| Lineage specific cells and phogenitor cells -> Monitor Keywords |
|
|
 
Lineage specific cells and phogenitor cellsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal CellLineage specific cells and phogenitor cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060003450, Lineage specific cells and phogenitor cells. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This invention relates to lineage specific cells and progenitor cells, methods of obtaining them and their uses. Pluripotent embryonic stem (ES) cells can be induced to differentiate in vitro into a mixture of cell types, comprising extraembryonic yolk sac and derivatives of all three embryonic germ layers. However, the disorganised and heterogeneous nature of the differentiation impedes manipulation and analysis of individual lineages. In particular, the invention provides a lineage-specific genetic selection technique to establish purified populations of neural precursors from differentiating ES cells. [0002] Embryonic stem (ES) cells are non-transformed cell lines derived directly from the pluripotent founder tissue in the mouse or human embryo, the epiblast (Evans and Kaufman, 1981; Martin, 1981; Brook and Gardner, 1997; Thomson et al, 1998; Shamblott et al, 1998). ES cells can be propagated and extensively genetically manipulated whilst retaining the capacity for multilineage differentiation, both in vivo and in vitro (Robertson, 1987). The differentiation of ES cells in culture closely reflects differentiative events in the embryo. In principal, therefore, ES cells provide in vitro access to the instructive and selective processes by which cellular diversification is generated in the mammalian embryo (Smith, 1992). [0003] Multilineage differentiation of ES cells can be initiated by simple aggregation (Martin and Evans, 1975; Doetschman et al., 1985). The aggregates form structures known as embryoid bodies, differentiation of which mirrors aspects of per- and early post-implantation mouse embryogenesis (Martin et al., 1977; Doetschman et al., 1985). A diverse array of cell types are subsequently found in outgrowths from embryoid bodies (Weiss and Orkin, 1996). Although representation of particular lineages can be diminished or enhanced by treatment with dimethyl sulphoxide or retinoic acid (RA), the differentiated products always consist of a mixture of cell types. This intrinsic disorganisation and complexity have limited the exploitation of in vitro ES cell differentiation for assignment of gene functions in developmental pathways. [0004] Several reports have documented the presence of neuronal cells and glia (Bain, 1995; Fraichard et al., 1995; Strubing et al., 1995; Okabe et al., 1996) in embryoid body outgrowths. This could be exploited to detect, characterise and manipulate factors that regulate neurogenesis and neuronal and glial differentiation. ES cells could also be used as a source of normal or genetically manipulated neural cells for biochemical and functional studies or for transplantation. The problem is that these objectives, however, are severely compromised by the abundance of non-neural cells in the cultures and because it is not currently possible to establish or maintain a culture predominantly containing neural cell progenitors. [0005] The art thus fails to provide a reliable method of obtaining a substantially pure population of a cell of any selected lineage, and progenitor cells of a selected lineage in particular. The art also fails to provide assays for developmental and other effects of factors on progenitor cells or differentiated cells of a selected lineage. [0006] The present invention aims to provide progenitor cells and/or differentiated cells of a selected lineage, assays for the effect of factors on such cells and uses of these cells, such as in transplantation. The present invention further aims to provide a genetic selection technique to eliminate non-neural cells from cultures and to enable purification from embryoid bodies of differentiation competent neural precursors. [0007] The present invention provides a method of obtaining a culture of cells of a selected lineage having a combination of two steps. One step is to select for cells that express a gene characteristic of cells of that lineage. The other is to so culture cells that they tend to differentiate into or proliferate as cells of that lineage. The effect is to yield a more highly purified culture of the selected-lineage cells than is otherwise possible. [0008] By way of example, one step of the combination is to select for cells that express a gene known to be expressed uniquely in haematopoietic progenitor cells, and the other is to culture cells in medium containing a nutrient known to promote differentiation of cells into haematopoietic progenitors. [0009] Accordingly, the invention provides a method for generating a culture that is purified or enriched in respect of cells of a selected lineage, comprising:-- [0010] (i) introducing into a multipotential cell a selectable marker that is differentially expressed in cells of the selected lineage compared with its expression in other cells; [0011] (ii) culturing the multipotential cell to induce (a) differentiation of the multipotential cell into a cell of the selected lineage or into a mixture of cells that is or includes cells of the selected lineage or (b) preferential survival of cells of the selected lineage; and [0012] (iii) selecting for those cells that express the selectable marker, [0013] The method is suitable for obtaining cells, optionally progenitors, of the selected lineage at high purity. Whilst following the art method for inducing differentiation of ES cells into a culture that includes neural cells can provide a maximum of about 50% neural progenitors, using the technique of the invention a purity in excess of 70% can be obtained, and in a specific embodiment described below the purity is substantially 100%. [0014] Step (ii) results in a pure population of cells or a mixed culture at least slightly purified or enriched in respect of desired cells and can suitably be carried out by culturing the multipotential cell in the presence of a factor that induces differentiation of the cell into a progenitor cell of the selected lineage. By way of example, a mitogen specific for neural progenitors is fibroblast growth factor. Reference herein to the term "factor" is not intended to be limited to protein or polypeptide factors but is intended to encompass any biologically active molecule or potentially biologically active molecule. [0015] The multipotential cell may be selected from embryonic stem (ES) cells, embryonic germ (EG) cells, embryonal carcinoma (EC) cells, a primary culture of foetal cells, a primary culture of post-natal cells and a primary culture of adult cells. The method may further comprise genetically modifying cells to delete, mutate, substitute or add genes in order to assay gene function in progenitor cells of the selected lineage or adapt a cell phenotype to render it more suitable for transplantation. The cells may thus be obtained by introducing a selectable marker into a multipotential cell line or a primary culture containing the cells of interest and then selecting out the cells of interest. The selectable marker is optionally introduced by transfection or viral infection via a transgenic animal from which the primary cultures are then established, suitably using the methods described in WO-A-94/24274. [0016] The invention thus advantageously enables a highly enriched population of cells and in particular all lineages of progenitors of a chosen lineage to be obtained. In an example below the population obtained is substantially 100% pure making it possible to isolate a single cell of known progenitor lineage. The invention is of application to all lineages of cells, particularly progenitor cells. A selectable marker expressed in cells that express a Sox gene leads to neural progenitor cells; the CD34, CD44 and SCL genes are suitable for obtaining haematopoietic progenitors, and the Nkx 2.5 or GATA-4 gene for cardiac progenitors. For generating myogenic progenitors, Myo0 or myE5 are suitable. Using retinoic acid induces differentiation of ES cells into neural cells, DMSO induces differentiation into haematopoietic cells and absence of retinoic acid induces a population enriched in cardiac progenitors. [0017] The method optionally further comprises:-- [0018] (iv) introducing into the multipotential cell a second selectable marker that is differentially expressed in progenitor cells or other cells of a selected sub-lineage compared with its expression in other cells, wherein cells of the selected sub-lineage are formed by differentiation of cells of the selected progenitor lineage; and [0019] (v) when a culture of progenitor cells of the selected lineage has been obtained, allowing or inducing differentiation of the cells and selecting for cells that express the second selectable marker. [0020] This aspect of the invention further enhances the purity of the obtained culture of cells, and is of advantage in cases that cells of the selected lineage differentially express a gene but only at a level slightly different from non-desired cells. [0021] In a preferred embodiment of the invention, described in more detail below, the selectable marker is a gene that codes for antibiotic resistance and selecting for those cells that express the selectable marker comprises introducing antibiotic into the culture. In use, application of the antibiotic selectively kills or ablates cells that do not express the marker, leaving behind a population of cells purified or enriched in respect of those expressing the antibiotic resistance, i.e. viable cells of selected lineage. At least two ways of introducing the selectable marker are known and suitable for the invention. The selectable marker may be introduced into the multipotential cell by targeted integration or gene trapping into a gene that is differentially expressed in progenitor cells of the selected lineage. Expression of the selectable marker is thereby operatively linked to a gene differentially expressed in a desired pattern. The selectable marker may also be introduced into the multipotential cell via random integration as a transgene wherein it is expressed under control of the regulatory elements of a gene that is differentially expressed in progenitor cells of the selected lineage. [0022] The selectable marker may more generally be a marker that when expressed results in preferential survival of cells expressing the marker, with antibiotic resistance being one such example. In this instance the marker is expressed in cells of the selected lineage. The selectable marker may also be a marker whose expression results in preferential killing of cells expressing the marker. In this instance the marker is expressed in cells other than those of the selected lineage. [0023] In a specific embodiment of the invention described in an example below, the multipotential cell is an ES, EC or EG cell and the method comprises inducing differentiation of the multipotential cell--one way is to form an embryoid body from the cell--and dissociating the cells. Trypsin is used in one example. It is an advantage that individual cells are thereby obtained. These are all exposed to the culture medium and not being attached to neighbouring cells are free of cell-to-cell influences that might affect the pattern of growth and/or differentiation of the cells, and hinder formation of progenitor cells according to the invention. [0024] A culture that is purified or enriched in respect of ventral progenitor cells is obtainable according to the invention, wherein the selectable marker is differentially expressed in neural progenitor cells and the second selectable marker is differentially expressed in ventral progenitor cells. The second selectable marker for this is suitably differentially expressed in cells that express Pax 6. [0025] A culture that is purified on enriched in respect of dorsal progenitor cells is obtainable according to the invention, wherein the selectable marker is differentially expressed in the neural progenitor cells and the second selectable marker is differentially expressed in dorsal progenitor cells. The second selectable marker for this is suitably differentially expressed in cells that express Pax 3. [0026] The invention also provides a cell, preferably a progenitor, of a selected lineage, obtainable according to the method of the invention. Hitherto, preparations of progenitors were too impure for certainty as to whether any chosen cell was a progenitor cell. With culture according to the invention that can give rise to substantially 100% pure preparations of progenitors, isolation of a single progenitor is achieved. [0027] The invention further provides a composition comprising a plurality of cells, wherein a majority of the cells are progenitor cells of a selected linage. Preferably, at least 60% of the cells are progenitor cells of the selected lineage. More preferably, at least 60% of the cells are neural progenitor cells. In addition, the invention provides an isolated neural progenitor cell. [0028] A significant application of the invention is in the field of assaying factors for the effects they may have on a selected progenitor. Accordingly, the invention still further provides an assay of the effect of a factor on a culture of progenitor cells of a selected lineage, comprising:-- [0029] (i) introducing into a multipotential cell a selectable marker that is differentially expressed in cells of the selected lineage compared with its expression in other cells; [0030] (ii) culturing the multipotential cell to induce differentiation of the multipotential cell into a cell or mixture of cells that includes cells of the selected lineage or to induce preferential survival of cells of the selected lineage; [0031] (iii) selecting for those cells that express the selectable marker; and [0032] (iv) culturing the thereby obtained cells of selected lineage in the presence of the factor. [0033] The assay method is preferably for assay of the effect of a factor on a culture of progenitor cells of selected lineage, wherein the selectable marker is differentially expressed in those progenitor cells. Reference to a "factor" in the assay, is intended to be a reference to any actual or potential biologically active molecule that may be introduced into culture of cells of the selected lineage, and is thus not intended to be limited to protein and polypeptide factors. The term is also intended to encompass a gene introduced or modified in a multipotential cell from which cells of the selected lineage are obtained. The assay can thus conveniently be used to assay for the effect of a particular gene, as well as for the effect of polypeptide and/or protein factors or small molecules of interest. Continue reading about Lineage specific cells and phogenitor cells... Full patent description for Lineage specific cells and phogenitor cells Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Lineage specific cells and phogenitor cells patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Lineage specific cells and phogenitor cells or other areas of interest. ### Previous Patent Application: Plant artificial chromosome compositions and methods Next Patent Application: Cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Lineage specific cells and phogenitor cells patent info. IP-related news and info Results in 0.33772 seconds Other interesting Feshpatents.com categories: Novartis , Pfizer , Philips , Polaroid , Procter & Gamble , 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
