Lineage restricted glial precursors from the central nervous...

Details Lineage restricted glial precursors from the central nervous... Lineage restricted glial precursors from the central nervous...

1997-11-29

2001-05-22

Kunz, Gary L. (Department: 1647)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S368000, C435S395000, C435S402000, C435S378000

Reexamination Certificate

active

06235527

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to lineage restricted glial precursors from the central nervous system (CNS). More particularly, the invention relates to a purified population of glial restricted precursor (GRP) cells capable of differentiating into oligodendrocytes and two types of astrocytes and methods of making and using thereof.
Relatively little is known about the origins of glial cells, which represent 90% of the cells in the central nervous system (CNS). Studies on late embryonic and postnatal rats have identified (1) cells apparently restricted to differentiation into astrocytes and (2) oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells able to generate oligodendrocytes and type-2 astrocytes. M. C. Raff et al., A Glial Progenitor Cell That Develops In Vitro into an Astrocyte or an Oligodendrocyte Depending on the Culture Medium, 303 Nature 390-96 (1983); M. C. Raff, Glial Cell Diversification in the Rat Optic Nerve, 243 Science 1450-55 (1989); R. K. Small et al., Evidence for Migration of Oligodendrocyte-type-2 Astrocyte Progenitor Cells into the Developing Rat Optic Nerve, 328 Nature 155-57 (1987); R. H. Miller et al., The Macroglial Cells of the Rat Optic Nerve, 12 Ann. Rev. Neurosci. 517-34 (1989); M. Noble et al., From Rodent Glial Precursor Cell to Human Glia Neoplasia in the Oligodendrocyte-type-2 Astrocyte Lineage, 15 Glia 222-30 (1995). In vivo labeling of dividing precursor cells with retroviruses has also indicated the existence of precursor cells that only generate astrocytes, and others that generate oligodendrocytes and astrocytes, J. Price et al., The Generation of Cellular Diversity in the Cerebral Cortex, 2 Brain Pathol. 23-29 (1992); J. E. Goldman, Lineage, Migration, and Fate Determination of Postnatal Subventricular Zone Cells in the Mammalian CNS, 24 J. Neurooncol. 61-64 (1995); J. R. Sanes, Analysing Cell Lineages with a Recombinant Retrovirus, 12 TINS 21-28 (1989), but the relationship of these cells to those characterized by in vitro experimentation is not yet known. How many classes of glial precursor cells there are and their developmental relationship to each other remain to be defined.
A critical missing component in understanding the development and generation of the glial cells of the central nervous system is to determine whether there exists any cell in vivo that is able to generate all three of the best-described glial populations, i.e. oligodendrocytes and cells with the characteristics of type-1 and type-2 astrocytes. Prior to the discoveries disclosed in the present patent application, the existence of such a cell was strictly hypothetical.
The ability to isolate and grow mammalian glial restricted precursor cells would allow for using pure populations of such cells for therapeutic transplantation, discovery of genes specific to selected stages of development, generation of cell-specific antibodies for therapeutic and diagnostic uses such as for targeted gene therapy, and the like. GRP's are important for transplantation therapy because of the ability of glial cells to migrate extensively after transplantation. Thus, a single injection can allow diffuse migration of cells to otherwise inaccessible sites. For example, multiple sclerosis is a disorder where oligodendrocytes and myelin sheaths are damaged. For example, transplantation of genetically modified GRPs for treating diseases of the CNS would allow cells to incorporate within the CNS parenchyma instead of forming an isolated mass of cells that provides a very high local concentration of biologically active factor(s) as a single large point source. As another example, in diseases where remyelination is required, the migration of GRP cells and/or their derivatives away from a lesion site might allow a reduced number of injections to be made in the CNS, thus simplifying surgical procedures. Dividing GRP's can also be made to express a variety of genes and can be used to deliver drugs that would not normally cross the blood-brain barrier. Exemplary of disorders that could be treated in this manner are glycogen storage disorders such as Gaucher's disease and Niemann-Pick disease. GRP's can also be used as a source for purification of trophic molecules, i.e. molecules that are selectively enriched in such cells. Further, GRP's can be used as a source of mRNA for generation of cDNA libraries that are specific for the stage of development that GRP's represent. Moreover, GRP's would provide a ready source of cells for high throughput screening of drugs.
In view of the foregoing, it will be appreciated that providing a population of tripotential glial precursor cells capable of differentiating into oligodendrocytes and two types of astrocytes, and methods of making and using thereof would be significant advancements in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide isolated (pure) populations of mammalian glial restricted precursor cells.
It is also an object of the invention to provide a method of isolating a pure population of mammalian glial restricted precursor cells from the central nervous system.
It is another object of the invention to provide mammalian CNS glial cells via generation and differentiation from multipotent GRP cells isolated from mammalian central nervous system.
It is still another object of the invention to provide a purified population of mammalian glial restricted precursor cells, wherein such cells are distinct from previously described cells including (1) neuroepithelial stem (NEP) cells, (2) neural crest stem cells, (3) neurospheres, (4) E-NCAM
+
neuronal progenitor cells, and (5) O-2A progenitor cells.
These and other objects can be addressed by providing an isolated, pure population of mammalian CNS glial restricted precursor cells that is capable of generating oligodendrocytes and at least two distinct populations of astrocytes. These glial restricted precursor cells are preferably isolated from a mammal selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, porcines, ovines, rats and lagomorphs. The glial restricted precursor cells express cell surface antigens recognized by the A2B5 monoclonal antibody, but do not express embryonic neural cell adhesion molecule (E-NCAM), i.e. the cells are E-NCAM
−
. The glial restricted precursor cells are capable of self renewal in adherent feeder-cell-independent culture medium and of differentiating into CNS glial cells, including oligodendrocytes, A2B5-immunoreactive process-bearing astrocytes, and A2B5-non-immunoreactive fibroblast-like astrocytes, but not into CNS neuronal cells.
A method of isolating a pure population of mammalian CNS glial restricted precursor cells comprises the steps of:
(a) removing a sample of CNS tissue from a mammal at a stage of development after closure of the neural tube;
(b) dissociating cells comprising the sample of CNS tissue removed from the mammal; and
(c) purifying from the dissociated cells a subpopulation expressing a selected antigen defining glial restricted precursor cells.
The purified subpopulation of cells can then be grown in feeder-cell-independent culture on a substratum and in a medium configured for supporting adherent growth of the glial restricted precursor cells and at a temperature and in an atmosphere conducive to growth of the glial restricted precursor cells. In a preferred embodiment of this method, the selected antigen defining glial restricted precursor cells is a ganglioside recognized by the A2B5 antibody. The glial restricted precursor cells can be purified through procedures well known in the art, such as specific antibody capture, fluorescence activated cell sorting, magnetic bead capture, and the like.
A method of obtaining glial cells comprises:
(a) providing glial restricted precursor cells; and
(b) plating the glial restricted precursor cells under differentiating conditions, thereby causing the glial restricted precursor cells to differentiate into glial cells. Non-process bearing A2

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