Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions.
OBJECTIVE
Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect.


METHODS
To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10- to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay).


RESULTS
These experiments revealed remarkable expansions of Nucleoporin98-Homeobox-transduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion.


CONCLUSIONS
These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.
Ohta
Hideaki
H
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
Sekulovic
Sanja
S
Bakovic
Silvia
S
Eaves
Connie J
CJ
Pineault
Nicolas
N
Gasparetto
Maura
M
Smith
Clayton
C
Sauvageau
Guy
G
Humphries
R Keith
RK
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D028861:Nuclear Pore Complex Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics*
D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins; C513262:Hoxb4 protein, mouse; D028861:Nuclear Pore Complex Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C093350:nuclear pore complex protein 98; C092119:Hoxa10 protein, mouse