Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

Abstract

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-Homeoboxtransduced 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.Conclusion-These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.The self-renewal function of hematopoietic stem cells (HSCs) is essential to their ability to sustain lifelong hematopoiesis and to regenerate the hematopoietic system after myeloablative treatments. This property is the basis of an increasing range of applications of HSC transplants to treat various malignant and genetic disorders [1,2]. Further improvements in the safety and therapeutic utility of HSC transplants can be readily envisaged if robust methods for largescale ex vivo expansion of HSCs were available. For example, the low absolute numbers of HSCs in most cord blood samples [3] [4] restrict the clinical utility of these products. A method for significantly expanding HSCs could not only address these insufficiencies but also broaden the exploitation of reduced conditioning regimens and associated therapeutic benefits.One approach that has allowed some HSC expansion in vitro to be achieved has focused on the identification of optimized combinations and concentrations of externally acting growth factors and related molecules [5][6][7][8][9][10][11][12]. A complementary approach has been to identify intrinsic regulators such as transcription factors [13] and key mediators of signaling pathways [14,15] that can be manipulated to activate or promote HSC self-renewal divisions. A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numb...

Hox genes in hematopoiesis and leukemogenesis

“…Intriguingly, both engineered NUP98-HOXB4 and NUP98-HOX-A10HD (NUP98 fused only to the homeobox of HOXA10) were extremely potent stimulators of ex vivo HSC expansion with net levels of 300-fold for NUP98-HOXB4 and >2000-fold for NUP98-HOXA10HD demonstrated in 7 days in culture (Ohta et al, 2007). Like Hoxb4, both fusions reconstituted normally both myeloid and lymphoid compartments in vivo without inducing leukemia.…”

Lentiviral Transfer of γ-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine β-Thalassemia

“…21 Transduction of a fusion gene encoding the N-terminal half of NUP98 and the 60 aa homeodomain of HOXA10 (NUP98-HOXA10HD or NA10HD) has proved to be extremely potent in promoting expansion of murine long-term hematopoietic stem cells both in vitro and in vivo. 22,23 It increased engraftment of human short-term and long-term repopulating cells in immunodeficient mouse models 24,25 and in non-human primate models. 26 The properties of NA10HD have thus provided a powerful new tool for manipulating and investigating the self-renewing behavior of primitive murine, non-human primate, or human hematopoietic cells.…”

Comprehensive microRNA expression profiling of the hematopoietic hierarchy

“…To achieve this goal, we developed and validated a protocol for miRNA profiling in rare cell types that is based on multiplexed stem-loop RT-qPCR (10), followed by high-throughput nanoliter volume qPCR on microfluidic arrays (48.48 Dynamic Array; Fluidigm). All protocol development and testing was performed by using freshly derived primitive adult mouse bone marrow cells engineered to express a NUP98-HOXD13 (ND13) fusion gene (11). These cells possess a phenotype that reflects immature (c-kit + ) and mature (Mac1 + ) murine bone marrow cells and, thus, are a suitable representative of the murine hematopoietic system for which miRNA expression has been characterized in depth (12).…”