WO2005095588A1

WO2005095588A1 - Cortex glutamatergic neuron precursor providing cortex glutamatergic neuron and cortex glutamatergic neuron precursor alone in vivo

Info

Publication number: WO2005095588A1
Application number: PCT/JP2005/006821
Authority: WO
Inventors: Nobuaki Tamamaki
Original assignee: Kyoto University
Priority date: 2004-03-31
Filing date: 2005-03-31
Publication date: 2005-10-13

Abstract

A method which comprises: the step of preparing a cell mass induced from a fetal cortex tissue, embryonic stem cells or nerve stem cells; the step of transferring an expression vector, which has a promoter selected from among NEX(Math2), NeuroD, NeuroD-related-factor, Neurogenin 1, Neurogenin 2, Svet 1, Otx1 and Tbr2 and a gene for confirming the expression of this molecule attached to the downstream of the promoter, into individual cells in the cell mass; the step of isolating cortex glutamatergic neurons and cortex glutamatergic neuron precursors with the use of the expression of the function of the expression vector as an indication; and the step of isolating the cortex glutamatergic neuron precursors with the use of the proliferation ability as an indication. According to this method, it is possible to obtain cortex glutamatergic neuron precursors for treating dysfunction caused by cortex injury, etc.

Description

Description Glutamate-operating neural cells of the cerebral cortex and glutamine-operated neural progenitor cells of the cerebral cortex that produce only progenitor cells in vivo

TECHNICAL FIELD The invention of this application relates to a cerebral cortical glutamatergic neural progenitor that produces only cerebral cortical glutamatergic neurons and itself (cerebral cortical glutamate neural progenitor cells) in vivo. It is about cells. More specifically, the invention of this application makes it possible to restore the area of the cerebral cortical gluten-mate nervous cells lacking or reduced to normal by cell transplantation, thereby enabling a therapeutic action for treating cerebral dysfunction. It relates to cells as medical materials and the like.

BACKGROUND ART Neurons in the central nervous system include excitatory neurons and inhibitory neurons. Both neurons are included in various ratios depending on the area of the central nervous system, and information processing is performed. In the cerebral cortex, excitatory neurons use glucan mate as a neurotransmitter. Excitatory neurons in the cerebral cortex are present in about 80% of the neurons, so that the neural circuit as a whole can maintain a moderate level of activity and perform smooth information processing. However, at times, traumatic disorders, cerebrovascular disorders, and disorders caused by genetic disorders result in loss of excitatory neurons in the cerebral cortex and, at the same time, loss of some brain functions. Attempts have been made to supplement this by injecting nerve cell growth factor into the brain or transplanting nerve cells derived from ES cells, but the effects of growth factors on the neocortex of the cerebrum are not known. No, the cells from the dorsal side of the neural tube are still made from ES cells Not in. In addition, a method of isolating neural stem cells from mammalian fetal cerebral cortex has been established.However, when neural stem cells are cultured and induced to differentiate in vitro, most of the differentiated neural cells become GABAergic neurons. In addition, astrocytes, oligodendrocytes and neural stem cells are mixed, and cerebral glucan mate-operating neurons have not been obtained yet. In the fetal cerebral neocortex, dal evening mate-operated neurons have been thought to be produced by division of neural stem cells in the ventricular zone (Non-Patent Documents 1, 3, 7, and 8). Recently, it has been suggested that some of these may be produced by Svetl-positive cells in the subventricular zone (Non-Patent Document 9). However, this Non-Patent Document 9 merely suggests the production of cerebral cortical glutamatergic neurons outside the ventricular zone, and that the subventricular zone Svetl-positive cells are cerebral cortical glutamatergic neurons. There is no evidence that they produced only neurons and cerebral cortical glutamatergic neural progenitors. Similarly, there is no description of a phenomenon in which Otxl-positive cells proliferate in the cerebral cortex and produce only cerebral cortex glumate-activated neurons and cerebral cortex glumate-activated neuronal precursor cells. Molecules that are expressed in mammalian cerebral cortical cells and are considered to be involved in the differentiation of neurons include, in addition to the aforementioned Svet and Otxl, proneural basic helix-loop-helix (abbreviated bHLH) transcriptional factor Neurogenin l neruogenin2, and bHLH differentiation gene that promotes neural differentiation, NeuroD, NeuroD-related-factor, NEX (also known as Math2) are known. Tbr2 is also known. Among the molecules listed here, those classified as bHLH are expressed in one of the daughter cells produced by asymmetric division of undifferentiated ventricular zone neural stem cells, and differentiate the expressed cells into neural cells. It is interpreted as a molecule that plays a role in promoting maturation. This interpretation stems from the fact that proneural bHLH transcriptional factor promotes the expression of cell division inhibitory molecules, and it has been speculated that cell division stops when expression of molecules belonging to proneural bHLH transcriptional factor or bHLH differentiation gene occurs ( Non-patent document 4). Therefore NEX (Non-Patent Documents 2, 5, Similarly, 6), NeuroD, NeuroD-related-factor, and Tbr2 have also been thought to be expressed in cells that do not undergo cell division, promoting neuronal differentiation. Non-Patent Document 1: Angevine, JB & Sidman, RL Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse.Nature 192, 766-768 (1961).

Non-Patent Document 2: Bartholoma, A. & Nave, KA NEX-1: a novel brain-specific helix-loop-helix protein with autoregulation and sustained expression in mature cortical neurons.Mech. Dev. 48, 217-228 (1994) Non-Patent Document 3: Noctor, SC, Flint, AC, Weissman, TA, Dammerman, RS & Kriegstein, AR Neurons derived from radial glial cells establish radial units in neocortex. Nature 409, 7 14-720 (200 1 ).

Non-Patent Document 4: Ross, S.E. and Greenberg, M.E.Basic helix-loop-helix factors in cortical development.Neuron 39, 12-25 (2003).

Non-Patent Document 5: Shimizu, C, Akazawa, C., Nakanishi, S. & Kageyama, R. MATH-2, a mammalian helix-loop-helix factor structurally related to the product of Drosophila proneural gene atonal, is specifically expressed in The nervous system. Eur. J. Biochem. 229, 239-248 (1995).

Non-Patent Document 6: Schwab, MH et al. Neuronal basic helix-loop-helix proteins (NEX, neuroD, NDRF): spatiotemporal expression and targeted disruption of the NEX gene in transgenic mice.J. Neurosci. 18, 1408-1418 ( 1998).

Non-patent literature: Takahashi, T. et al. Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall. J. Neurosci. 19, 10357-10371 (1999).

Non-Patent Document 8: Tamamaki, N., Nakamura, K., Okamoto, K. & Kaneko, T. Radial glia is a progenitor of neocortical neurons in the developing cerebral cortex. Neurosci. Res. 41, 51-60 (2001) .

Non-Patent Document 9: Tarabykin, V., Stoykova, A., Usman, N., & Gruss, P. Cortical upper layer neurons derive from the subventricular zone as indicated by Svetl gene expression. Development 128, 1983-1993 (2001). Non-Patent Document 10: Wu, S. et al., Pyramidal neurons of the neocortex generated from progenitor cells in the mantle layer, (submitted).

DISCLOSURE OF THE INVENTION The present application relates to cerebral cortical cells for transplantation used for treating dysfunction caused by cerebral cortical damage and the like. The task is to provide cells that produce only sexual neural progenitor cells. Another object of the present invention is to provide a method for separating and obtaining the cells. This application provides the following inventions (1) to (20) to solve the above problems.

(1) The following characteristics:

(a) included in the cell population containing cerebral cortical daltamatergic neurons and cerebral cortex glutamate-nergic neural progenitors;

(b) has a proliferative capacity or can be made to proliferate by stimulation; and

(c) expressing one or more molecules selected from the group consisting of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogenin2, Svetl, Otxl, Tbr2,

A cerebral cortical glutamate-active neural progenitor cell that specifically produces cerebral cortical glutamate-active neuronal cells and cerebral cortical glutamate-active neural progenitor cells in vivo. (2) The cell of the above invention (1), which is derived from a human. (3) The cell of the invention (2), which is derived from a human embryonic stem cell.

(4) The cell of the invention (1), which is derived from a mammal closely related to human.

(5) The cell of the above-mentioned invention (4), which is derived from an embryonic stem cell of a human closely related mammal.

(6) A method for separating cerebral cortical glumate-operating neural progenitor cells that specifically produces cerebral cortical glumate-operating neurons and cerebral cortex gulatmate-operating neural progenitors in vivo. The following steps:

(a) preparing a cell population containing cerebral cortex glumate-operating neurons and cerebral cortex glumate-operating neural progenitor cells;

(b) A reporter that emits a signal that can be detected in vivo in the downstream of the gene promoter of any of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogenin l, Neurogenin2, Svetl, Otxl or Tbr2 Introducing an expression vector linked to a protein gene into each cell of the cell population;

(c) a step of isolating cerebral cortex dal mate-operated neurons and cerebral cortical glue mate-operated neural progenitor cells from other cells based on the presence or absence of a signal generated by the repo overnight protein;

A method comprising:

(7) A method for separating cerebral cortex gluten-mate-operating neural progenitor cells that specifically produces cerebral cortical gluten-mate-operating neural cells and cerebral cortical gluten-mate-operating neural progenitor cells in vivo. The following steps:

(a) preparing a cell population containing cerebral cortical dalmatergic neurons and cerebral glutamate nervous progenitor cells;

(b) NEX (Math2), NeuroD, NeuroD-related-factor, Neurogenin l, Neurogenin2, Svetl, Otxl or Tbr2 Introducing into each cell of the cell population; (c) isolating cerebral cortical glumate-operating neurons and cerebral cortical glumate-operating neural progenitor cells from other cells depending on the presence or absence of drug resistance. . (8) A method for separating cerebral cortex gluten-mate-operating neural progenitor cells that specifically produces cerebral cortex gluten-mate-operating neurons and cerebral cortex gluten-mate-operating neural progenitor cells in vivo. The following steps:

(a) preparing a cell population containing cerebral cortical glutamatergic neurons and cerebral glutamate neuronal precursor cells;

(b) an expression vector in which a recombinase gene is ligated to the downstream of any one of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogenin2 Svetl, Otxl or Tbr2 gene promoter Introducing, into each cell of the cell population, an expression vector linked to a reporter protein gene that emits a signal detectable in a living body after genetic recombination downstream of the forced expression promoter;

(c) a step of isolating cerebral cortical glutamate nervous cells and cerebral cortical glutamate nervous progenitors from other cells based on the presence or absence of a signal generated by the reporter protein;

A method comprising:

(9) A method for separating cerebral cortical glumate-operating neural progenitor cells that specifically produces cerebral cortical glumate-operating neural cells and cerebral cortical glumate-operating neural progenitor cells in vivo. The following steps:

(a) preparing a cell population containing cerebral cortex dalmatergic neurons and cerebral cortex glumate nervous progenitor cells;

(b) NEX (Math2), NeuroD, NeuroD-related-factor, Neurogenin l, Neurogenin2, Svetl, Otxl or Tbr2 Any gene promoter linked to the downstream of the gene promoter and forced expression A step of introducing, into each cell of the cell population, an expression vector linked downstream of the promoter with a drug resistance gene imparting drug resistance properties after genetic recombination; (c) isolating cerebral cortical glumate-operating neurons and cerebral cortical glumate-operating neural progenitor cells from other cells depending on the presence or absence of drug resistance. (10) The method according to any one of the inventions (6) to (9), wherein the cell population prepared in the step (a) is a cell population derived from embryonic stem cells or neural stem cells.

(11) The method according to the above-mentioned invention (10), wherein the embryonic stem cells or neural stem cells are derived from a mammal.

(12) The method according to the invention (11), wherein the mammal is a human.

(13) The cell population prepared in step (a) is a cell population prepared by dispersing tissue containing cerebral cortical glumate-operating neural cells and cerebral cortex glumate-operating neural progenitor cells of the donor animal. The method according to any one of the inventions (6) to (9).

(14) The method according to the invention (13), wherein the donor animal is a mammal.

(15) The method according to the invention (14), wherein the mammal is a human.

(16) The method according to any one of the above-mentioned inventions (6) to (9), wherein in the step (b), the expression vector is introduced by virus-mediated transformation.

(17) The method according to any one of the inventions (6) to (9), wherein the expression vector is introduced by a physical method in the step (b).

(18) The method according to any one of the inventions (6) to (9), wherein in the step (b), the expression vector is introduced by ribosome-mediated transformation. (19) The cerebral cortical glue isolated by the method according to any one of the inventions (6) to (18). A method for specifically separating cerebral cortical glutamatergic neural progenitor cells from evening mate neuronal cells and cerebral cortical glutamate neural progenitor cells. A method comprising: (20) The method according to any one of the inventions (6) to (19), wherein the cell specifically produces in vivo a cerebral cortical glumate-operated neural progenitor and a cerebral cortex glumate-operated neural precursor cell. Reagents and cell kits used to obtain The cells provided by the invention of this application proliferate when cultured under appropriate conditions, and when transplanted into the human cerebral neocortex, continue to produce gluten-mate-operating neurons, and produce the dalnymate-operated neurons. Nerve cells are incorporated into neural circuits of the cerebral cortex and can restore dysfunction due to a lack of glutamatergic neurons. In the present invention, “cerebral cortical glutamate-operating neural progenitor cells” are referred to as “cerebral cortical glutamate-active neural progenitors” and “cerebral cortical glutamate-operating neural progenitor cells” in vivo. Are cells produced by cell proliferation. This means that the daughter cells resulting from the division differentiate into “cerebral cortical glutamatergic neurons” or stay in “cerebral cortical glutamatergic neural progenitor cells” in vivo. Cell division of cerebral cortex gluten-mate neural progenitor cells is constantly regulated by growth factors secreted by surrounding cells. Therefore, when cerebral cortical gluten-mate-operating neural progenitor cells are taken out and grown in vitro, an appropriate culture solution is required (Non-Patent Document 10). In vivo, expression of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogeninl, Neurogenin2, Svetl, Otxl, Tbr2 is halted when the secretion of growth factors decreases with the development of the fetus In some cases, the properties of the cells may change, and the properties of the cells may be altered. Other terms and concepts in the present invention are described in the description of the embodiments of the invention and examples. And will be described in detail. Various materials and techniques used for carrying out the present invention can be easily and reliably obtained by those skilled in the art based on publicly known documents and the like, except for the technique whose source is clearly indicated, It is feasible. For example, genetic engineering and molecular biology techniques and materials therefor are described in Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al "Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows that a recombinant adenovirus (CApromoter-loxP-CAT-loxP-GGFP) is infected into the E14 fetal ventricle of a NEX-Cre knock-in mouse and stained at 3 weeks of age Cerebral cortex gluten mate-operated neurons. In a study of 1000 GFP-positive cells, all cells exhibited the characteristics of cerebral cortical gluten-mate neurons. Fig. 2a shows NEX-GFP positive and phosphorylated histone H3 positive cells in the E15 fetal cerebral cortex. Figure 2b shows NEX-GFP positive and phosphorylated histone H3 positive cells. Figure 2c shows isolated NEX-GFP positive and Ki-67 positive cells. Figure 3 shows that NEX-GFP-positive cells were taken from the cerebral cortex tissue of the half of the cortical plate immediately after BrdU labeling, dispersed, transplanted into the cerebral cortex of a wild-type mouse immediately after birth, and GFP found one week later. Cerebral cortical pyramidal cells where label and BrdU label overlap. Figure 4a shows NEX-Cre-positive and tva800-positive cells that became GFP-positive by infection with recombinant ASLV. FIG. 4b is an explanatory diagram in which up to four or one pyramidal cell is stained in three days. Figure 4c is an illustration of the cell lineage. Figure 5 shows that the isolated E15 fetal cerebral cortex NEX-GFP positive was run on a cell sorter. Isolate only GFP-positive cells, add EGF, bFGF, and BrdU to a conditioned medium in which the fetal brain was cultured overnight with NeuroBasal, culture for 2 days, extract DNA from the cells, attach them to the membrane, and add BrdU. Detected by immunochemical method. Also shown is the case where DNA synthesis was inhibited with AraC using 293 cells as a control group.

BEST MODE FOR CARRYING OUT THE INVENTION The cerebral cortical glutamate-operating nerve of the present invention, which produces only cerebral cortical glutamate-operated neurons and itself (cerebral cortical glutamate-operated neural progenitor cells) in vivo. Neural progenitor cells are characterized by having the following characteristics.

(I) included in a cell population containing cerebral cortex dal mate-operating neurons and cerebral cortex glutamine-operated neural progenitors;

(Ii) capable of proliferating or capable of proliferating by stimulation; and (HI) NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl,

Expressing one or more molecules selected from the group consisting of Neurogenin2, Svetl, Otxl, Tbr2,

It is a cerebral cortical glumate-operating neural progenitor that produces only cerebral cortical daltamatergic neurons and itself (glutamate-activated neural progenitor cells) in vivo. Characteristics (I μ こあるあるダル夕夕集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団集団.) Cell populations isolated from mammalian fetal cerebral tissues at specific times (particularly in the middle and subventricular zones) also include cerebral cortical gluten mate neurons and cerebral cortical gluten mate neurons. The characteristic (Π) is a cell population having proliferative ability or a stimulus in the cell population of (前記). Are identified as proliferating cells. “Stimulation” in this case refers to the administration of unknown and known growth factors, and the degree of stimulation differs depending on the combination, concentration, timing, and the like of the growth factors. Furthermore, the characteristic (ΠΙ) is selected from the group consisting of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogemn2, Svetl, Otxl, and Tbr2 among the cells having the proliferative ability of the above (Π). Identified as cells expressing one or more selected molecules. Whether or not these molecules are expressed can be determined, for example, by examining the presence of the transcript (mRNA) of the gene encoding these molecules, for example, by hybridization using a probe DNA prepared based on the nucleotide sequence. It can be confirmed by measurement and quantification by the PCR method, the PCR method using primer DNA, the RT-PCR method, or the method using a DNA microarray. Expression can also be confirmed by using an antibody that recognizes each molecule. The information such as the amino acid sequence of each molecule and the gene sequence encoding it is stored in an existing database (eg, GenBank: http://www.ncbi.nlm.nih.gov/Genbank). /index.html) You can know exactly what you are doing. The cells identified by the above characteristics (1), (D), and (m) are cerebral cortical glutamate-operating neurons and cerebral cortex that produce in vivo the Dalmes-mate-operating neural progenitor cells themselves. It is a glutamatergic neural progenitor cell. The cells as described above (cerebral cortex glumate-operated neural progenitor cells that produce cerebral cortex glumate-operated neural progenitors and cerebral cortex glutamate-operated neural progenitors in vivo) are the invention of this application. (6) It can be isolated by the method of (9). That is, the methods of the inventions (6) to (9) are characterized by including the following steps. (a) a step of preparing a cell population containing cerebral cortex dalmatergic neurons and cerebral cortex glumate nervous neural progenitor cells;

(b) expression of any of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogenin2, Svetl, Otxl or Tbr2 (hereinafter, these may be described as "specifically expressed molecules") Introducing an expression vector designed to identify the cells into each cell of the cell population.

(c) a step of isolating cerebral cortical glutamatergic neurons and cerebral cortical glutamatergic neural progenitors from other cells by expressing the function of the expression vector. The cell population of step (a) can be prepared as described above, for example, by deriving from embryonic stem cells or neural stem cells of mammals including humans, or at a specific time in mammals including humans. It can be prepared as a cell population isolated from cerebral tissue. The “expression vector designed to identify any expression of a specific expression molecule” in step (b) can be detected in vivo, for example, downstream of the gene promoter encoding each molecule. This is an expression vector linked to a signal gene for a repo that emits a signal. As the repo overnight protein gene, a gene (cDNA or the like) encoding a green fluorescent protein (GFP or EGFP) can be used. Further, the above-mentioned expression vector is an expression vector in which a drug resistance protein gene is linked downstream of a gene promoter encoding each molecule. As the drug resistance protein gene, for example, a known gene such as a neomycin resistance gene can be used. Further, in this step (b), the following expression vector can be used. That is, an expression vector is constructed in which a DNA recombinase (eg, ere recombinase) gene is ligated to DNA containing the promoter region of the specific expression molecule gene. Separately, a DNA containing a stop codon sequence is placed downstream of the forced expression promoter between two DNA sequences recognized by DNA recombinase (for example, ΙοχΡ) in the forward direction. Then, an expression vector in which a repo overnight DNA (for example, GFP) or a drug resistance gene DNA (for example, neo mycin resistance gene) is placed downstream of the DNA sequence recognized by the second DNA recombinase. I do. These two expression vectors are introduced into each cell of the cell population prepared in the step (a). Specific if the expression vector-transduced cells are cerebral cortical glumate-operating neural progenitors that produce cerebral cortical glutamate-active neural progenitors and cerebral cortical glutamate-active neural progenitors in vivo. Expression molecule The DNA recombinase is expressed by the promoter activity of the gene, recombination occurs between the DNA sequences recognized by the two DNA recombinases, and the DNA containing the stop codon sequence between them is removed, Yuichi Protein genes (eg, GFP gene) and drug resistance genes (eg, neomycin resistance gene) are placed downstream of the forced expression promoter and are expressed. In order to introduce the expression vector into cells, the expression vector may be introduced by transformation via a viral vector such as adenovirus or retrovirus, or a physical method such as electroporation, ultrasound, or microinjection. A known method such as a method for introducing an expression vector by a method such as a method for introducing an expression vector by a transformation via a lipid such as ribosome, and the like can be employed. In the step (c), the expression of the function of the expression vector introduced in the step (b) (for example, the expression of a repo protein or a drug resistance trait) is used as an index, and the cerebral cortical glutagenic activity is determined. Separate neurons and cerebral cortical gluten-mate neural progenitors from other cells. For example, fluorescent protein (GFP) -positive cells can be separated on a cell saw. Neomycin-resistant dividing cells can be selected by culturing by adding Geneticin (G418) to the culture solution. The invention (19) of the present application further comprises isolating cells having a proliferative ability from the cerebral cortical gluten-mate-operating neurons and the cerebral cortex Dalmat-mate-operating neural progenitor cells isolated in the above invention. In this method, only Dalmat mate neural progenitor cells of the cerebral cortex are separated. Further, in a preferred embodiment, the isolated cells are transplanted into a recipient animal. In this case, “proliferating cells” specifically refer to somatic cells that completely replicate genome DNA and divide, and the two daughter cells after division maintain both cell functions. It has the ability to divide and can select cells based on the point that the number of cells is increased with time in a culture solution containing growth factors. Invention (20) is a kit of reagents and cells used in the method of the invention, which is used for obtaining cells that produce only cerebral cortical glutamate-nergic neurons and cerebral cortical glutamate-nergic neural progenitors in vivo. It is. Such a kit can be composed of, for example, cerebral cortical gluten-mate-operating neural progenitor cells and a growth factor mix thereof.

EXAMPLES Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited to the following examples.

Example 1

Prove that all NEX-positive cells become cerebral cortical pyramidal cells

A recombinant adenovirus that expresses GFP only when Cre-recombinase acts on the fetal lateral ventricle of E14 of NEX-Cre knock-in mouse with Cre recombinase DNA inserted downstream of the NEX gene promoter region (CApromoter- loxP-CAT-loxP-GGFP) and infected cells were observed 3 weeks after birth. Examination of 1000 GFP-positive cells revealed that all cells had a dense spine on the dendritic surface, a characteristic of cerebral cortical gluten-mate neurons, and 95% of cells had The apical dendrites were confirmed, confirming that they were pyramidal cells (Fig. 1). In this experiment, the fate of NEX-positive cells was examined by extracting 1000 cells at the third week after birth. It means that the fruits were 100% cerebral cortical glutamate-operated neurons.

Example 2

Cerebral cortex Evidence that NEX-positive cells have proliferating cells 1

First, all Cre-positive cells were labeled with GFP by crossing NEX-Cre knock-in mouse with GFP reporter mouse for Cre-recombinase detection (CApromoter-loxP-CAT-loxP-GGFP). NEX-positive cells are labeled with GFP.E15 Mouse fetal cerebral cortex, immunohistochemistry using an antibody against phosphorylated histone H3, which is positive in all cells during cell division, and cells double-labeled with GFP I was looking for Indeed, some cells were double-labeled (Figure 2a). To examine the frequency of double-labeled cells, tissues from the ventricular half of the cerebral cortex obtained from the above mice were taken and separated into individual cells with a protease. The separated cells were spread on a slide glass, dried, and then subjected to immunocytochemical staining for GFP and phosphorylated histone H3. As a result, 1.2% of GFP-positive cells (NEX-positive cells) were in the cell division phase (Fig. 2b) Double labeling of Ki-67, which is often used as a marker molecule for the cell growth phase, by immunocytochemistry revealed that 14% of GFP-positive cells were in the cell growth cycle (Fig. 2c). ).

Example 3

Proof of existence of proliferative cells in cerebral cortex HEX-positive cells 2 All Cre-positive cells were labeled with GFP by crossing NEX-Cre knock-in mouse with GFP reporter mouse for Cre detection Mice were injected with thymidine analog BrdU, and 30 minutes later, the mice were decapitated to remove the cerebral cortical tissue on the half of the cortical plate, and separated into individual cells using protease. The isolated cells were transplanted into the cerebral cortex of the mouse immediately after birth of the wild type, and one week later, when cells were searched for overlapping GFP-labeled and BrdU-labeled cells, more than 60% of the double-labeled cells showed two cones So that the cells form a pair Found in. All GFP-positive cells, including the remaining BudU-positive cells, were dal cortical mate-operated neurons with many spines. This is because cerebral cortical gluten-mate-operating neural progenitor cells, which were in the DNA synthesis phase in the cortical plate of GFP-labeled mice, took up BrdU and were transplanted to divide and produce two pyramidal cells. (Figure 3).

Example 4

Cerebral cortex HEX-positive cells prove that there are cells with the ability to reproduce 3

In order to express the avian sarcoma- leukosis virus (ASLV) receptor (tva800) only in Cre-recombinase positive cells, it must be expressed in the fetal cerebral cortex ventricular zone of NEX-Cre knock-in mouse. Kuta (pCA-loxP-CAT-loxP-tva800) was introduced by electroporation. The solution was mixed with recombinant ASLV (RCAS-CMV-GGFP) at the same time. Several hours later, expression of tva800 began only in the subventricular zone, and GFP-positive cells also appeared in the subventricular zone. On day 3 post-transfection, the stained cells also contained juvenile pyramidal cells with apical and basal dendrites, extending the axons toward the intermediate zone. Figure 4a). The known cell division cycle of the subventricular zone is 15 hours, and the cell cycle of the ventricular zone is 17 hours and, due to the nature of the retrovirus, it starts from one cell, assuming that cell division continues without stopping. The estimated number of cells was 2 at 2 days and 4-8 at 3 days. In actual experiments, the number of cells found in labeled cell clones was up to 2 on day 2 and up to 4 on day 3, so the predictions and experiments were consistent and some cerebral cortex It was found that mate-operated neural progenitors continued to divide at the highest frequency (Fig. 4b, c).

Example 5

Proof that isolated NEX-positive cells in the cerebral cortex can be cultured in vitro Multiply NEX-Cre knock-in mouse with GFP reporter mouse for Cre detection As a result, all Cre-positive cells were extracted from the mouse cerebral cortex tissue labeled with GFP, and separated into individual cells using protease. The separated cells were subjected to celso overnight using GFP fluorescence as an index, and only GFP-positive cells were collected and cultured in vitro for 2 days. The culture medium used was a conditioned medium obtained by culturing the fetal brain with NeuroBasal plus EGF, bFGF, and BrdU. Two days later, DNA was extracted from the cultured cells, attached to the membrane, and BrdU was detected by immunochemistry (FIG. 5). The figure also shows the case where DNA synthesis was inhibited with AraC using 293 cells as a control group. This example showed that only NEX-GFP-positive cells were taken out and that cerebral cortical gluten-mate-operating neural progenitor cells could be cultured and grown in vitro without feeder cells.

Industrial applicability Until now, various cells including GABAergic neurons have been induced by adjusting the culture conditions of embryonic stem cells and neural stem cells. However, glutamic nervous progenitor cells of the cerebral cortex could not be induced so far. In addition, under any conditions, there was no system that produced only a single type of cells, and it was necessary to separate cells again before use for treatment, which impaired cell activity. According to the invention of this application, cerebral cortical glutamergic neural progenitor cells can be obtained with high purity. Furthermore, since the cells proliferate in the brain of the recipient transplanted with the cells, more effective transplantation treatment can be performed. '

Claims

The scope of the claims

1. The following characteristics:

(a) included in the population containing cerebral cortex dal mate-operating neurons and cerebral cortex glutamine-operated neural progenitors;

(b) capable of proliferating or capable of being proliferated by stimulation; and ic) NEX (Math2), NeuroD, NeuroD-related-factor, Neurogenin l,

A cerebral cortical glumate-operating neural progenitor cell which specifically produces cerebral cortical glumate-operating neural progenitor cells in vivo.

2. The cell of claim 1, which is of human origin.

3. The cell of claim 2, which is derived from a human embryonic stem cell.

4. The cell of claim 2, which is derived from a mammal closely related to humans.

5. The cell according to claim 4, which is derived from embryonic stem cells of a closely related mammal.

6. A method for separating cerebral cortical glutamatergic neural progenitor cells that specifically produces cerebral cortical glutamatergic neural progenitor cells in vivo, comprising the following: Process of:

(a) preparing a cell population containing cerebral cortex dal mate-operating neurons and cerebral cortex dal mate-operated neural progenitor cells;

(b) A reporter that emits a signal that can be detected in vivo in the downstream of the gene promoter of any of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogenin2, Svetl, Otxl or Tbr2. Introducing an expression vector linked to the target protein gene into each cell of the cell population; (C) a step of isolating the cerebral cortex dal nergic neurons and the cerebral cortex glutamic nervous progenitors from other cells based on the presence or absence of a signal generated by the reporter protein;

A method comprising:

7. A method for separating cerebral cortical glumate-operating neural progenitor cells that specifically produces cerebral cortical glumate-operating neural cells and cerebral cortical glutamate-active neural progenitor cells in vivo. Process of:

(a) preparing a cell population containing cerebral cortical dal mate-operating neurons and cerebral cortical dal mate-operating neural progenitor cells;

(b) An expression vector obtained by ligating a drug resistance protein gene to the downstream of any one of NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogenin2, Svetl, Otxl or Tbr2 gene promoter. Introducing into each cell of the population;

(c) isolating cerebral cortex dal mate nervous neurons and cerebral cortex glu mate nervous progenitor cells from other cells depending on the presence or absence of drug resistance. .

8. A method for separating cerebral cortical glumate-operating neural progenitor cells which specifically produces cerebral cortical glumate-operating neural cells and cerebral cortical glumate-operating neural progenitor cells in vivo. The following steps:

(a) preparing a cell population containing cerebral cortex dalmatergic neurons and cerebral cortex glutaminergic neural progenitor cells;

ib) NEX (Math2), NeuroD, NeuroD-related-factor, Neurogeninl, Neurogenin2, Svetl, Otxl or Tbr2 Any gene promoter linked to the downstream of the gene promoter and downstream of the forced expression promoter Transfecting each cell of a cell population with an expression vector linked to a repo overnight protein gene that emits a signal detectable even in a living body after genetic recombination;

(c) The presence or absence of a signal generated by the repo overnight protein Isolating the nervous system neural cells and the cerebral cortical glutenergic neural progenitor cells from other cells,

A method comprising:

9. A method for isolating cerebral cortical glumate-operating neural progenitor cells which specifically produces cerebral cortical glumate-operating neural progenitors in vivo. The following steps:

(a) preparing a cell population containing cerebral cortex dal mate-operating neurons and cerebral cortex glu-mate-operated neural progenitor cells;

(b) NEX (Math2), NeuroD, NeuroD-related-factor, Neurogenin l, Neurogenin2, Svetl, Otxl or Tbr2 Introducing a DNA fragment linked downstream of the forced expression promoter to a drug resistance gene imparting drug resistance properties after gene recombination into each cell of the cell population;

(c) isolating cerebral cortical glutamatergic neurons and cerebral cortical glutamatergic neural progenitor cells from other cells depending on the presence or absence of drug resistance.

10. The method according to claim 6, wherein the cell population prepared in the step (a) is a cell population derived from embryonic stem cells or neural stem cells.

1 1. The method of claim 10, wherein the embryonic stem cells or neural stem cells are derived from a mammal.

12. The method of claim 11, wherein the mammal is a human. -

13. The cell population prepared in step (a) is a cell population prepared by dispersing tissues containing cerebral cortical dal mate-operating neurons and cerebral cortical glu-mate-operating neural progenitor cells of the donor animal. 10. The method of any one of claims 6 to 9.

14. The method of claim 13, wherein the animal is a mammal.

15. The method of claim 14, wherein the mammal is a human.

16. The method according to any one of claims 6 to 9, wherein in the step (b), the expression vector is introduced by virus-mediated transformation.

17. The method according to any one of claims 6 to 9, wherein in step (b), the expression vector is introduced by a physical method.

18. The method according to any one of claims 6 to 9, wherein in step (b), the expression vector is introduced by ribosome-mediated transformation.

19. From the cerebral cortical glutamatergic neurons and the cerebral cortex glutamatergic neural progenitor cells isolated by any one of claims 6 to 18, the cerebral cortex Dalmeimate neural progenitor cells are obtained. A method for specifically separating cells, which comprises isolating cells capable of proliferating.

20. The method according to any one of claims 6 to 19, wherein the method comprises obtaining cells that specifically generate cerebral cortical glumate-activated neurons and cerebral cortical glumate-operated neural progenitors in vivo. Kit of reagents and cells to be used.