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Novel methods for high level ex vivo expansion of hematopoietic stem cells

University of British Columbia

The development of strategies to extensively expand HSCs ex vivo could greatly improve the utility of hematopoietic stem cell (HSC) - based therapies. In addition to potential clinical applications, such an advance would provide an invaluable tool for studying the mechanisms underlying HSC self-renewal. Engineered overexpression of the homeobox transcription factor HOXB4 has emerged as a powerful stimulator of hematopoietic stem cell (HSC) expansion ex vivo (>40-fold net increase in 2 weeks). More recent studies of the properties of natural and engineered NUP98-HOX fusion genes, initially of interest to us for their role in human AML, suggested these molecules might have similar effects on HSCs. To examine whether specific NUP98 and HOX fusion genes stimulate murine HSC expansion in short term liquid cultures, 3x10⁶ marrow cells from mice given 5-fluorouracil 4 days previously were prestimulated with IL-3, IL-6 and SF, retrovirally transduced with MSCV-IRES-GFP retroviral vectors also encoding NUP98-HOXB4, NUP98-HOXA10, or HOXB4 (only) or nothing as controls and then cultured for another 6 days with the same growth factors. Limiting dilution assays were used to determine the frequency and hence number of Competitive long-term (>4months) lympho-myeloid Repopulating Units (CRU) present before and after culture. The results of these experiments showed that the CRU content of the cultures of NUP98-HOXB4-, and NUP98-HOXA10- transduced cells increased 290-fold and >2000-fold, respectively, i.e. ~4 and >25x the effect obtained with HOXB4 and >10⁴ and >10⁵x the yield of CRU in the control cultures. Similar results were obtained in cultures of NUP98-HOXA10- transduced cells that were initiated with limiting numbers of CRUs (1-2), demonstrating that the cells targeted were not a rare subset of HSCs. Additional studies of the same design showed that the effect of NUP98-HOXA10 on HSC expansion was preserved when sequences flanking the homeodomain were removed, thus identifying the homeodomain as the key HOX gene sequence required in concert with the N-terminal region of NUP98. These findings demonstrate a greater potency of NUP98-HOX fusions as novel agents for HSC expansion ex vivo, reveal the essential contribution of the DNA-binding homeodomain to achieve this effect and set the stage for the design of minimal HOX-based fusion proteins for future studies.

Text

2009-12-15

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http://hdl.handle.net/2429/16702

Medical Genetics

University of British Columbia

UBCV

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