JP2002051775A - Method for concentrating and separating dopaminergic neuron - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for concentrating and separating dopaminergic neuron, by which the dopaminergic neuron can be concentrated and separated from a cell group comprising various cells at a high rate. SOLUTION: This method for concentrating and separating the dopaminergic neuron from the cell group, characterized by transducing reporter nucleic acid molecules expressing a fluorescent protein under the control of the promoter/ enhancer of a gene expressing the dopaminergic neuron into the cells of the cell group, respectively, and then separating fluorescent light-emitting cells from the cell group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for concentrating and separating dopaminergic neurons. More specifically, the present invention relates to a method for identifying, efficiently and surely enriching and isolating dopaminergic neurons useful as transplant cells for treating Parkinson's disease and the like and as research materials for developing therapeutic methods for those diseases. It is.

[0002]

2. Description of the Related Art Parkinson's disease is a disease caused by the selective degeneration and loss of dopaminergic neurons in the substantia nigra. For the treatment, the feasibility of transplanting fetal midbrain tissue, which is rich in dopaminergic neurons (or cells capable of differentiating into dopaminergic neurons), into the brain (striatum) of patients has been proven. ing.

[0003] However, it is practically impossible to secure a sufficient amount of fetal brain tissue for normal clinical use. For this reason, there is a need for donor cells to replace the fetal midbrain.

For example, it has been studied to use cells differentiated from a large number of undifferentiated nervous system cells into dopaminergic neurons as donor cells for transplantation. Furthermore, the use of cells differentiated from non-neural cells such as ES cells and bone marrow stromal cells into dopaminergic neurons as donor cells for transplantation is also being studied. Since these cells can be induced to differentiate after being expanded in a test tube, they can be a means for solving the problem of donor shortage. Furthermore, since bone marrow stromal cells can be safely collected from adults, dopaminergic neurons for transplantation can be prepared from the patient's own cells. Therefore, the realization of such a treatment method would not only solve the technical problems such as the shortage of donors and rejection, but also the ethical problems of obtaining dopaminergic neurons from aborted fetuses. Is done.

However, a method for efficiently differentiating dopaminergic neurons from an undifferentiated cell population has not been sufficiently established. In addition, various cells other than dopaminergic neurons are differentiated from the undifferentiated cell population. In addition, there is a risk that some of the undifferentiated cell populations include cells that form tumors after transplantation. Therefore,
In order to use in vitro differentiated dopaminergic neurons for transplantation, it is necessary to selectively separate dopaminergic neurons from various cell populations.

[0006]

As described above, concentrated dopaminergic neurons are expected to be useful as transplant donor cells for treating Parkinson's disease and the like. Enriching and isolating dopaminergic neurons is also extremely useful for identifying novel proteins and genes specifically expressed in these neurons. These proteins and their genes are expected to be novel therapeutic agents.

[0007] It is also extremely important to identify factors that differentiate dopaminergic neurons from undifferentiated cells in vitro. Such a factor is not only useful for efficiently inducing dopaminergic neurons from undifferentiated cells, but also is expected to be a novel therapeutic agent by itself.

However, a method for separating dopaminergic neurons from living tissue or a cell population under culture conditions has not yet been established. Furthermore, not only a method for searching for a factor that induces dopaminergic neurons in vitro, but also a method for visualizing a live dopaminergic neuron required for such a search has not yet been established.

The invention of this application provides a method for visualizing a live dopaminergic neuron in a cell population composed of a wide variety of cells, and enriching and separating the dopaminergic neuron at a high rate. The task is to provide.

Another object of the present invention is to provide a dopaminergic neuron separated by the method. Another object of the invention of this application is to provide a method for specifying a factor that induces undifferentiated cells to differentiate into dopaminergic neurons.

[0011]

The present invention relates to, as a first invention, a method for separating dopaminergic neurons from a cell population, the method comprising controlling the promoter / enhancer of a gene expressed in the dopaminergic neurons. The present invention provides a method for concentrating and separating dopaminergic neurons, which comprises introducing a reporter nucleic acid molecule expressing a fluorescent protein into each cell of a cell population, and separating cells emitting fluorescence from the cell population.

This application also provides, as a second invention, cells that have been concentrated and separated by the method of the first invention and that have been maintained under culture conditions. The present invention also provides, as a third invention, a method for visualizing and identifying a living dopaminergic neuron present in a cell population, wherein the fluorescent light is controlled under the control of a promoter / enhancer of a gene expressed in the dopaminergic neuron. Provided is a method for identifying a dopaminergic neuron, which comprises introducing a reporter nucleic acid molecule expressing a protein into each cell of a cell population and measuring the fluorescence distribution of the cell population.

The present invention further provides, as a fourth invention, a method for identifying a factor that induces a cell capable of differentiating into a dopaminergic neuron into a dopaminergic neuron, which is expressed in the dopaminergic neuron. A reporter nucleic acid molecule that expresses a fluorescent protein under the control of a gene promoter / enhancer is introduced into a cell, and the cell and a candidate substance are allowed to coexist. Using the fluorescence of the cell as an index, whether or not the candidate substance is a dopaminergic neuron inducing factor And a method for identifying a dopaminergic neuron inducing factor.

In the first and third invention methods, the following are preferred embodiments. The gene expressed in dopaminergic neurons is a tyrosine hydroxylase gene.

The fluorescent protein is a green fluorescent protein. Each cell of the cell population is derived from the brain. Each cell of the cell population is an ES cell.

[0016] Each cell of the cell population is derived from bone marrow stromal cells. Each cell of the cell population is of human origin.
Each cell of the cell population is a cell into which a recombinant vector carrying a reporter nucleic acid molecule has been introduced.

[0017] The cell population is derived from an animal in which totipotent cells of a non-human animal into which a reporter nucleic acid molecule has been introduced, or an animal progeny thereof. Further, in the first aspect of the present invention, it is a preferable aspect that the cells emitting fluorescence are concentrated and separated using a cell sorter.

Further, in the third invention, the following are preferred embodiments. The gene expressed in dopaminergic neurons is a tyrosine hydroxylase gene.

The fluorescent protein is a green fluorescent protein. The cells capable of differentiating into dopaminergic neurons are neural stem cells.

The cells capable of differentiating into dopaminergic neurons are ES cells. The cells capable of differentiating into dopaminergic neurons are bone marrow stromal cells.

The cells capable of differentiating into dopaminergic neurons are of human origin. The cell capable of differentiating into a dopaminergic neuron is a cell into which a recombinant vector carrying a reporter nucleic acid molecule has been introduced.

The cells capable of differentiating into dopaminergic neurons are derived from an animal in which a reporter nucleic acid molecule-introduced totipotent cell of a non-human animal or an offspring animal thereof.

Hereinafter, embodiments of the present invention will be described in detail.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The first invention is to introduce a reporter nucleic acid molecule expressing a fluorescent protein under the control of a promoter / enhancer of a gene expressed in a dopaminergic neuron into each cell of a cell population derived from an animal, The method is characterized by separating cells that emit fluorescence from the cell population.

The reporter nucleic acid molecule to be introduced into the cells of the cell population comprises a polynucleotide sequence encoding the promoter / enhancer of a gene expressed in dopaminergic neurons, and a polynucleotide encoding a fluorescent protein downstream of the polynucleotide sequence. Are fused nucleic acid molecules.

As a promoter / enhancer of a gene expressed in dopaminergic neurons, a promoter sequence of a tyrosine hydroxylase (TH) gene of various animal species can be used.
Gene promoters are preferred. This rat TH gene promoter sequence is based on GenBank Accession No. AF069036.
A method for screening a rat genomic library using a probe synthesized based on this known sequence, or PCR using synthetic primers
It can be obtained and used by law.

As the fluorescent protein, green fluorescent protein (GFP) derived from Oan jellyfish and red fluorescent protein (RFP) derived from sea anemone can be used. In particular, GFP or a GFP derivative (eg, Curr
ent Biology 6 (2): 178-182, 1996). As the polynucleotide encoding GFP, cDNA (Gene 111 (2): 229-233, 1990: G
enBank No. M62654) is known. Also, EGFP cDNA
(EGFP Poly (A): manufactured by Clontech) can also be used.

The cells into which the reporter nucleic acid molecule is introduced can be differentiated nervous cells derived from the brains of animals including humans. Alternatively, dopaminergic neurons in which neural stem cells, ES cells, bone marrow stromal cells, and the like, which have the ability to differentiate into dopaminergic neurons, are induced in vitro can also be used. In order to induce the differentiation of dopaminergic neurons from these undifferentiated cells, known methods (for example, neural stem cells: Nat. Neurosci.
1: 290-295, 1998, ES cells: Nat. Biotechnol. 18: 675
-679, 2000 and Neuron 28: 31-40, 2000).

In order to introduce a reporter nucleic acid molecule into a cell, a method of introducing an expression vector incorporating the reporter nucleic acid molecule into individual cultured cells can be adopted. As the expression vector, a plasmid vector for animal cell expression can be used. To introduce such a plasmid vector into cells, an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be employed. In addition, a method of infecting cells with a viral vector such as an adenovirus vector can also be used.

Alternatively, when targeting a non-human animal, the reporter nucleic acid molecule may be introduced into cells by preparing a transgenic animal into which the reporter nucleic acid molecule has been introduced. Transgenic animals can be prepared by known methods (eg, Proc. Natl. Acad.
Scl. USA 77; 7380-7384, 1980). Since such a transgenic non-human animal has a reporter nucleic acid molecule in all somatic cells, it extracts dopaminergic neurons by removing its central nervous system tissue and isolating cells that emit a fluorescent signal. Can be obtained in large quantities.

In order to concentrate and separate dopaminergic neurons from a cell population into which a reporter nucleic acid molecule has been introduced by the above-described method, cells that emit a fluorescent signal can be separated from cultured cells one by one using a fluorescence microscope. However, a method using a cell sorter (fluorescence-activated cell sorter: FACS) is preferred for greatly improving the efficiency of the operation. With this cell sorter, dopaminergic neurons can be automatically concentrated and separated.

[0032] In the method of the third invention, the reporter nucleic acid molecule is introduced into each cell of a cell population, and the fluorescence distribution of the cell population is measured to visualize the dopaminergic neurons existing in the cell population as alive. It is a method of identifying. The material and the method for introducing nucleic acid molecules can be basically the same as in the first invention. By observing the cell population into which the reporter acetate molecule has been introduced under a microscope, dopaminergic neurons can be visualized and identified as a fluorescence distribution.

[0033] The method of the fourth invention is to introduce a reporter nucleic acid molecule into a cell capable of differentiating into a dopaminergic neuron, cause the cell to coexist with the candidate substance, and use the fluorescence of the cell as an index to identify the candidate substance. This is a method for identifying a dopaminergic neuron inducing factor for determining whether or not it is a dopaminergic neuron inducing factor. Cells capable of differentiating into dopaminergic neurons include neural stem cells, ES cells, bone marrow stromal cells, and the like. The same reporter nucleic acid molecule as in the first invention is introduced into these undifferentiated cells, and a candidate substance is added to the cell culture medium. Whether or not a candidate substance induces undifferentiated cells into dopaminergic neurons can be easily confirmed by the method of the second invention.

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 by the following examples.

[0035]

EXAMPLES Example 1 Dopaminergic neurons were concentrated and separated from transgenic mice as follows.

A vector (RTH-GFP) expressing GFP under the control of the promoter sequence of the rat TH gene was constructed.
That is, a promoter sequence of 10 kb upstream of the rat TH gene which is known to be specifically expressed in dopaminergic neurons (Mol. BrainRes. 27: 281-289, 1994; M
ol. Cells 7: 394-398, 1997) was inserted upstream of EGFP cDNA (Clontech) to construct an introduction vector. next,
This recombinant vector was opened to make it linear, and injected into the pronuclei of fertilized eggs derived from F1 mice of C57BL / 6J mice and DBA / 2J mice. The transgenic fertilized eggs were transplanted into the fallopian tubes of a foster parent according to a conventional method, developed into individuals, and TH-EGFP transgenic mice were prepared.

The obtained male TH-EGFP mouse was mated with a wild-type mouse, the ventral midbrain of the fetus on the 12th day of embryo was cut out, and this tissue was treated in a trypsin / EDTA solution.
The cells were dispersed by pipetting. 24 cells
After incubation for 2 hours, anti-TH antibody and Texas Red labeled 2
As a result of reaction with the secondary antibody and analysis, it was confirmed that about half or more of the GFP-positive antibodies were TH-positive dopaminergic neurons.

After adding propidium iodide to the obtained cell dispersion and removing undigested tissue pieces through a nylon mesh, analysis was performed using a cell sorter (FACS Vantage: Becton Dickinson). As a result, as shown in FIG. 1, 7 of the cells contained in the cell dispersion were
% Of the cells showed a fluorescent signal.

Next, propidium iodide negative (live cells)
The cells emitting GFP fluorescence were collected in a test tube. The collected cells were adhered to a cover glass, and the reactivity with the antibody was analyzed in the same manner as in the above 2. As a result, FIG.
, Almost all cells were GFP-positive, and about 60% of the cells were confirmed to be TH-positive (dopaminergic neurons). Example 2 The cells obtained in Example 1 were transplanted into the striatum of a Parkinson model rat prepared by 6-OHDA. After 5 weeks, the rotational movement caused by the administration of amphetamine was analyzed, and significant improvement in symptoms was observed in all individuals. Example 3 The same TH-EGFP recombinant vector as that used in the preparation of the transgenic mouse in Example 1 was introduced into mouse ES cells by a transfection method, and an ES cell line having a transgene was established. 10,000 of these ES cell lines were cultured on a 3 cm-diameter culture dish to which the bone marrow stromal cell line PA6 was adhered in a saturated state with 10% KSR, 2 mM glutamine, 1 mM pyruvate, and 0.1 mM pyruvate.
Using a G-MEM medium containing 1 mM non-essential amino acids and 0.1 mM 2 mercaptoethanol, the cells were cultured at 37 ° C.

The ES cell line into which TH-EGFP has been introduced does not express EGFP in an undifferentiated state, but after starting the mixed culture with PA6,
From the day, cells emitting green fluorescence appeared, and the number gradually increased. After 12 days, the expression of TH, Hu protein, and dopamine β-hydroxylase in EGFP-positive cells was examined by indirect immunofluorescence. As a result, most of EGFP-positive cells (FIG. 3, left) were TH-positive (FIG. 3, right). It was confirmed that the protein was positive for Hu protein and negative for dopamine β-hydroxylase.

TH is involved not only in dopamine but also in the synthesis of noradrenaline and adrenaline. However, dopamine β-hydroxylase is required to synthesize noradrenaline and adrenaline from dopamine. The majority of EGFP positive cells are TH positive,
Being negative for dopamine β-hydroxylase and expressing the neuron-specific marker Hu protein, these cells are confirmed to be dopaminergic neurons rather than noradrenergic or adrenergic neurons. Was done.

From the above results, it was confirmed that cells differentiated from undifferentiated ES cells into dopaminergic neurons can be reliably identified. Further, the dopaminergic neurons thus identified can be separated by the same method using a cell sorter as in Example 1.

[0043]

As described above in detail, according to the invention of this application, a method for concentrating and separating dopaminergic neurons from a cell population composed of various types of cells, and a method for dopamine highly concentrated by this method Operative neurons are provided. These cells are useful not only as therapeutic materials (transplantation cells) for human Parkinson's disease and the like, but also for the etiology and pathological analysis of Parkinson's disease, and for the development of therapeutic techniques and therapeutic agents. Further, the invention of the present application provides a method for visualizing and identifying dopaminergic neurons alive, and a method for identifying a dopaminergic neuron differentiation inducer using this method. By these methods, cells for transplantation such as Parkinson's disease can be efficiently obtained from undifferentiated cells. In addition, dopaminergic neuron differentiation inducers are useful for the development of new therapeutic agents.

[Brief description of the drawings]

FIG. 1 shows the results of FACS analysis of a cell dispersion obtained from the fetal midbrain of a TH-EGFP transgenic mouse.

FIG. 2 shows the results of analyzing GFP fluorescence and TH gene expression of all cells (A) and cells (B) obtained by FACS in the cell dispersion.

[FIG. 3] ES cells into which TH-EGFP gene has been introduced are mixed and cultured with PA6 cells, fixed 12 days later, and cells that emit green fluorescence are observed (left), and cells stained with anti-TH antibody Is the result of observing.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/48 G01N 33/58 A 33/50 C12R 1:91) 33/58 C12N 15/00 A // (C12N 5/10 5/00 B C12R 1:91) C12R 1:91) (72) Inventor Natsuki Matsushita 6-10-30 Nodacho, Fukushima City, Fukushima Prefecture Sante Lumiel 202 F-term (reference) 2G045 BA13 BB03 BB13 BB20 BB24 CB17 CB26 CB30 DA12 DA13 DA14 DA80 FA37 FB02 FB03 4B024 AA11 AA20 CA01 CA04 CA11 DA02 EA04 FA02 FA06 FA10 GA12 GA27 HA13 4B063 QA01 QA18 QQ08 QR32 QR35 QR60 QR77 QR80 QS03 QS01 QS05 QS03 QS05 QS03 QS05 QS05 4B065 AA91X AA93X AB01 BA04 BA25 CA46 CA60

Claims (29)

[Claims] 1. A method for separating dopaminergic neurons from a cell population, comprising the steps of: isolating a reporter nucleic acid molecule expressing a fluorescent protein under the control of a promoter / enhancer of a gene expressed in the dopaminergic neuron; A method for concentrating and separating dopaminergic neurons, comprising introducing cells into cells and separating cells that emit fluorescence from the cell population. 2. The method according to claim 1, wherein the gene expressed in dopaminergic neurons is a tyrosine hydroxylase gene. 3. The method according to claim 1, wherein the fluorescent protein is a green fluorescent protein. 4. The method of claim 1, wherein each cell of the cell population is derived from a brain. 5. The method of claim 1, wherein each cell of the cell population is derived from an ES cell. 6. The method of claim 1, wherein each cell of the cell population is derived from bone marrow stromal cells. 7. The method of claim 1, 4, 5 or 6, wherein each cell of the cell population is of human origin. 8. The method according to claim 1, wherein each cell of the cell population is a cell into which a recombinant vector having a reporter nucleic acid molecule has been introduced. 9. The method according to any one of claims 1 to 6, wherein each cell of the cell population is derived from an animal in which a totipotent cell of a non-human animal into which a reporter nucleic acid molecule has been introduced, or an offspring thereof. 10. The method according to claim 1, wherein the cells that emit fluorescence are concentrated and separated using a cell sorter. A cell that has been concentrated and separated by the method according to any one of claims 1 to 10, and has been maintained under culture conditions. 12. A method for identifying a living dopaminergic neuron present in a cell population, comprising the steps of:
A method for identifying a dopaminergic neuron, comprising introducing a reporter nucleic acid molecule that expresses a fluorescent protein under the control of an enhancer into each cell of a cell population, and measuring the fluorescence distribution of the cell population. 13. The method according to claim 12, wherein the gene expressed in dopaminergic neurons is a tyrosine hydroxylase gene. 14. The method of claim 12, wherein the fluorescent protein is a green fluorescent protein. 15. The method of claim 12, wherein each cell of the cell population is derived from a brain. 16. The method of claim 12, wherein each cell of the cell population is derived from an ES cell. 17. The method of claim 12, wherein each cell of the cell population is derived from a bone marrow stromal cell. 18. The method of claim 12, 15, 16 or 17, wherein each cell of the cell population is of human origin. 19. The method according to claim 12, wherein each cell of the cell population is a cell into which a recombinant vector having a reporter nucleic acid molecule has been introduced. 20. The method according to any one of claims 12 to 17, wherein each cell of the cell population is derived from an animal in which a totipotent cell of a non-human animal into which a reporter nucleic acid molecule has been introduced, or an offspring animal thereof. 21. A method for identifying a factor that induces a cell capable of differentiating into a dopaminergic neuron into a dopaminergic neuron, wherein the factor is controlled under the control of a promoter / enhancer of a gene expressed in the dopaminergic neuron. A reporter nucleic acid molecule expressing a fluorescent protein is introduced into a cell, and the cell and a candidate substance coexist,
A method for identifying a dopaminergic neuron inducing factor, which determines whether or not a candidate substance is a dopaminergic neuron inducing factor using cell fluorescence as an index. 22. The method according to claim 21, wherein the gene expressed in dopaminergic neurons is a tyrosine hydroxylase gene. 23. The method according to claim 21, wherein the fluorescent protein is a green fluorescent protein. 24. The method according to claim 21, wherein the cells capable of differentiating into dopaminergic neurons are neural stem cells. 25. The method according to claim 21, wherein the cells capable of differentiating into dopaminergic neurons are ES cells. 26. The method according to claim 21, wherein the cells capable of differentiating into dopaminergic neurons are bone marrow stromal cells. 27. The method according to claim 21, 24, 25 or 26, wherein the cell capable of differentiating into a dopaminergic neuron is derived from a human. 28. The method according to any one of claims 21 to 27, wherein the cell capable of differentiating into a dopaminergic neuron is a cell into which a recombinant vector having a reporter nucleic acid molecule has been introduced. 29. The cell according to claim 21, wherein the cell capable of differentiating into a dopaminergic neuron is derived from an animal in which a totipotent cell of a non-human animal into which a reporter nucleic acid molecule has been introduced has been ontogenously developed or a progeny animal thereof. Either way.