight loss and DAI score,low levels of serum inflammation cytokines and reduced inflammatory cell infiltration in colon tissue.(2)Intravenously administrated embryonic stem cell derived MSCs mostly locate in lung and liver,only a very small portion of cell can distribute to the lower part of intestines.Conclusions1.Human embryonic stem cell have the potential to be induced to MSC under certain condition and stimulation.The induction includes a“trophoblast”intermediate stage.2.Human embryonic stem cell derived MSCs can escape the killing of NK cell and inhibit the proliferation of T lymphocytes.3.Human embryonic stem cell derived MSCs obtains a therapeutic potential of mouse DSS colitis and the distribution of MSC administrated intravenously includes lung,liver and the lower part of small intestines(only a small portion).