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  Cultures of human cns neural stem cellsUSPTO Application #: 20080107633Title: Cultures of human cns neural stem cells Abstract: The invention provides a cell culture including proliferating human neural stem cells with a doubling rate faster than thirty days. The invention also provides a cell culture media for proliferating mammalian neural cells including a standard defined culture medium, a carbohydrate source, a buffer, a source of hormones, one or more growth factors that stimulate the proliferation of neural stem cells, and LIF. The invention also provides a method for protecting, repairing or replacing damaged tissue comprising transplanting mammalian neural stem cells formed into neurospheres. The invention also provides a cell culture of differentiated human neural stem cells where the cells are glioblasts. The invention also provides a method of differentiating human neural stem cells in culture media. (end of abstract) Agent: Ivor R. Elrifi Mintz, Levin, Cohn, Ferris, Glovsky And Popeo, P.c - Boston, MA, US Inventor: Melissa Carpenter USPTO Applicaton #: 20080107633 - Class: 424093210 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.), Eukaryotic Cell The Patent Description & Claims data below is from USPTO Patent Application 20080107633. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD OF THE INVENTION [0001] This invention relates to isolation of human central nervous system stem cells, and methods and media for proliferating, differentiating and transplanting them. BACKGROUND OF THE INVENTION [0002] During development of the central nervous system ("CNS"), multipotent precursor cells, also known as neural stem cells, proliferate, giving rise to transiently dividing progenitor cells that eventually differentiate into the cell types that compose the adult brain. Stem cells (from other tissues) have classically been defined as having the ability to self-renew (i.e., form more stem cells), to proliferate, and to differentiate into multiple different phenotypic lineages. In the case of neural stem cells this includes neurons, astrocytes and oligodendrocytes. For example, Potten and Loeffler (Development, 110:1001, 1990) define stem cells as "undifferentiated cells capable of a) proliferation, b) self-maintenance, c) the production of a large number of differentiated functional progeny, d) regenerating the tissue after injury, and e) a flexibility in the use of these options." [0003] These neural stem cells have been isolated from several mammalian species, including mice, rats, pigs and humans. See, e.g., WO 93/01275, WO 94/09119, WO 94/10292, WO 94/16718 and Cattaneo et al., Mol. Brain. Res., 42, pp. 161-66 (1996), all herein incorporated by reference. [0004] Human CNS neural stem cells, like their rodent homologues, when maintained in a mitogen-containing (typically epidermal growth factor or epidermal growth factor plus basic fibroblast growth factor), serum-free culture medium, grow in suspension culture to form aggregates of cells known as "neurospheres". In the prior art, human neural stem cells have doubling rates of about 30 days. See, e.g., Cattaneo et al., Mol. Brain. Res., 42, pp. 161-66 (1996). Upon removal of the mitogen(s) and provision of a substrate, the stem cells differentiate into neurons, astrocytes and oligodendrocytes. In the prior art, the majority of cells in the differentiated cell population have been identified as astrocytes, with very few neurons (<10%) being observed. [0005] There remains a need to increase the rate of proliferation of neural stem cell cultures. There also remains a need to increase the number of neurons in the differentiated cell population. There further remains a need to improve the viability of neural stem cell grafts upon implantation into a host. SUMMARY OF THE INVENTION [0006] This invention provides novel human central nervous system stem cells, and methods and media for proliferating, differentiating and transplanting them. In one embodiment, this invention provides novel human stem cells with a doubling rate of between 5-10 days, as well as defined growth media for prolonged proliferation of human neural stem cells. In another embodiment, this invention provides a defined media for differentiation of human neural stem cells so as to enrich for neurons, oligodendrocytes, astrocytes, or a combination thereof. The invention also provides differentiated cell populations of human neural stem cells that provide previously unobtainable large numbers of neurons, as well as astrocytes and oligodendrocytes. This invention also provides novel methods for transplanting neural stem cells that improve the viability of the graft upon implantation in a host. BRIEF DESCRIPTION OF THE DRAWINGS [0007] FIG. 1 shows a representation of spheres of proliferating 9FBr human neural stem cells (passage 6) derived from human forebrain tissue. [0008] FIG. 2, Panel A, shows a growth curve for a human neural stem cell line designated 6.5Fbr cultured in (a) defined media containing EGF, FGF and leukemia inhibitory factor ("LIF") (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds); Panel B shows a growth curve for a human neural stem cell line designated 9Fbr cultured in (a) defined media containing EGF, FGF and LIF (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds); Panel C shows a growth curve for a human neural stem cell line designated 9.5Fbr cultured in (a) defined media containing EGF, FGF and LIF (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds); Panel D shows a growth curve for a human neural stem cell line designated 10.5Fbr cultured in (a) defined media containing EGF, FGF and leukemia inhibitory factor ("LIF") (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds). [0009] FIG. 3 shows a growth curve for a human neural stem cell line designated 9Fbr cultured in (a) defined media containing EGF and basic fibroblast growth factor ("bFGF") (shown as open diamonds), and (b) defined media with EGF but without bFGF (shown as closed diamonds). [0010] FIG. 4 shows a graph of cell number versus days in culture for an Mx-1 conditionally immortalized human glioblast line derived from a human neural stem cell line. The open squares denote growth in the presence of interferon, the closed diamonds denote growth in the absence of interferon. DETAILED DESCRIPTION OF THE INVENTION [0011] This invention relates to isolation, characterization, proliferation, differentiation and transplantation of CNS neural stem cells. [0012] The neural stem cells described and claimed in the applications may be proliferated in suspension culture or in adherent culture. When the neural stem cells of this invention are proliferating as neurospheres, human nestin antibody may be used as a marker to identify undifferentiated cells. The proliferating cells show little GFAP staining and little .beta.-tubulin staining (although some staining might be present due to diversity of cells within the spheres). [0013] When differentiated, most of the cells lose their nestin positive immunoreactivity. In particular, antibodies specific for various neuronal or glial proteins may be employed to identify the phenotypic properties of the differentiated cells. Neurons may be identified using antibodies to neuron specific enolase ("NSE"), neurofilament, tau, beta-tubulin, or other known neuronal markers. Astrocytes may be identified using antibodies to glial fibrillary acidic protein ("GFAP"), or other known astrocytic markers. Oligodendrocytes may be identified using antibodies to galactocerebroside, O4, myelin basic protein ("MBP") or other known oligodendrocytic markers. Glial cells in general may be identified by staining with antibodies, such as the M2 antibody, or other known glial markers. [0014] In one embodiment the invention provides novel human CNS stem cells isolated from the forebrain. We have isolated 4 neural stem cell lines from human forebrain, all of which exhibit neural stem cell properties; namely, the cells are self renewing, the cells proliferate for long periods in mitogen containing serum free medium, and the cells, when differentiated, comprise a cell population of neurons, astrocytes and oligodendrocytes. These cells are capable of doubling every 5-10 days, in contrast with the prior art diencephalon-derived human neural stem cells. Reported proliferation rates of diencephalon-derived human neural stem cells approximate one doubling every 30 days. See Cattaneo et al., Mol. Brain. Res., 42, pp. 161-66 (1996). [0015] Any suitable tissue source may be used to derive the neural stem cells of this invention. Neural stem cells can be induced to proliferate and differentiate either by culturing the cells in suspension or on an adherent substrate. See, e.g., U.S. Pat. No. 5,750,376 and U.S. Pat. No. 5,753,506 (both incorporated herein by reference in their entirety), and prior art medium described therein. Both allografts and autografts are contemplated for transplantation purposes. [0016] This invention also provides a novel growth media for proliferation of neural stem cells. Provided herein is a serum-free or serum-depleted culture medium for the short term and long term proliferation of neural stem cells. [0017] A number of serum-free or serum-depleted culture media have been developed due to the undesirable effects of serum which can lead to inconsistent culturing results. See, e.g., WO 95/00632 (incorporated herein by reference), and prior art medium described therein. [0018] Prior to development of the novel media described herein, neural stem cells have been cultured in serum-free media containing epidermal growth factor ("EGF") or an analog of EGF, such as amphiregulin or transforming growth factor alpha ("TGF-.alpha."), as the mitogen for proliferation. See, e.g., WO 93/01275, WO 94/16718, both incorporated herein by reference. Further, basic fibroblast growth factor ("bFGF") has been used, either alone, or in combination with EGF, to enhance long term neural stem cell survival. [0019] The improved medium according to this invention, which contains leukemia inhibitory factor ("LIF"), markedly and unexpectedly increases the rate of proliferation of neural stem cells, particularly human neural stem cells. Continue reading... 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