HOXB4-Mediated Immortalization of Murine Hematopoietic Stem/Progenitor Cells.

HOXB4 overexpression has been shown to be able to expand hematopoietic stem/progenitor cells (HSPCs) in short-term ex vivo culture. Here we found that HOXB4-transduced cells could expand continuously in up-to-eight-month long-term liquid culture. In 3 independent experiments, mouse bone marrow (BM) Sca1⁺ cells were transduced with HOXB4 or YFP vectors and kept culture in IMDM/10%FCS containing SCF, Flt3-L, and TPO each at 50 ng/ml. After 3–4 weeks of culture, all YFP-expressing cells differentiated and died. However, HOXB4-transduced cells continuously expanded and percentage of HOXB4-expressing cells increased to more than 95% after 4 weeks of culture. Without cytokine support, these cells stopped growing, while a single cytokine, SCF, was able to support the expansion of these cells, albeit at a lower rate. In the presence of SCF, TPO and Flt3-L, these cells expanded robustly with cell doubling time of around 2 days. These cells are morphologically immature by cytospin and Wright-Giemsa staining. CFU assay showed a clonogenicity of 2%. Phenotyping by FACS showed that all the cells expressed c-kit, while some cells expressed Gr-1, Mac-1 and CD3, indicating that HOXB4-immortalized cells spontaneously differentiate into myeloid and lymphoid cells in ex vivo culture. To test the repopulating capability of these cells, 1x10⁷ cells were injected into each sublethally irradiated NOD/SCID mice via tail vein. These cells engrafted in the recipients with 2% in BM and 10% in spleen. Transplantation into lethally irradiated congenic CD45.1 mice is in progress. Immortalization of human CD34⁺ by HOXB4, however, failed. To determine the mechanisms under which HOXB overexpression immortalizes mouse rather than human HSPCs, we performed Western blot assay with HOXB4-specific antibody. HOXB4 protein levels in human cells decreased continuously from 1–3 weeks posttransduction, whereas expression in mouse cells remained stable. Furthermore, we found that HOXB4 transduced in mouse cells was tyrosine phosphorylated, which is evidenced by treatment of cells with sodium orthovanadate, a protein tyrosine phosphatase inhibitor, treatment of cell extracts with l-phosphatase and immunoprecipitation with phosphorylated tyrosine-specific antibody. Consistent with other reports, no leukemogenesis was observed in our studies as yet. It would be interesting to compare the differences in gene expression profiles between the HOXB4- and HOXA9-immortalized hematopoietic cells, which may unveil the molecular mechanisms governing leukemogenesis. In conclusion, murine HSPCs can be immortalized by HOXB4 overexpression and tyrosine phosphorylation of HOXB4 may be responsible for the self-renewal and immortalization of HSPCs.