Ectopic HTERT Gene Expression In Human Bone Marrow Mesenchymal Stem Cell

Besides hemopotoietic stem cells, there is another type of stem cells called mesenchymal stem cells (MSCs) in human bone marrow. It has been reported that MSCs differentiated into not only osteoblasts, chondroblasts, adipocytes, but also into neural cells, hepatocyte and endothelial cell etc. As a good differentiation model, MSCs can be used to study molecular mechanism of signal transduction, and the control of gene expression.However, the amount of MSCs among mononuclear cells (MNCs) of human bone marrow is limited. Proliferating MSCs in vitro without losing their multipotent differentiation capacity is important.It has been reported recently that ectopic expression of hTERT gene reconstitutes telomerase activity, maintains telomere length, and expands the replicative life span in target cells, which implies that ectopic expression of the hTERT gene in human mesenchymal stem cell might cause the cells to overcome replication senescence while maintaining their normal characteristics.The aim of this study is to investigate the role of telomerase activity in the proliferation of human MSCs, and extends the replicative life span in human MSCs with multipotent differentiation capacity. Methods:1. Human MSCs were isolated from healthy donator and transfected with a pEGFP-hTERT plasmid by Liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR.2. Telomerase activity of transfected MSCs was detected by PCR ELISA, that of untransfected MSCs was tested simultaneously as control.3. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers(NF-M,MAP2) were detected by RT-PCR. Results:1. Target fragment (3 lObp fragment) was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs, which demonstated the integration and mRNA expression of the exogenous hTERT gene in hTERT-transfeced cells.2. The absorbance(A450nm-A690nm) of the hTERT-transfected cells was lower than that of positive control(provided by the Telo TAGGG Telomerase PCR ELISA kit ) but obviously higher than that of the untransfected human MSCs. As Samples are regarded as telomerase-positive if the difference in absorbance(A450nm-A690nm) is >0.2, we concluded that the untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. These results indicated that ectopic expression of the hTERT gene in human mesenchymal stem cell can reconstitute telomerase activity. And in following culture, the telomerase-negative MSCs entered a nondividing state after about 20 to 25 passages and senesced. While in test group, telomerase-positive MSCs to date have undergone 35 passages. Telomerase-positive MSCs retained normal karyotypes and exhibited morphologic and functional characteristics of human MSCs.3. RT-PCR analysis showed that telomerase-positive MSCs can express neuron-specific markers, such as NF-M, MAP2 after induceing with EGF and bFGF in vitro. Conclusions:.1. The introduction of hTERT gene into human MSCs leads to expression of hTERT mRNA and reconstitutes telomerase activity.2. The introduction of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.