By Donald Zuhn --
On Monday, Neuralstem, Inc. announced that the European Patent Office had granted European Patent No. EP 0 915 968 to the Rockville, Maryland-based biotech company. The '968 patent, entitled, "Isolation, Propagation and Directed Differentiation of Stem Cells from Embryonic and Adult Central Nervous System of Mammals," is directed to methods of expanding, culturing, and differentiating mammalian central nervous system stem cells in vitro; in vitro mammalian central nervous system (CNS) stem cell cultures produced from such methods; methods of generating mature neurons from mammalian multipotential CNS stem cells; and mature neurons generated from such methods. According to Neuralstem's press release, the European patent has been validated in France, Germany, Ireland, Spain, Sweden, Switzerland, and the United Kingdom.
Neuralstem's technology allows for the production of commercial quantities of neural stem cells from the human brain and spinal cord, and for the differentiation of such cells into mature, physiologically relevant human neurons and glia. Neuralstem is utilizing this technology to treat diseases of the major central nervous system, including ischemic paraplegia, traumatic spinal cord injury, and ALS (Lou Gehrig's disease). According to Neuralstem's website, the company's technology has been used to extend the life of rats with ALS, as well as to reverse paralysis in rats with ischemic spastic paraplegia.
Neuralstem CEO Richard Garr called the '968 patent a "core technology patent," adding that "the European Patent Office has now joined with the U.S. Patent Office in rejecting any arguments that the body of Stem Cells Inc. patents and publications, noted in the [European] examinations, in any way prevents Neuralstem's patents from issuing." As we reported last week, StemCells, Inc. filed a patent infringement suit against Neuralstem, asserting that Neuralstem had infringed four of StemCells' U.S. patents (see "StemCells' Patents Survive Reexam -- StemCells and Neuralstem Differ on Extent of Changes"). Neuralstem countered by seeking a reexamination of the asserted patents, and earlier this month, StemCells announced that the Patent Office had "upheld" two of the patents with "only minor amendments." Neuralstem released its own statement, contending that StemCells had "completely mischaracterized the meaning of the US Patent and Trademark Office's most recent action." While Mr. Garr's comments suggest that Neuralstem believes it has the better argument in its ongoing patent dispute with StemCells, StemCells has indicated that it plans to move forward with the case.
Independent claims 1, 6, 14, 17, and 24 of the '968 patent recite:
1. A method for expansion and long-term culture in vitro of stem cells of the central nervous system of a mammal, wherein the stem cells maintain the multipotential capacity to differentiate into neurons, astrocytes, and oligodendrocytes, comprising the steps of:
a) dissociating cells from central nervous system tissue by mechanical trituration;
b) culturing the dissociated cells adhered onto a plate in a chemically defined serumfree culture medium;
c) plating dissociated cells at a density not exceeding 20,000 cells/cm2 and, in subsequent passages, replating the cultured cells at a density not exceeding 10,000 cells/cm2;
d) adding daily to the cultured cells a growth factor selected from the group consisting of
i) bFGF at a concentration of at least 10 ng/ml,
ii) EGF at a concentration of at least 10 ng/ml,
iii) TGF-alpha at a concentration of at least 10 ng/ml, and
iv) aFGF at a concentration of at least 10 ng/ml plus 1yg/ml heparin;
e) replacing the culture medium with fresh medium within every two days;
f) passaging the cultured cells within four days after plating so as not to exceed 50% confluence; and
g) passaging the cultured cells by treating the cultured cells with saline solution and scraping cells from the plate.6. An in vitro culture of stem cells of the central nervous system of a mammal, wherein the stem cells maintain the multipotential capacity to differentiate into neurons, astrocytes, and oligodendrocytes.
14. A method for differentiation of an in vitro culture of stem cells of the central nervous system of a mammal, wherein the stem cells maintain the multipotential capacity to differentiate into neurons, astrocytes, and oligodendrocytes, comprising the steps of:
a) dissociating cells from central nervous system tissue by mechanical trituration;
b) culturing the dissociated cells on a plate in complete absence of serum;
c) adding daily to the cultured cells a first growth factor selected from the group consisting of
i) bFGF at a concentration of at least 10 ng/ml,
ii) EGF at a concentration of at least 10 ng/ml,
iii) TGF-alpha at a concentration of at least 10 ng/ml, and
iv) aFGF at a concentration of at least 10 ng/ml plus 1Hg/ml heparin;
d) passaging the cultured cells by treating the cultured cells with saline solution and scraping the cells from the plate; and
e) removing the first growth factor from the cultured cells.17. A method for in vitro generation of region-specific, terminally differentiated, mature neurons from cultures of mammalian multipotential
CNS stem cells, comprising the steps of:
a) culturing multipotential CNS stem cells from a specific region in a chemically defined serum-free culture medium containing a growth factor;
b) replacing the medium with growth factor-free medium;
c) harvesting the stem cells by trypsinization;
d) plating the stem cells at a density of between 100,000 to 250,000 cells per square centimeter; and
e) culturing the cells in a glutamic acid-free chemically defined serum-free culture medium.24. An in vitro culture of region-specific, terminally differentiated, mature neurons derived from cultures of mammalian multipotential CNS stem cells from a specific region.
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