Besides progressive telomere shortening, human cells undergo senescence in response to various types of stress. The stress-related signaling pathways triggered converge to varying extents on the p53 and retinoblastoma (Rb) tumor suppressors, and the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p16INK4a. We therefore attempted to extend the life-span of human CD34+ cord blood (CB) progenitors by ectopically expressing the human papillomavirus type 16 (HPV16) E6 and E7 genes, which accelerate the degradation of p53 and Rb, respectively, separately and in conjunction with the gene encoding the reverse transcriptase catalytic subunit of human telomerase (hTERT). CD34+ CB cells were transduced with VSV-G glycoprotein-pseudotyped self-inactivating lentiviral vectors that express HPV16 E6/E7 or hTERT linked to downstream GFP/YFP reporter genes on bicistronic transcripts. Sorted GFP+ and/or YFP+ CB cells were maintained under serum-free conditions in the presence of stem cell factor (100 ng/ml), Flt3 ligand (100 ng/ml), thrombopoietin (20 ng/ml) plus interleukin-3 (20 ng/ml). In all cases (n = 3), control CD34+ CB cells differentiated into macrophage-like cells and underwent senescence-associated proliferation arrest after ~4 months in culture. Constitutive expression of hTERT did not increase CB cell replicative capacity in repeated attempts (n = 3), and macrophage-like cells were also the predominant cell type that accumulated in these cultures. In contrast, CB cells constitutively expressing HPV16 E6/E7 alone (n = 2) or in concert with hTERT (n = 5) continued to proliferate beyond 4 months, eventually giving rise to permanent (> 2 years) cell lines with a CD45+CD34−CD44+CD235a+CD71+ CD203+CD33+CD13+ myeloerythroid/mast cell progenitor phenotype. Interestingly, two CB cell lines coexpressing E6/E7 plus hTERT were also positive for the CD133 hematopoietic stem/progenitor cell antigen. When the immortalized CB cells were cultured in the presence of erythropoietin, slight up-regulation of the CD36 erythroid marker was observed. Moreover, when selected CB cells were subjected to a myeloid differentiation regimen, up to 90% of the cells acquired the ability to adhere to fibronectin in the best example, of which ~30% were capable of superoxide-dependent nitroblue tetrazolium reduction reflective of terminal monocytic differentiation. Notably, CB cell cultures expressing only HPV16 E6/E7 went through a crisis period and, when analyzed by spectral karyotyping, G-banding and fluorescence in situ hybridization with centromere-specific probes, were found to be highly aneuploid. By comparison, the CB cell lines obtained by coexpression of HPV16 E6/E7 plus hTERT exhibited near diploid karyotypes with minimal chromosomal aberrations, concomitant with stabilization of telomere length. Upon injection of the two CD133+ E6/E7 plus hTERT-expressing CB cell lines subcutaneously (5 x 106 cells) or intravenously (1 x 107 cells) into sublethally-irradiated (250 cGy) NOD/SCID mice, no tumors developed after 7 months of observation (n = 11), with tumorigenic conversion requiring cooperative enforced expression of a v-H-ras or BCR-ABL oncogene (n = 12). These findings establish the feasibility of bypassing senescence in human hematopoietic progenitors through genetic engineering, providing proof of principle for gene-based or other approaches that might eventually allow establishment of permanent human hematopoietic stem cell lines.