The Characteristics Of SSEA4~+ HUC-MSC And The Role And Mechanism Of HUC-MSC Expressing T Cell Costimulatory Molecules In Tumor Therapy

Objective:Human umbilical cord mesenchymal stem cell(hUC-MSC)has the capacity for multi-potential differentiation and the potential to repair multiple tissue injury.Screening a sub-population with a better multi-potential ability from the heterogeneous hUC-MSC will help to expand the potential clinical application for tissue injury repair.As MSC has a tumor homing capacity,using MSC as a transport carrier to enter and express T cell costimulatory molecules in tumor microenvironment will promote tumor killing effect,which indicates a new method for tumor treatment.The aim of this study is to screen SSEA4+ hUC-MSC by SSEA4 magnetic beads and analyze its characteristics;and investigate the effect and mechanism in tumor therapy of the hUC-MSC with expression of T cell costimulatory molecules.It will provide a new strategy for a better application of hUC-MSC in tissue injury repair,and using MSC as a transport carrier in tumor therapy.Methods:The hUC-MSC was separated from the umbilical cord of the newborn,and the SSEA4+hUC-MSC was screened by SSEA4 magnetic beads.Antibody flow analysis including OCT-4,cell cycle assay,and cell proliferation curve analysis was performed later.Through RT-PCR and qRT-PCR,we analyzed the expression of stem cell totipotent markers of Nanog,OCT4,Sox2 and C-myc.And the SSEA4+hUC-MSC at P1 and P3 were cultured under the condition of paving gelatin for embryoid body induction experiment Then the formed embryoid body was analyzed by immunofluorescence staining and RT-PCR of?-?-tubulin,AFP,?-actin,which are markers of the three types of germ layers.We conducted the osteoblasts and adipoblasts induction experiments of hUC-MSC,SSEA4-cells and SSEA4+cells.In addition,qRT-PCR detection of osteoblasts and lipid-related markers was performed respectively at d0 and d14.The skin damage model of mice with second-degree burn was constructed,and then we injected the hUC-MSC,SSEA4-and SSEA4+hUC-MSC cells to observe the repair of skin injury.The lentivirus plasmid vectors of murine OX40L and 4-1BBL were constructed respectively.hUC-MSC was infected after lentivirus packaging.The tumor migration experiment of hUC-MSC was carried out by transwell culture plate.The tumor model of C57 mice and nude mice were established respectively by injecting the prostate cancer cell line RM-1.The hUC-MSC expressing murine OX40L and 4-1BBL were injected into C57 mouse model and nude mouse tumor model to observe the changes of tumor size and survival rate.Two weeks after MSC injection in the C57 mouse model,the tumor tissue was separated to make paraffin section,then anti-CD4 and CD8 immunofluorescence staining was performed.The lentivirus plasmid vectors of human OX40L were conducted and hUC-MSC was infected after virus packaging.After packaging the lentiviurs of CD19 chimeric antigen receptor(CAR),T cells originated from cord blood were infected by CAR lentiviurs to cultivate CD19-CART cells.In vitro killing experiment of CD19-CART was performed and analyzed by flow cytometry.The leukemia mouse model was established by injecting Raji cell expressing Luciferase in NOG mice.MSC+CART?CART and PBS were injected into the NOG mouse mode respectively,and the changes of tumor size were observed through the in vivo imaging,and the survival of mice were also observed.The IFN-? level in mice serum was detected by Elisa method.Results:During the culture of hUC-MSC,we found a group of small cells with high expression of SSEA4.The SSEA4+ hUC-MSC were screened by magnetic beads,which showed a better proliferation ability than the unselected hUC-MSC.In addition,the proportion of its original cells was higher through the cell cycle detection.The immunofluorescence staining,RT-PCR and qRT-PCR analysis showed that SSEA4+cell gained a persistent expression of stem cell totipotency markers of OCT4,Nanog,SOX2,and c-myc,which is significantly higher than the unscreened hUC-MSC and SSEA4-MSC.The expression level is similar to that of the hESC.Thus,it can be preliminarily confirmed that SSEA4+ hUC-MSC was similar to embryonic stem cells in the aspect of the expression of totipotency markers.The multidifferentiation experimental showed SSEA4+hUC-MSC had the better differentiation ability.After embroid body induction culture of the SSEA4+hUCMSC,the high-level expression of the three germ layers markers ?-?-tubulin,AFP,?-actin,was detected through immunofluorescence and RT-PCR method,which further confirmed the totipotency-like characteristics of the SSEA4+ hUC-MSC.In the burn wound healing experiment,SSEA4+cell treatment group showed a large increase of dermal cells and epidermal cells in the damaged skin areas,which confirmed the repair ability of SSEA4+hUC-MSC for skin tissue wound healing.After injection of hUC-MSC infected with murine OX40L(MSC-O)and hUC-MSC infected with murine 4-1BBL(MSC-4),into the prostate cancer C57 mouse model respectively,the tumor cells proliferation was effectively inhibited by MSC-O and MSC-4.After observation of tumor section in mice after treatment,the CD4 and CD8 T cells in the tumor site were significantly increased.While in the treatment of prostate cancer nude mouse model,the inhibition effect of tumor was not found.It can be initially confirmed that hUC-MSC carrying T cell co-stimulatory molecules of OX40L or 4-1BBL can promote the tumor killing effect,which is achieved by recruiting and activating tumor infiltrating T cells.In this research,CD19-CART cell with a 4-1BB intracellular signal region was successfully prepared,and it showed an effective in vitro killing ability to CD19 positive Raji cells.hUC-MSC expressing human OX40L was also prepared by gene transfection of OX40L.After establishment of the leukemia NOG mice model and tumor intervention treatment,we found that the combination of MSC and CD19-CART showed a more stable tumor inhibition effect.At d29,the tumor cell proliferation was fundamentally controlled in the combination therapy group.And the survival rate of NOG mice of the combination therapy group was also higher than the group with CD19-CART injection only.Through the IFN-y level detection,we found that the combination therapy group has a higher IFN-? level in mice serum.Thus,hUC-MSC expressing human OX40L can promote the killing effect of CD19-CART to B cell tumor.Conclusions:SSEA4+ cells derived from hUC-MSC have the possibility to act as an excellent source for multipotent stem cells.SSEA4+ cells may serve as a promising source for tissue wound healing therapies.Based on the tumor migration ability of MSC,it is confirmed that hUC-MSC expressing T cell costimulatory molecules can inhibit tumor cells through stimulating T cells,which indicates a new method for promoting tumor therapy effect by using MSC as a transport carrier.In the CART treatment,through utilizing hUC-MSC as an artificial antigen presenting cell to provide T cell costimulatory signal,it showed an enhancement for tumor killing effect of CD19-CART in the leukemia mouse model.It also provides a new strategic reference for improving the effect of CART in treating tumors,especially solid tumors.