GM-CSF And IL-4 Induce The Malignant Transformation Of The Bone Marrow-derived Human Adult Mesenchymal Stem Cell

Part.1 GM-CSF and IL-4 induce the malignant transformation of the bone marrow-derived human adult mesenchymal stem cellObjective:To examine the effects of GM-CSF and IL-4 on the bone-marrow-derived human adult mesenchymal stem cells (MSCs).Methods:The MSCs were isolated and cultured with GM-CSF and IL-4 for a period above one month. A single colony of transformed mesenchymal stem cells (TMCs) was then isoloated and their phenotype were characterized by morphology, surface marker expression, c-myc gene and tomelerase was detected by western blot.Results:After about 6-8 weeks culture, TMCs exhibited the morphology and phenotype similar to those of tumor cells. They also expressed higher level of the surface markers CD 133 and VEGFR2 but lower level of CD13 and CD105 than MSCs. The content of temolerase of TMCs was about 16.7 times more than that of MSCs and the expressiong of c-myc increased about 172.2% while MSCs were transformed.Conclusion:Cytokines-driven malignant transformation of MSCs may be a useful model for studying signaling pathways initiating malignant transformation of MSC. Part.2 Tumorigenicity of transformed mesenchymal stem cell in vivo.Objective:To evaluate the tumorigenicity of transformed mesenchymal stem cells (TMCs) in vivo.Methods:200μl TMCs or MSCs (2.5×105 cells/ml) were suspended and administrated into 45 immunodeficient NOD/SCID mices via the tail vein and into 16 male BALA/C mices subcutaneously. At 3,7,14,21 and 25 days post-injection,3 NOD/SCID mices in each group were sacrificed. The lungs of the NOD/SCID mices were harvested and the tumor nodules with a diameter greater than 1mm were counted. The BALA/C mices were sacrificed and the subcutaneous tumors were harvested at 25 days post-injection, the size and weight of the tumors were measured. All the tumors were fixed and embedded in paraffin, sectioned, and stained with HE before histological and immunohistochemical examination.Results:Control animals injected with MSCs show no signs of tumor development neither via the vein nor in the subcutis. The clustered tumor nodules were predominantly found in the lungs of mice injected with TMCs with an increase in lung weight at 21 days post-injection. The specimen from the mice injected with TMCs showed higher percentage of CD133 positive cells compared with that from the control group. All the mices injected with TMCs subcutaneously were found solid tumor after 14.2 days. The tumors were 3.0cm3 hudge and 1.37g weight when harvested at 25days after injection. The subcutaneous tumors showed Vim(+),Des(+),SMA(+),CD133(+),CD34(+),S100(+),NSE(-), PA-CK(-),EMA(-),GFAP(-)in the immunohistochemical examination.Conclusion:TMCs can form tumors in mices in ViVO either being injected via the vein or administrated in the subcutis. Objective:To investigate the effect of inhibiting human telomerase reverse transcriptase (hTERT) gene by small interfering RNA (siRNA) interference on the proliferation and the tumorigenicital ability of the transformed mesenchymal stem cells (TMCs)Methods:Small interference RNA (siRNA) targeting hTERT sequences were synthesized chemically in vitro. TMCs were transfected with three different concentrations of siRNA (50nmol/L, 100nmol/L and 200nmol/L) by LipofectamineTM 2000. The expression of hHERT was detected by Reverse transcript polymerase chain reaction (RT-PCR). The content and the activity of the telomerase were tested by western blot and TRAP-ELISA staining. The proliferation activity of TMCs was determined using methylthiazol tetrazolium (MTT) and the cell apoptosis of TMCs were analyzied by flow cytometry. The ability of the tumorigenicity in nude mice of TMCs was also measured.Results:The content of temolerase of TMCs transfected by three different concentrations of siRNA (50nmol/L, 100nmol/L and 200nmol/L) was obviously reduced than that of untransfected control group (46.2%, 69.5% and 91.5% ). As detected by RT-PCR, mRNA level of hTERT in TMCs transfected with siRNA was reduced 19.2%,47.2% and 86.4% than that of control group, respectively. Telomerase activities of TMCs transfected by siRNA in all groups were also decreased. The proliferation and the abilities of tumorigenesis of TMCs transfected by siRNA in all groups were significantly reduced.Conclusion:hTERT expression in TMCs can be inhibited significantly with siRNA and the down-regulation of hTERT expression can cause the reduction of the proliferation of TMCs in vitro and inhibit the ability of tumorigenesis in vivo. Telomerase is involved in the malignant transformation of MSCs.