Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-02-20
2003-09-30
Marx, Irene (Department: 1651)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C435S029000, C435S026000, C435S372000
Reexamination Certificate
active
06627759
ABSTRACT:
TECHNICAL FIELD
The present invention relates, in general, to stem cells, and in particular, to a method of isolating stem cells and to reagents suitable for use in such a method. The invention further relates to stem cell populations isolatable in accordance with the present method.
BACKGROUND
The most primitive hematopoietic stem cells (HSC) will reconstitute all of the hematopoietic lineages or an entire lifespan. These pluripotent hematopoietic stem cells (PHSC) are the transplantable cells that are ultimately the targets for gene delivery in stem cell-based gene therapies. One defining characteristic for PHSC is that they will survive most cytoablative conditioning regimens. The mechanisms for their resistance to these toxic agents suggest potential strategies by which these cells can be selected in vitro. One mechanism for drug resistance lies in the ability to efflux toxic substances out of the cell via the multiple drug resistance (MDR) pump. Fluorescent substrates for the MDR pump have permitted the isolation of PHSC based on their high capacity or dye efflux in a variety of assay systems. Drug resistances may also be conferred by more specific mechanisms. For example, a cytosolic aldehyde dehydrogenase (ALDH) mediates resistance to cyclophosphamide (CPA), an alkylating agent used in cytoreductive regimens in preparation for bone marrow transplant. Thus, expression of ALDH can be considered a selectable marker for true PHSC.
The therapeutic effectiveness of CPA has been attributed largely to the ability of PHSC and intestinal crypt cells to survive the drug regimen. Human hematopoietic progenitors express a cytosolic ALDH and primitive human HSC derived from mobilized peripheral blood stem cells can be selected when placed in culture with cyclophosphamide for 7 days. Jones et al have demonstrated that long-term reconstituting murine PHSC can be isolated by providing a membrane-permeable fluorescent substrate for ALDH and by then selecting cells with the highest levels of ALDH activity (Jones, Blood 85:2742 (1995); Jones et al, Blood 88:487 (1996)). In these studies, dansyl aminoacetaldyde (DAAA) was used to stain murine bone marrow cells prepared by countercurrent elutriation.
Preliminary studies using DAAA indicate that this reagent is unusable on preparations of human hematopoietic cells because the signal intensity of the reagent is too high to resolve discrete cell populations by flow cytometry. The present invention provides a fluorescent ALDH substrate that is free of the problems associated with DAAA and that can be used in the purification of primitive human hematopoietic cells.
SUMMARY OF THE INVENTION
The present invention relates to a novel reagent and method for isolating stem cells, including human stem cells. The reagent is a fluorescent substrate for ALDH. The method comprises staining a cell population that includes primitive stem cells with the substrate in the presence of an inhibitor of MDR activity. ALDH present in the cells converts the substrate to a product that is trapped within the cells. Since primitive stem cells have higher levels of ALDH activity than other cell types, these cells stain brighter than other cell Hypes. The presence of the MDR inhibitor reduces the efflux of the substrate from the stem cells.
Objects and advantages of the present invention will be clear from the description that follows.
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Jones et al, “Assessment of aldehyde dehydrogenase in viable cells”, Blood 85(10):2742-2746 (1995).
Jones et al, “Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity”, Blood 88(2):487-491 (1996).
Lindahl, “Aldehyde dehydrogenase in 2-acetamidofluorene-induced rat hepatomas”, Biochemical Journal 164(1):119-123 (1977).
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Kaiser et al, “Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorecence quencing”, Biochimica et Biophysica Acta 1375:13-22 (1998).
Storms et al, “Aldehyde dehydrogenase activity as a selectable phenotype for hematopoietic progenitgors”, Blood 92(10) Suppl 1 Part 1-2,:59A (1998), Nov. 15, 1998.
Colvin Michael
Ludeman Susan M.
Smith Clayton A.
Storms Robert W.
Afremova Vera
Duke University
Marx Irene
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