Targeting Eradication Of Malignant Cells Derived From Human Bone Marrow Stem Cells

Background: To many patients, transplantation of human bone marrow stem cells not only brings hopes, but also brings the risks. Of all of the risks of stem cell therapy, the most serious, and the risk that has gotten the most attention, is the risk for tumor formation. Human bone marrow stem cells do not exhibit the telomerase activation, but tumor cells will do. It is the responsibility of the community of stem cell scientists and clinicians to develop strategies to control the therapies after transplantation. However, this strategy must be faced many challenges, including high efficiency of tumor-targeting promoter design of mechanisms, obtaining a number of hBMSC-derived malignant cells for functional identification of designed promoters and tests of cytotoxicity in vitro and in vivo, and low survival rate of malignant cells after heterogenic implantation, etc. Until now, a reliable method has not been found to prevent neoplastic transformation of engrafted hBMSC.Purpose: 1. Investigate the improvement of targeting transcriptional activities in hBMSC-derived malignant cells; 2. Seek a method for obtaining enough number of hBMSC-derived malignant cells; 3. In order to establish a research model for prevention of malignant transformation and targeting eradication of malignant cells derived from hBMSC.Methods:1. Core hTERT promoter modified by c-Myc binding element: wild-type core hTERT (WThTERT) promoter fragment (from -268 to -10 upstream of the initiating ATG start codon) and the modified hTERT (Mod1hTERT) promoter fragment (a triplet of E-boxes were ligated to the 3′terminal of the WThTERT promoter) were generated through synthesis. The two reporter genes of enhanced green fluorescent protein (EGFP) and luciferase were expressed and assayed in 293FT, HepGⅡ, SGC7901, U2OS, and the primary culture human bone marrow stem cells (hBMSC) under the control of the WThTERT promoter and Mod1hTERT promoter, respectively.2. In vitro amplification of PALA resistant malignant cells derived from human bone marrow stem cells: isolated culture hBMSC from human bone marrow and assess their phaenotype by flow cytometer, FCM; after the treatment by carcinogenic reagent of BPDE and lentivirus infection, malignant cells could be sieved by selection of PALA; detect expression of lentivirus infection,cell cycle regulation related gene and telomerase activation by PCR, RT-PCT and west blot.3. Modification of hTERT promoter for down-regulation of transcriptional activities in human bone marrow stem cells: in this section, a tetrad of MZF-2 binding elements were ligated to the 5′terminal of the Mod1hTERT promoter, the new generated promoter was named as Mod2hTERT prmoter; Cytosine deaminase (CD) gene and enhanced green fluorescent protein (EGFP) gene from E.coli K12 and plasmid of pEGFP-N3 respectively are subcloned into plasmid of pLenti6/V5-D-TOP; The wild hTERT promoter fragment(WT-hTERT), Mod1hTERT, and Mod2hTERT promoters were generated by chemical synthesized; EGFP and luciferase gene code sequence into the lentivirus pLenti6/V5-D-TOP; CMV promoter in pLenti6/V5-D-TOP was replaced by above hTERT promoters, respectively; The hBMSC, BR-hBMSC and PBR-hBMSC were transfected by lentivirus; 6.The expression of EGFP under the controls of modified hTERT promoters were observed by fluorescence contrast phase microscope and the luciferase activity was detected by Kit.4. In vitro and in vivo cytotoxicity test: PBR-hBMSCs with fusion genes of WThTERT-luciferase-IRES-CD, Mod1hTERT-luciferase-IRES-CD, Mod2hTERT-luciferase-IRES-CD, CMV-luciferase-IRES-CD and prmoter(lack)-luciferase-IRES-CD were treated in the mediums with different concentration gradient of 5-FC, respectively; 2.MTT reduction assay were used for cytotoxicity detections of PBR-hBMSCs with different fusion genes in different concentration gradient of 5-FC; PBR-hBMSCs with fusion genes of WThTERT-luciferase-IRES-CD, Mod2hTERT-luciferase-IRES-CD, and CMV-luci-ferase-IRES-CD were transplanted into hypo of athymic mice; After one week, each mouse were detected with animal in vivo fluorescence imaging system were given a injection after given a injection of 5㎎ 5-FC once a day. The signal of survive engraft cells were detected once a week.Results:1. Core hTERT promoter modified by c-Myc cis element: in the Mod1hTERT groups, EGFP could be observed in the hTERT-positive groups of 293FT, HepGⅡ, and SGC7901 cells, but not in the hTERT-negative groups of U2OS and hBMSC. The luciferase activity of the Mod1hTERT promoter groups was significantly higher than that of the WThTERT promoter groups in the hTERT positive cell lines of 293FT, HepGⅡ, and SGC7901(P<0.01), however, no significant difference was found in U2OS and hBMSC (P>0.05). 2. In vitro amplification of PALA resistant malignant cells derived from human bone marrow stem cells:3. Modification of hTERT promoter for down-regulation of transcriptional activities in human bone marrow stem cells: in the Mod1hTERT groups, EGFP could be observed in the hTERT-positive groups of 293FT, HepGⅡ, and SGC7901 cells, but not in the hTERT-negative groups of U2OS and hBMSC. The luciferase activity of the Mod1hTERT promoter groups was significantly higher than that of the WThTERT promoter groups in the hTERT positive cell lines of 293FT, HepGⅡ, and SGC7901(P<0.01), however, no significant difference was found in U2OS and hBMSC (P>0.05).4. In vitro and in vivo cytotoxicity test: MTT assays results shown that the relative survival rates of cells were only 5.79% in the Mod1hTERT group, 6.53% in the Mod2hTERT group, and 1.97% in the CMV group, when treated with 100μM 5-FC; 2. Bioluminescence imaging system test showed that after the administration of 4-5 weeks, in grope of malignant cells derived from hBMSC Mod2hTERT promoter and malignant cells derived from hBMSC with CMV mice bioluminescence signal was vanished. It is suggested that the malignant cell neoplasia be cleared.Conclusions:1. 3'terminal extra E-boxes can improve the tumor-targeting transcription activity of the core hTERT promoter.2. Malignant transformation of hBMSC can be co-induced by transfection of lentivirus and treatment of BPDE. The malignant cells derived from hBMSC could be amplification in vitro with IC50 selection culture of PALA.3. 5′terminal extra MZF-2 binding sites of core hTERT promoter can effectively degrade transcriptional activity of hTERT promoter in MZF-2-positive cells of hBMSC.4. Transcriptional activity of modification with c-Myc and MZF-2 binding elements is stronger than the normal cell. While in vitro and in vivo, the expression of the CD promoter and luciferase gene in PBR-hBMSC has been cleared by 5-FC.In briefly, this study has not just provided a method for the research of targeting eradication of malignant cells derived hBMSC, but may also provide a strategy for bio-safety research in future clinic stem cell therapy.