Abstract
Expanding hematopoietic stem cells (HSCs) ex vivo remains a major challenge due to differentiation. Previous studies have shown that engineered overexpression of HOXB4 increases HSCs >40-fold in short term liquid culture. Most recently we have demonstrated that overexpression of Hox genes of different paralogs fused to the N-terminal region of the nucleoporin98 (NUP98) gene, a common fusion partner of Hox in AML, causes a strong block in differentiation as reflected by marked increases in CFU-S output and lineage negative cell expansion in vitro (Pineault et al, MCB, 2004). NUP98 fusions of Abd-B like HOX genes, HOXA10 and HOXD13 (NA10 and ND13), are more potent in these effects than those of Antennepedia-like-HOX genes, HOXB4 and HOXB3 (NB4 and NB3), prompting us to examine the HSC expanding potential of NUP98 fusions. Following in vitro culture of BM cells transduced with such fusions, we observed that the HSC expanding ability of HOXB4 can be augmented some 10-fold by fusion to NUP98 gene (i.e. NB4) perhaps due to the strong transactivation properties of the NUP98 fragment. Moreover we documented that NA10 has even more potent HSC expansion activity (>1,000-fold net HSC increase in 10 days) (Ohta et al, ISEH 2004 abstract # 24). To further examine NA10’s HSC expansion potency at a clonal level, multiple replicate cultures were initiated with limiting number of 5-FU treated BM cells estimated to contain ~1-2 CRU (5,000 cells per culture). After 2 days of pre-stimulation, individual wells were retrovirally transduced with NA10 for 2 days using an MSCV-based vector and expanded for a further 6 days. After a total 10-day culture, various fractions of individual wells (ranging from 1/2 to 1/250th of a well) were transplanted in limiting dilution assay for lympho-myeloid competitive repopulating cells (CRU). All recipients from individual GFP control wells (initiated with 25,000 cells) were not reconstituted. In marked contrast, all wells assayed for NA10, were positive for lympho-myeloid reconstituting cells at all dilutions tested. At the highest transplant doses (1/2 of a well), 100% of recipients from 4 wells tested were strongly positive for donor cells, averaging 71.5%, 9.0%, 29.0%, 16.4% for myeloid, B-lymphoid, T-lymphoid, RBC for GFP+ donor derived cells respectively. Most strikingly, transplantation of 1/250th of a well yielded 23.4% reconstitution of 5 positive mice (total of 2 wells assayed) and all recipients at this dilution were positive revealing more than a 250-fold increase of HSCs. In support of this, Southern blot analysis showed similar band patterns among different recipients transplanted with cells from the same wells consistent with clonal expansion from 1-2 starting HSC. We further tested the HSC expanding potential of NA10 using highly enriched c-kit+Sca-1+Lin− starting cells, demonstrating >7,000-fold expansion of short-term repopulating cells at 5 weeks post-transplant, and longer term follow-up is in progress. Taken together these results provide strong evidence of the potent ability of NA10 to induce the ex vivo expansion of HSCs at a clonal level. Although the NA10 induced expansion of HSC has not associated with leukemia with observations over 10 months, further development of protein-based delivery systems for NA10 such as TAT-fusion proteins (Krosl et al, Nat Med, 2003) are in progress as a possible novel stem cell expanding agent for safe therapeutic application.
Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo