A NUP98-HOX Fusion Gene Containing the Homeodomain of HOXA10 Promotes Significant Expansion of Primitive Human Hematopoietic Cells in Extended Cultures.

Expanding human hematopoietic stem cells (HSCs) in vitro is a major goal in clinical hematology but remains a major challenge due to the potent differentiating activity of known cytokines. We recently demonstrated that a NUP98-HOX fusion gene containing only the homeodomain (hd) of HOXA10 (NUP98-HOXA10hd) is a powerful stimulator of murine HSC expansion in vitro - causing >1000-fold net HSC increases in 10 days (Sekulovic et al, ISEH 2005). To investigate the proliferative effect of NUP98-HOXA10hd on primitive human hematopoietic cells, highly enriched CD34+ cord blood cells were prestimulated overnight and exposed to self-inactivating MNDUSNUP98-HOXA10hd pgkGFP or control pgkGFP lentiviruses for 6h. The gene transfer efficiency into CD34+ cells determined 4 days after infection was 56 ± 5% for NUP98-HOXA10hd and 66 ± 5% for the GFP control. GFP+ cells were sorted on day 5 and then maintained for another 5 days in serum-free cultures containing Flt3-ligand, Steel factor, IL-3, IL-6 and G-CSF. An aliquot of each was then plated into “primary” colony-forming cell (CFC) assay cultures. No difference was detected in either the numbers or the types of colonies generated in these primary CFC assays of the 10-day cultured cells from the NUP98-HOXA10hd and control arms. However, when these primary CFC assays were replated into secondary CFC assays, the number of colonies obtained from the NUP98-HOXA10hd-transduced cells was 5-fold higher as compared to the GFP-control transduced cells and, upon replating into tertiary CFC assays, this difference increased to over a 100-fold. To determine the effect of NUP98-HOXA10hd on more primitive hematopoietic cells, 104 day-10 GFP+ cells were co-cultured on mouse fibroblast feeders engineered to produce human SF, IL-3 and G-SCF. At the end of 6 weeks, 13-fold more cells were recovered from the cultures initiated with NUP98-HOXA10hd-transduced cells than from the control cultures (474,000 ± 190,000 vs 37,000 ± 16,000, 3 experiments). CFC outputs were also greatly enhanced (21-fold more CFC than in the controls cultures, range=20–80, 3 experiments). Moreover, the proportion of progenitors in the assays of the cultures initiated with NUP98-HOXA10hd cells that were multi-lineage (CFU-GEMM) was >10-fold higher as compared to the CFCs obtained from the control cultures (8 ± 3% vs 0.7 ± 0.7%). When this experiment was repeated using limiting dilutions of initial day-10 cells, the frequency of NUP98-HOXA10hd-transduced cells able to generate CFCs another 6 weeks later was 10-fold higher as compared to the day-10 GFP control-transduced cells. These findings document an unprecedented potency of NUP98-HOXA10hd for stimulating the ex-vivo expansion of very primitive pluripotent human hematopoietic cells.