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Characterization of mouse hematopoietic stem cells primed to actively self-renew by NUP98-HOXA10hd fusion gene

University of British Columbia

High-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Recently, it has been demonstrated that engineered NUP98-HOXA10hd expression stimulates >1,000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin⁻Sca-1⁺c-kit⁺ cells. In this thesis I coupled such ability of engineered NUP98-HOXA10hd expression, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. I discovered that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at near unit efficiency in cultures initiated with single cells. The clonally expanded HSCs showed preservation of normal proliferation kinetics in vitro and consistent balanced contributions long-term to the lymphoid and myeloid lineages in vivo without evidence of leukemogenic transformation. Preservation of a normal proliferating HSC phenotype allowed their re-isolation in large numbers at 25% purity. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process. Although there is growing excitement about the prospect of in vitro expansion of HSCs and their use to enhance the safety and application of transplant-based therapies, deleterious consequences of such manipulations remain unknown. Thus, I further examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. I observed ~10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert⁻/⁻ HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type or Tert⁻/⁻ HSCs, suggesting that HSC replication post-transplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity upon prolonged self-renewal stimulation.

Text

2011-07-08

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/35927

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/