Maintenance and expansion of hematopoietic stem cells in vitro using genetic methods.

Abstract

Chromosomal translocations encoding fusion proteins between N-terminal nucleoporin 98 (NUP98) and some members of the homeodomain protein superfamily have been observed recurrently in certain types of leukemia. Subsequent studies have shown that the ectopic expression of these leukemogenic or similar fusion genes in primitive bone marrow (BM) cells can induce hematopoietic stem cell (HSC) activity in these cells. We have extensively surveyed different branches of the homeodomain superfamily as partners to NUP98 in order to find fusion genes that could sustain HSC activity in BM cells in long-term continually expanding in vitro cultures. Remarkably, we found that CDX1, extended HOX subfamily member, when fused to NUP98, was able to sustain balanced HSC activity with high frequencies in massively expanding cultures as measured by robust long-term competitive in vivo transplantation assays. Interestingly, closely related NUP98-CDX4 induced leukemia with the shortest latency ever reported for NUP98-Homeodomain fusions unaided by MEIS1 transcription factor. Since HSC and leukemic stem cells (LSC) share similar self-renewal mechanisms, these pre-leukemic stem cells generated by the first genetic hits can provide useful models for elucidating the self-renewal mechanisms of HSC as well as LSC.

We also found that NUP98-HOXA10 was able to reprogram B220+ cells in the bone marrow and conferred them long-term reconstituting multipotentiality in vivo. Transcriptional profiling of NUP98-HOXA10 transduced BM cells revealed that the aryl hydrocarbon receptor (AhR) pathway was the most suppressed pathway. Inhibition of this pathway using small molecule AhR inhibitor in combination with cytokine cocktail allowed 30-100 fold expansion of HSC in vitro. This provides an additional component to the elusive “magic” cocktail that allows maximal expansion of HSC ex vivo.