Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics

Uchida, Naoyuki; Dykstra, Brad; Lyons, Kristin; Leung, Frank Y.K; Eaves, Connie J.

doi:10.1016/j.exphem.2003.09.001

Cited by 100 publications

(114 citation statements)

References 60 publications

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Section: Discussionsupporting

confidence: 84%

“…The elimination of nontarget cells that along with HSC may undergo extensive expansion in culture would also allow studying the relationship between changes in HSC numbers and phenotype of NUP98-HOX-expanded cells. Consistent with this, in preliminary experiments initiated with single lin − Rho dull SP cells [27] followed by NUP98-HOXA10HD transduction and in vitro expansion, we have observed the generation of almost entirely lineage-negative cultures that include cells with robust in vivo HSC activity.In summary, we have developed a new strategy for consistently achieving very high expansion of HSCs ex vivo using a single agent. Together with recent publication revealing the potency of another NUP98-HOX fusion to increase the numbers of human long-term culture-initiating cells [43,44], our findings raise optimism for future extensions of this technology to enable even greater levels of HSC expansion to be obtained in vitro after longer times and/or different growth factor conditions.…”

supporting

confidence: 84%

“…The average magnitude of the HSC expansions achieved by overexpression of the NUP98-HOXB4 fusion gene was ~300-fold, i.e., ~4 times the effect of HOXB4 alone using the same vector. The greater than 2000-fold expansions of HSCs obtained using NUP98-HOXA10 fusion genes are unprecedented and come close to the theoretical limit in a maximum period of 7 to 8 days of gene expression, assuming no significant shortening of the reported 12-to 14-hour cell cycle time for these cells [26,27]. Even further levels of HSC expansion could be anticipated in more prolonged cultures (~10-fold expansion every 2 additional days).…”

supporting

confidence: 52%

“…Assuming a HSC cell cycle time of 12 to 14 hours [26,27], the continuous execution of symmetric self-renewal divisions, and no cell death, the maximum expansion of HSCs that would be predicted to occur over a period of 5 to 8 days is 1000-fold, i.e., greater than 10 times that found to be achieved by overexpressing HOXB4. In a related paper, we show that HSC expansions of this magnitude can be realized by combining the overexpression of HOXB4 with the suppressed expression of the Hox cofactor, Pbx1.…”

mentioning

confidence: 99%

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

Ohta

Sekulovic

Bakovic

et al. 2007

Experimental Hematology

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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...

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Section: Discussionsupporting

confidence: 84%

supporting

confidence: 84%

supporting

confidence: 52%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Ohta

Sekulovic

Bakovic

et al. 2007

Experimental Hematology

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“…Like Hoxb4, both fusions reconstituted normally both myeloid and lymphoid compartments in vivo without inducing leukemia. The level of ex vivo HSC expansion obtained for NUP98-HOXA10HD and Hoxb4 (high)-Pbx1 (low) are unprecedented and, according to the reported doubling time of HSCs (Uchida et al, 2003), are near-maximal symmetrical expansions.…”

Section: Hox As Potent Stimulators Of Hsc Expansionmentioning

confidence: 89%

Hox genes in hematopoiesis and leukemogenesis

2007

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Characterization of Mouse Hematopoietic Stem and Progenitor Cells

2008

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The unit describes functional assays for the quantification of mouse hematopoietic stem cells and progenitor cells. The competitive repopulating unit (CRU) assay detects transplantable mouse hematopoietic stem cells with the capacity to regenerate all of the blood cell lineages for extended time periods in vivo. The long-term culture-initiating cell (LTC-IC) assay, founded on the bone marrow long-term culture system, measures primitive hematopoietic progenitors based on their capacity to produce myeloid progeny for at least four weeks. Colony-forming cell (CFC) assays, performed in semisolid medium cultures to assess mouse pre-B, megakaryocyte, erythroid, granulocyte-monocyte, and multipotential hematopoietic progenitors are also described. These assays are powerful tools for evaluating human stem cell (HSC) and progenitor content in various hematopoietic tissues, during development as well as in the adult animal, and in cell populations manipulated ex vivo.

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Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics

Cited by 100 publications

References 60 publications

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

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

Hox genes in hematopoiesis and leukemogenesis

Characterization of Mouse Hematopoietic Stem and Progenitor Cells

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