Immortalized Human Placenta Derived Mesenchymal Cells by Transfection with Bmi-1 Lost the Abilitiy To Differencitate into Cell Types of Osteocytic, Condoricytic and Neural Lineages.

Abstract

Introduction: We previously reported that mesenchymal cells derived from chorionic villi of human placenta could differentiate into various cell types including osteocytic, condoricytic and neural lineages. However, these cells can slowly proliferate with limited life span. In the present study, we transduced human placenta-derived mesenchymal cells (PDMCs) with cDNA encoding Bmi-1 and telomerase reverse transferase (hTert), respectively or simultaneously by lentiviral vector, and tested transduced cells for their ability to differentiate into multi-lineages and to support expansion of CD34+ cells in cord blood as feeder cells in the presence of cytokine cocktails.Method: The lentivairal vector expressing either Bmi-1, hTert or hrGFP was prepared as previously. The human PDMCs were isolated from chorionic villi of human placenta using the explant culture, and were cultured in DMEM (low glucose) with 10% FBS. The established cells were infected with either of the three viruses or a combination of Bmi-1 and hTert virus at the multiplicity of infection that allowed almost 100% of infection efficiency based on hrGFP fluorescence by flow cytometry. Those infected cells and uninfected cells served as control were then continuously propagated. These cells were then transferred into the specific culture media to differentiate them to osteocyte, condorocyte and neural cells as reported previously.Results: The control and PDMC/hTert cells showed a broad and flat shape suggesting that these cells were senescence and ceased proliferation. Cells infected with Bmi-1 and Bmi-1/hTert escaped the crisis and continued proliferation. These cells have been maintained in culture for more than 1 year and continued to proliferate near to 100PD. We found that Bmi-1 over-expression could induce endogenous telomerase activity and elongate telomere length in PDMCs. The expression of p16 mRNA and protein were down-regulated in prolonged culture cells, but p14ARF was not. The differentiation potential of transduced cells was gradually lost as culture was prolonged as well as the ability to support the expansion of CB CD34+ cells. Gene expression analysis on Bmi-1- infected cells at different PD revealed that more than 100 genes were induced or repressed in several different functional categories.Extensive increase in expression of the IGFBP2 mRNA and higher concentration of IGFBP2 was observed.Conclusion: Bmi-1 induced Telomerase activity and immorlized human placenta derived mesenchymal cells. The ability of transduced cells to differentiate into various cell types and support expansion of CB CD34+ cells were lost with propagation suggesting that subtle transformation has occurred in the Bmi-1 transduced cells.