Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1999-10-21
2002-10-22
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S343000
Reexamination Certificate
active
06468794
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to enriched neural stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for neural stem and progenitor cells, particularly central nervous system neural stem cells and progenitor cells, and most particularly to enriched populations of neurosphere initiating cells (NS-IC).
BACKGROUND OF THE INVENTION
Stem cell populations constitute only a small percentage of the total number of cells, but are of immense interest because of their ability to repopulate the body. The longevity of stem cells and the dissemination of stem cell progeny are desirable characteristics. There is significant commercial interest in these methods because stem cells have a number of clinical uses. There is also medical interest in the use of stem cells as a vehicle for gene therapy.
Proteins and other cell surface markers found on stem cell and progenitor cell populations are useful in preparing reagents for the separation and isolation of these populations. Cell surface markers are also useful in the further characterization of these important cells.
Yin et al., U.S. Pat. No. 5,843,633, incorporated herein by reference, describes a monoclonal antibody called AC133, which binds to a surface marker glycoprotein on hematopoietic stem and progenitor cells. The AC133 antigen is a 5-transmembrane cell surface antigen with a molecular weight of 117 kDa. Expression of this antigen is highly tissue specific, and has been detected on a subset of hematopoietic progenitor cells derived from human bone marrow, fetal bone marrow and liver, cord blood, and adult peripheral blood. The subset of cells recognized by the AC133 antibody is CD34
bright
, and contains substantially all of the CFU-GM activity present in the CD34
+
population, making AC133 useful as a reagent for isolating and characterizing human hematopoietic progenitor and stem cells.
However, surface markers specific to non-hematopoietic stem cells and progenitor cells, and particularly central nervous system neural stem cells and progenitor cells have not been identified. Further, the AC133 antibody has not been used in methods for identifying, isolating, or enriching for non-hematopoietic stem cells or progenitor cells, particularly central nervous system (CNS) neural stem cells and progenitor cells. There remains a need for tools, such as monoclonal antibodies that are useful in isolating and characterizing human non-hematopoietic progenitor and stem cells, and particularly central nervous system (CNS) neural stem cells and progenitor cells.
SUMMARY OF THE INVENTION
This invention provides methods for identifying, isolating, and enriching for human non-hematopoietic progenitor and stem cells, and particularly central nervous system (CNS) neural stem cells which can initiate neurospheres (NS-IC) and progenitor cells. The invention also provides for enriched populations containing CNS neural stem cells that can initiate neurospheres, and progenitor cells. A “neurosphere initiating cell (NS-IC)” is a cell that can initiate long-term neurosphere culture. A “neurosphere”, in turn, is an aggregate or cluster of cells which includes neural stem cells and primitive progenitors. The identification, culture, growth, and use of neurospheres is disclosed in Weiss et al., U.S. Pat. No. 5,750,376 and Weiss et al., U.S. Pat. No. 5,851,832, both incorporated herein by reference. While the term “NS-IC” is defined by the ability or capacity of that cell to form a neurosphere, these cells may be appropriately grown in adherent culture (see, for example, Johe, U.S. Pat. No. 5,753,506, incorporated herein by reference), and it should be noted that the methods and populations described herein are not to be limited to suspension cultures of NS-IC. An NS-IC is nestin
+
and has the capability to differentiate, under appropriate differentiating conditions, to neurons, astrocytes, and oligodendrocytes.
According to one embodiment of this invention, enriched populations of non-hematopoietic stem cells and progenitor cells, preferably CNS neural stem cells including NS-ICs, and progenitor cells, and method of identifying, isolating, or enriching for such cells, is achieved by contacting a population of cells containing at least one stem cell or NS-IC, or progenitor cell with a reagent that binds to surface marker glycoprotein antigen (“AC133 antigen”) recognized by the AC133 antibody. In a preferred embodiment the reagent is the AC133 antibody (the AC133 antibody is alternately referred to herein as “5F3”). Use of traditional techniques for cell sorting, such as by immunoselection (e.g., FACS), then permits identification, isolation, and/or enrichment for cells in which contact between the reagent and the AC133 antigen has been detected.
In another embodiment, this invention provides a novel antibody, herein called 5E12, that may be used to provide enriched populations of non-hematopoietic stem cells and progenitor cells, preferably CNS neural stem cells that can initiate neurospheres and progenitor cells, and may be used in methods of identifying, isolating, or enriching for such cells, by contacting a population of cells containing at least one stem cell NS-IC, or progenitor cell with the 5E12 antibody, which binds to a surface marker glycoprotein antigen other than the AC133 antigen.
In a preferred embodiment, the cells of this invention, preferably the CNS neural stem cells, are additionally characterized as lacking cell surface markers for CD45 and CD34.
In a further embodiment, this invention provides a novel antibody, herein called 8G1, believed to recognize CD24, which permits subselection between populations of CNS neural stem cells (characterized as 8G1
−/lo
) and populations of CNS progenitor cells (characterized as 8G1
+
).
REFERENCES:
patent: 5750376 (1998-05-01), Weiss et al.
patent: 5753506 (1998-05-01), Johe
patent: 5843633 (1998-12-01), Yin et al.
patent: 5851832 (1998-12-01), Weiss et al.
patent: 5968829 (1999-10-01), Carpenter
patent: WO 94/02593 (1994-02-01), None
patent: WO 00/12683 (2000-03-01), None
Geysen et al. J. Molecular Recognition 1: 32-41, 1988.*
Corbeil et al., AC133 Hematopoietic Stem Cell Antigen: Human Homologue of Mouse Kidney Prominin or Distinct Member of a Novel Protein Family? BLOOD 91:2625-2626 (1998).
De Wynter et al., Characteristics of MACS Isolated CD34+AC133+ Hematopoietic Progenitor Cells, MACS & MORE 2:8-9 (1998).
Miraglia et al., AC133, A New Antigen for Human Hematopoietic Stem Cells. Is Prominin the Murine Homologue? MACS & MORE 2:6-7 (1998).
Miraglia et al. A Novel Five-Transmembrane Hematopoietic Stem Cell Antigen: Isolation, Characterization, and Molecular Cloning, BLOOD 90:5013-5021 (1997).
Miraglia et al., A Response to AC133 Hematopoietic Stem Cell Antigen: Human Homologue of Mouse Kidney Prominin or Distinct Member of a Novel Protein Family? BLOOD 91:4390-4391 (1998).
Yin et al., AC133, A Novel Marker for Human Hematopoietic Stem and Progenitor Cells, BLOOD 90:5002-5012 (1997).
Uchida et al. Society for Neuroscience Abstracts 25 (1-2): 1767 (1999).
Weigmann et al. Proc. Natl. Acad. Sci. USA 94:12425-12430 (Nov. 11, 1997).
Corbeil et al. Journal of Cell Science, 112:1023-1033 (1999).
CD133 Cell Isolation Kit, MACS, (Miltenyi Biotec, Auburn, CA) (downloaded Dec. 11, 2001).
Buck David W.
Uchida Nobuko
Weissman Irving
Elrifi, Esq. Ivor R.
Hayes Robert C.
Karnakis, Esq. Christina V.
Kunz Gary L.
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
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