Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-08
2001-12-04
Martin, Jill D. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S069100, C435S320100, C435S325000, C424S093210, C514S04400A
Reexamination Certificate
active
06326205
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to myeloproliferative receptors (mpl)f specifically, the use of mpl ligands to expand primitive human stem cell subpopulations with minimal differentiation.
BACKGROUND OF THE INVENTION
Thrombopoietin (TPO) is a recently isolated ligand of mpl (Bartley et al. (1994) Cell 77:1117; Kaushansky et al. (1994) Nature 369:568; Lok et al. (1994) Nature 269:565; Kuter et al. (1994) Proc. Natl. Acad. Sci. USA 91:11104-11108; Kuter & Rosenberg (1994) Blood 84:1464; Wendling et al. (1994) Nature 369:571), first identified as the proto-oncogene transduced by the murine myeloproliferative leukemia (MPL) virus (Wendling et al. (1989) Blood 73:1161-1167; Souyri et al. (1990) Cell 63:1137-1147; Vigon et al. (1992) Proc. Natl. Acad. Sci. USA 89:5640-5644; Skoda et al. (1993) EMBO J. 12:2645-2653; Methia et al. (1993) Blood 82:1395-1401). TPO has been shown to independently stimulate megakaryocyte (MK) progenitor division and MK maturation in vivo and in vitro (Bartley et al. (1994) supra; Kuter et al. 1994) supra; Kuter & Rosenberg (1994) supra; Wendling et al. (1994) supra; de Sauvage et al. (1994) Nature 369:533; Broudy et al. (1995) Blood 85:1719-1726; Lok & Foster (1994) Stem Cells 12:586-598; Zeigler et al. (1994) Blood 84:4045). In vivo administration of TPO to thrombocytopenic rodents was found to significantly boost the platelet count as well as increase the number and ploidy of maturing MKs in the bone marrow (Lok et al. (1994) supra; Lok et al. (1994) supra; Wendling et al. (1994) supra; de Sauvage et al. (1994) supra). Absence of a c-mpl (TPO receptor) gene in mice was reported to result in thrombocytopenia (Guerney et al. (1994) Science 265:1445). More recently, it has been demonstrated that MKs can be primed to produce functional platelets in culture after exposure to TPO (Choi et al. (1995) Blood 85:402).
Hematopoietic stem cells are rare cells that have been identified in fetal bone marrow, umbilical cord blood, adult bone marrow, and peripheral blood, which are capable of differentiating into each of the myeloerythroid (red blood cells, granulocytes, monocytes), megakaryocyte (platelets) and lymphoid (T-cells, B-cells, and natural killer cells) lineages. In addition, these cells are long-lived, and are capable of producing additional stem cells, a process termed self-renewal. Stem cells initially undergo commitment to lineage restricted progenitor cells, which can be assayed by their ability to form colonies in semisolid media. Progenitor cells are restricted in their ability to undergo multi-lineage differentiation and have lost their ability to self-renew. Progenitor cells eventually differentiate and mature into each of the functional elements of the blood. This maturation process is thought to be modulated by a complex network of regulatory factors including erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), thrombopoietin (TPO), steel factor (St1), Flk-2 ligand and interleukins (IL) 1-15.
Recently, in vitro assays have been developed to identify human hematopoietic stem cells having self-renewal and multi-lineage differentiative capacity. One assay is the cobblestone area-forming cell (CAFC) assay, based on freshly isolated stromal cells or established stromal cell lines. In the mouse system, late-appearing cobblestone area formation on fresh marrow-derived stroma (Ploemacher et al. (1991) Blood 78:2527) or on a cloned stromal cell line (Neben et al. (1993) Exp. Hematol. 21:438) has been shown to correlate with in vivo hematopoietic repopulating ability. Correlation of CAFC and long term culture-initiating cell (LTCIC) frequencies using the mouse stromal cell line SyS1 has been demonstrated (Reading et al. (1994) Exp. Hematol. 22:406). In addition, the in vivo severe combined immunodeficiency (SCID)-hu bone assay has been used to measure long term engraftment of candidate stem cell populations (Kyoizumi et al. (1992) Blood 79:1704; Baum et al. (1992) Proc. Natl. Acad. Sci. USA 89:2804; Chen et al. (1993) Blood 82 (Suppl. 1):180a). Both the in vitro CAFC assay and the SCID-hu bone model permit analysis of B-cell and myeloid cell generation from candidate pluripotent hematopoietic stem cells (PHSC).
It is becoming increasingly apparent that distinct subpopulations of stem cells may be responsible for different phases of engraftment post transplantation. As early as 1964, differences in the ability of murine spleen colony forming units (CFU-S) to generate secondary CFU-S were defined (Ploemacher & Brons (1994) Exp. Hematol. 17:263-266). Although evidence now indicates that most CFU-S are not involved in repopulating lethally irradiated hosts (Jones et al. (1990) Nature 347:188-189; Jones et al. (1989) Blood 73:397-401), heterogeneity in transplantation potential appears to exist even within subpopulations of radioprotective cells. This has been demonstrated with serial bone marrow transplantations. The long-term repopulating ability of the grafts are lost with serial transfers, while a cell population survives which contributes to short-term reconstitution (Jones et al. (1989) supra). Further support for the concept that both short-term and long-term reconstituting stem cell populations exist have been derived from studies in which isoenzyme analysis and retroviral gene marking of hematopoietic cells have been utilized to track the fate of stem cells. A mathematical analysis of correlations and variances of donor reconstitution with isoenzyme variants in lethally irradiated mice indicates that a large number of multi-lineage clones are active immediately after reconstitution but rapidly decline, with the majority being inactive 12 weeks post-transplantation (Harrison & Zhong (1992) Proc. Natl. Acad. Sci. USA 89:10134-10138; Harrison et al. (1993) Exp. Hematol. 21:206-219). These observations indicate the existence of a population of cells with multi-lineage short-term engrafting potential in donor murine bone marrow. Similar observations have been made in a large animal transplantation model, where isoenzyme differences have indicated the contribution of multiple clones to short-term engraftment followed by sustained contributions by relatively few stem cell clones (Abkowwitz et al. (1990) Proc. Natl. Acad. Sci. USA 87:9062-9066). These findings have been confirmed by an eloquent analysis of clonal development after transplantation with retrovirally marked stem cells (Jordan et al. (1990) cell 61:953; Capel et al. (1990) Blood 75:2267).
Theoretically, subsets of cells with differing proliferative potentials may also differ with regards to physical characteristics, and therefore may be isolated and functionally defined. Bone marrow cells responsible for reconstitution following lethal irradiation can be isolated using centrifugation techniques exploiting cell size and density fractionation, or fluorescence-activated cell sorting (FACS) based on uptake of fluorescent vital dyes, lectin binding, or cell surface antigen expression (Sprangrude (1989) Immunol. Today 10:344-350; Visser & Van Bekkum (1990) Exp. Hematol. 18:248-256). FACS isolated murine cells that are responsible for engraftment lack lineage markers for B-cells, T-cells, myelomonocytes and erythrocytes and are termed lineage negative (Lin
−
) (Spangrude et al. (1988) Science 241:58-62). The Lin
−
fraction of murine bone marrow can be further subdivided based on low levels of Thy-1 and expression of the Sca-1 antigen (Visser & Van Bekkum (1990) supra; Spangrude at al. (1988) supra; Szilvassay et al. (1989) Blood 74:930-939; Szilvassay & Cory (1993) Blood 81:2310-2320; Spangrude & Scollay (1990) Exp. Hematol. 18:9920-926; Jurecic et al. (1993) Blood 82:2673-2683). Murine cells that are Thy-1.1
lo
Lin
−
Sca-1
+
are 1000-fold enriched in radioprotective ability, and contain all of the radioprotective cells found in the bone marrow of syngeneic C57BL/thy-1.1 mice (Uchida & Weissman (1992) J. Exp. Med. 175:175-184). As few as 100 cells that are Thy-1.1
lo
Lin
−
Sca-1
+
Murray Lesley J.
Young Judy C.
Karny Geoffrey M.
Martin Jill D.
Systemix, Inc.
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