The establishment of hematopoiesis in culture from murine embryonic stem (ES) cells provides a powerful approach for studying early commitment steps as hematopoietic precursors develop from pre-hematopoietic mesoderm. Analysis of developing embryoid bodies has demonstrated that hematopoietic differentiation in these cultures recapitulates many aspects of the onset of hematopoiesis in utero, including vasculogenesis and the switch from the primitive to the definitive program. We previously used CCE ES cells transduced with our MSCV retroviral vector overexpressing the diverged homeobox gene HOX11 of T-cell acute lymphoblastic leukemia to establish factor-dependent hematopoietic precursor cell lines arrested at novel stages of primitive and definitive hematopoietic development. These results, demonstrating proof-of-concept, provide the rationale for the present proposal to extend this strategy to obtain conditionally-blocked murine hematopoietic precursor cell lines with a variety of differentiative potentials. We are particularly interested in the possibility of conditionally arresting common endothelial/hematopoietic precursors (i.e., hemangioblasts) as well as hematopoietic stem cells with long- term in vivo repopulating activity. To this end, our specific aims are to develop two systems for conditional immortalization: the first, based on site-specific excision of HOX11 by adenovirus-mediated transient expression of Cre recombinase; and the second, based on a tetracycline-regulatable HOX11 retroviral vector. While both approaches will permit precursor-progeny relationships to be unequivocally established, we hypothesize that the latter strategy will allow clonal descendants of precursor cells to be arrested and amplified at successive stages of the hematopoietic hierarchy. It is envisioned that the multipotent precursor lines obtained will have broad utility for molecular biological investigations of hematopoietic differentiative progression and lineage restriction. Toward this goal, homogenous populations of clonally-related precursor cells will be used as starting material for identification by cDNA microarray technology of differentially expressed genes as candidate regulators of hematopoietic cell fate decisions. It is anticipated that the information gained from these studies will also provide insight into the potential roles of those homeobox genes implicated in the transcriptional control of hematopoiesis as well as lead to a better understanding of the underlying mechanism of leukemia initiation mediated by HOX11. Successful realization of these goals will provide a basis for future research endeavors involving primate ES cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066305-05
Application #
6921361
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Thomas, John
Project Start
2001-08-15
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2008-06-30
Support Year
5
Fiscal Year
2005
Total Cost
$402,849
Indirect Cost
Name
George Washington University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043990498
City
Washington
State
DC
Country
United States
Zip Code
20052
Zweier-Renn, Lynnsey A; Riz, Irene; Hawley, Teresa S et al. (2013) The DN2 Myeloid-T (DN2mt) Progenitor is a Target Cell for Leukemic Transformation by the TLX1 Oncogene. J Bone Marrow Res 1:
Hawley, Teresa S; Riz, Irene; Yang, Wenjing et al. (2013) Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1. Am J Hematol 88:265-72
Hawley, Robert G; Chen, Yuzhong; Riz, Irene et al. (2012) An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates. Open Biol J 5:6-16
Ramezani, Ali; Zweier-Renn, Lynnsey A; Hawley, Robert G (2011) Factor VIII delivered by haematopoietic stem cell-derived B cells corrects the phenotype of haemophilia A mice. Thromb Haemost 105:676-87
Riz, Irene; Hawley, Teresa S; Hawley, Robert G (2011) Lentiviral fluorescent protein expression vectors for biotinylation proteomics. Methods Mol Biol 699:431-47
Riz, Irene; Zweier-Renn, Lynnsey A; Toma, Ian et al. (2011) Apoptotic role of IKK in T-ALL therapeutic response. Mol Cancer Res 9:979-84
Ramezani, Ali; Hawley, Robert G (2010) Strategies to insulate lentiviral vector-expressed transgenes. Methods Mol Biol 614:77-100
Riz, Irene; Hawley, Teresa S; Luu, Truong V et al. (2010) TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells. Mol Cancer 9:181
Zweier-Renn, Lynnsey A; Hawley, Teresa S; Burkett, Sandra et al. (2010) Hematopoietic immortalizing function of the NKL-subclass homeobox gene TLX1. Genes Chromosomes Cancer 49:119-31
Riz, Irene; Hawley, Teresa S; Johnston, Helen et al. (2009) Role of TLX1 in T-cell acute lymphoblastic leukaemia pathogenesis. Br J Haematol 145:140-3

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