Immortalization of Human HSPC by a Self-Renewal Signal Coupled with Telomere Maintenance.

Expression of the leukemia-associated fusion protein AML1-ETO (AE) in human hematopoietic stem and progenitor cells (HSPC) results in long-term cytokine-dependent cells that maintain CD34 expression, multipotential differentiation and the ability to engraft NOD/SCID mice. These cells gradually lose telomere length and are not immortal. To determine whether expression of telomerase would cooperate with AE in immortalization of human HSPC, we transduced AE-expressing CD34+ cells with the telomerase gene, hTERT. In four independent AE cell cultures, hTERT expression allowed the cells to proliferate beyond the crisis point. Cells retained B lymphoid, myeloid and erythroid differentiation potential, displayed continuous replating ability in methylcellulose assays and engrafted in NOD/SCID mice. The hTERT-expressing cells had a significant increase in progenitor cell numbers, more cells in S phase, and increased resistance to gamma radiation-induced apoptosis compared to control cells. No genetic changes were identified by G-banding or comparative genomic hybridization during the passage through crisis, implying that these signals are sufficient for the immortalization. Similar results were obtained using a long-term culture established with the related core binding factor (CBF) fusion gene CBFβ-MYH11 (CM). By microarray analysis we identified a number of genes implicated in stem cell self-renewal, including HOXB4, GATA3 and STAT3, that are upregulated in CBF-expressing cells when compared to control CD34+ cells. Ectopic hTERT expression did not affect the level of expression of these self-renewal genes. We hypothesize that the continual rescue of progenitor cells during culture, as a result of telomerase maintenance of telomeres, is responsible for the proliferation and survival phenotype observed in hTERT expressing AE and CM cells. This is supported by our findings that control cultures contained significantly more DNA damage foci as detected by immunofluorescence staining for phosphorylated H2AX. In addition, signal-free chromosome ends were significantly more abundant in control cultures as demonstrated by a telomeric FISH probe when compared to the hTERT cultures.

Senescence-associated β-gal staining was prominent in control cells as they slowed proliferation after 7 months of growth, but hTERT-expressing cells did not display SA-β-gal staining and continued proliferating indefinitely, for over two years at this point in time. We conclude that the self-renewal signal supplied by a CBF oncogene cooperates with telomere maintenance initiated by hTERT for the immortalization of human HSPC. This system permits a molecular dissection of the signals involved in HSPC self-renewal and leukemogenesis and in the contribution of hTERT expression to this process.