Isolation and Characterization of Living Cord Blood CD34+ Cells with Telomerase Reverse Transcriptase (TERT) Expression.

In the hematopoietic system, telomerase activity is suggested to be differentiation- and proliferation status-dependent. High telomerase activity has been demonstrated in bulk CD34+ progenitor cells. The telomerase activity is mainly controlled at the transcriptional level of the telomerase reverse transcriptase (TERT) gene. In this study, we functionally characterized living cord blood (CB) CD34+ cells with TERT promoter activity by using a TERT reporting adenoviral vector with Ad35 tropism. Such fiber retargeted Ad5F35 vectors allow efficient gene transfer into hematopoietic cells by using the ubiquitously expressed CD46 as a cellular receptor. An adenoviral vector (cTERTdGFP) encoding destabilized EGFP (half-life of 2 hours) under the control of the human TERT promoter and the chicken b-like globin gene insulator was constructed. The background expression of GFP from cTERTdGFP was assessed in the telomerase(−) CB CD15/33+ cells, WI-38 cells and fibroblast cells. Less than 3 percent of these cell types expressed low levels of GFP following transduction with cTERTdGFP in comparison to the control vector Ad5F35-GFP (PGK-promoter) transduced cells (78 to 95% expressed GFP). Under similar conditions, more than 95% of the telomerase(+) A549 cells expressed GFP+ following the cTERTdGFP vector transduction. Therefore, the cTERTdGFP vector allowed d2GFP expression in a telomerase activity-dependent manner. Telomerase activity levels were quantified using the TRAP assay. All transductions were performed at an MOI of 100. The CB CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin. When transduced with the Ad5F35-GFP vector at an MOI of 100, 47±6.7% of the CD34+ cells expressed GFP after two days, while 17±4.3% of the cells expressed GFP following the cTERTdGFP vector transduction. Sorted GFP+ cells following transduction with the cTERTdGFP (TERT sorted) or the Ad5F35-GFP (control sorted) vector were assessed for cell cycle distribution, colony forming capacity and repopulating capacity. Staining for the Ki-67 antigen and 7-AAD revealed that the TERT sorted cells had a greater proportion of cells in the S/G2/M phase of the cell cycle compared to the control sorted cells (55±1.2% versus 37±3.6%, p<0.01), and fewer cells in G0 phase (8.7±2.3% versus 21±3.7%, p<0.05). The colony forming capacity of TERT- and control-sorted cells was similar. Fourteen days following plating of 500 TERT sorted cells, 99±28 BFU-E and 59±18 CFU-G/M colonies were scored compared to the control sorted cells that formed 92±28 BFU-E and 59±11 CFU-G/M colonies. To further assess whether the TERT expressing cells contained repopulating primitive progenitor cells, 1x10⁵ TERT sorted cells were transplanted via tail vein injection into NOD/SCIDBeta2m−/− mice. Human cell reconstitution in the bone marrow was examined at 6 weeks post-transplant in two independent experiments. The TERT sorted cells showed an average of 34±18% (n=9) engraftment with both B- and myeloid-lineage differentiation. Similar engraftment was observed for control sorted cells (35±11%, n=8). In summary, the cTERTdGFP vector allowed isolation of single living primitive hematopoietic progenitor cells with TERT expression. This cell population is enriched for cells in the S/G2/M phase of cell cycle and contains colony-forming progenitor cells and NOD/SCIDBeta2m−/− repopulating progenitor cells.