Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-28
2003-07-01
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S325000, C435S366000, C435S372000, C436S501000, C436S503000, C530S351000
Reexamination Certificate
active
06586192
ABSTRACT:
BACKGROUND OF THE INVENTION
Hematopoiesis in mammals is maintained by a pool of self-renewing hematopoietic stem cells (HSCs) (Ogawa, 1993, Blood 81:2844-2853). HSCs feed into lineage(s)-committed undifferentiated hematopoietic progenitor cells (HPCs) with little or no self-renewal capacity (Ogawa, 1993, Blood 81:2844-2853). The HPCs in turn generate morphologically recognizable differentiated precursors and terminal cells circulating in peripheral blood.
Human HSCs are identified on the basis of their capacity for long-term hematopoietic repopulation in vitro and in vivo. Specifically, in vitro repopulation of an irradiated allogeneic stromal adherent layer in long term culture (LTC) of Dexter type has been observed. In Dexter type LTC, primitive HPCs and HSCs are assessed as five to eight week and twelve week LTC initiating cells (LTC-ICs; Sutherland et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:3584-3588; Valtieri et al., 1994, Cancer Res. 54:4398-4404; Hao et al., 1996, Blood 88:3306-3313), or cobblestone area forming cells (CAFCs; Breems et al., 1996, Blood 87:5370-5378). Particularly, short term repopulating primitive HPCs have been identified in five to eight week LTC (Sutherland et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:3584-3588; Larochelle et al., 1996, Nature Med. 2:1329-1337), whereas long-term repopulating putative HSCs have been identified in twelve week LTC (Hao et al., 1996, Blood 88:3306-3313). Moreover, in vivo repopulation of severe combined immunodeficiency (SCID) mice at two months (Nolta et al., 1994, Blood 83:3041-3047) or non-obese diabetic SCID (SCID-NOD) mice at one and a half months (Bock et al., 1995, J. Exp. Med. 182:2037-2043) after irradiation and HSC injection has been observed.
In murine embryonic life (day 7.5 of gestation), a close developmental association of the hematopoietic and endothelial lineages takes place in the yolk sack blood islands, leading to the hypothesis that the two lineages share a common ancestor referred to as the hemoangioblast (Flamme et al., 1992, Development 116:435-439; Risau et al., 1995, Ann. Rev. Cell. Dev. Biol. 11:73-91).
Vascular endothelial growth factor (VEGF) and one of its receptors, VEGFRII termed Flk1 in mice and KDR in humans, play a key role in early hemoangiogenesis. In fact, Flk1
−
knock-out mice are unable to form blood islands and blood vessels (Shalaby et al., 1995, Nature 376:62-66). Differentiated murine embryonic stem cells treated with VEGF and the ligand for c-kit receptor at the embryoid stage give rise to primitive blast cells which generate the various hematopoietic lineages (Kennedy et al., 1997, Nature 386:488-492; Kabrun et al., 1997, Development 124:2039-2048): these data suggest a role for VEGF at the level of primitive HPCs in murine embryonic hematopoiesis. There are no data concerning the effect of expression or the function of KDR in human embryonic/fetal HSCs.
In post-fetal life, the VEGF/KDR system plays an important role in the endothelial lineage. Indeed, KDR and CD34 antigens are expressed on progenitors of human adult endothelial cells (Ashara et al., 1997, Science 275:964-967). Again, there are no data concerning the effect of expression or the function of KDR in human post-fetal HSCs, particularly long-term repopulating HSCs. Most studies have focused on examination of the effect of VEGF on partially purified HPCs. The results of these studies suggest that VEGF exerts an enhancing or inhibitory effect on bone marrow (BM) HPC colony formation stimulated by diverse hematopoietic growth factors (HGFs; Broxmeyer et al., 1995, Int. J. Hematol. 62:203-215) and a stimulatory effect on hematopoietic cells in normal mice (Gabrilovich et al., 1998, Blood 92:4150-4166). In addition, KDR mRNA is expressed in cord blood (CB) and BM partially purified HPCs, while VEGF does not affect CB HPC colony formation but exerts an anti-apoptotic action on irradiated HPCs (Katoh et al., 1995, Cancer Res. 55:5687-5692).
There is a need in the art for efficient methods of purifying and characterizing long term repopulating HSCs and for methods of ex vivo expansion of these cells. In addition, there is a need in the art for methods of treating a variety of diseases using HSCs. The present invention satisfies these needs.
BRIEF SUMMARY OF THE INVENTION
The invention includes a method of obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue and isolating a population of KDR
+
cells therefrom, thereby obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells.
In one aspect, the human hematopoietic tissue is selected from the group consisting of embryonic hematopoietic tissue, fetal hematopoietic tissue, and post-natal henlatopoietic tissue.
In another aspect, the embryonic hematopoietic tissue is selected from the group consisting of yolk sac, and embryonic liver.
In yet another aspect, the fetal hematopoietic tissue is selected from the group consisting of fetal liver, fetal bone marrow and fetal peripheral blood.
In a further aspect, the post-natal hematopoietic tissue is selected from the group consisting of cord blood, bone marrow, normal peripheral blood, mobilized peripheral blood, hepatic hematopoietic tissue, and splenic hematopoietic tissue.
In yet a further aspect, the KDR
+
cells are isolated using a reagent which specifically binds KDR.
In one aspect, the reagent is an antibody is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.
In another aspect, the antibody is a monoclonal antibody.
In yet another aspect, the monoclonal antibody is 260.4. In a further aspect, the KDR
+
cells are isolated using a conjugated vascular endothelial growth factor or a molecule derived therefrom.
In yet a further aspect, the cells are starvation resistant long-term repopulating human hematopoietic stem cells.
The invention includes an enriched population of long-term repopulating human hematopoietic stem cells obtained using a method of obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue and isolating a population of KDR
+
cells therefrom, thereby obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells. The invention also includes a cell isolated using this method. The invention also includes the cell isolated using this method wherein the cell comprises an isolated nucleic acid.
In one aspect, the cell comprising an isolated nucleic acid comprises an isolated nucleic acid selected from the group consisting of a nucleic acid encoding adenosine deamininase, a nucleic acid encoding &bgr;-globin, a nucleic acid encoding multiple drug resistance, an antisense nucleic acid complementary to a human immunodeficiency virus nucleic acid, an antisense nucleic acid complementary to a nucleic acid encoding a cell cycle gene, and an antisense nucleic acid complementary to a nucleic acid encoding an oncogene.
In another aspect, the isolated nucleic acid is operably linked to a promoter/regulatory sequence.
In even another aspect, the promoter/regulatory sequence is selected from the group consisting of a retroviral long terminal repeat, and the cytomegalovirus immediate early promoter.
The invention includes a method of obtaining a purified population of long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue, isolating a population of hematopoietic progenitor cells therefrom, and isolating a population of KDR
+
cells from the population of hematopoietic progenitor cells, thereby obtaining a purified population of long-term repopulating human hematopoietic stem cells.
In one aspect, the human hematopoietic tissue is selected from the group consisting of embryonic hematopoietic tissue, fetal hematopoietic tissue, and post-natal heniatopoie
Peschle Cesare
Ziegler Benedikt L.
Akin Gump Strauss Hauer & Feld L.L.P.
Belyavskyi Michail A
Chan Christina
Thomas Jefferson University
LandOfFree
Compositions and methods for use in affecting hematopoietic... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Compositions and methods for use in affecting hematopoietic..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compositions and methods for use in affecting hematopoietic... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3086559