KR20070089089A - A composition for de-differentiating astrocytes into neural stem cell comprising human bmi-1 protein or a nucleic acid encoding and the same and a process of de-differentiation using said composition - Google Patents

Abstract

A composition comprising a Bmi-1 protein or a nucleic acid coding the Bmi-1 protein is provided to de-differentiate the astrocytes into neural stem cell-like cell which has the self-regeneration capability and the differentiation capability into astrocyte, neuron, and oligodendrocyte. The composition for inducing de-differentiation of astrocytes into neural stem cells comprises a Bmi-1 protein or a nucleic acid material including a nucleotide sequence coding a Bmi-1 protein, wherein the nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein is a vector allowing the Bmi-1 protein to be expressed. The method for inducing the de-differentiation of astrocytes comprises the steps of: (a) culturing astrocytes in a medium; (b) treating the astrocytes with the Bmi-1 protein or the nucleic acid material; and (c) inducing the astrocytes to differentiate into neural stem cells.

Description

A composition for de-differentiating astrocytes into neural stem cell comprising human Bmi-1 protein or a nucleic acid encoding and the same and a process of de-differentiation using said composition}

1 is a result of increasing the growth rate of ^astrosite by reducing the expression of p16 ^Ink4a and p19 ^Arf by the Bmi-1 gene.

Figure 1a is a result of confirming the astrosite isolated from the mouse and the astrosite overexpressed Astrosite-specific markers (GFAP, S100β) by immunocytochemistry (Immunocytochemistry). The left side is the astrosite isolated from the mouse, the right side is the astrosite overexpressed the Bmi-1 gene.

1b confirms that the Bmi-1 gene is overexpressed by Western blot analysis, and that the expression of target genes p16 ^Ink4a and p19 ^Arf is reduced by the Bmi-1 gene. α-tubulin indicates loading of the same amount.

Figure 1c is the result of comparing the growth rate of the astrosite (control) and the astrosite overexpressed Bmi-1 gene introduced only the vector (Vector).

Figure 2 shows that astrocytes overexpressing the Bmi-1 gene induces differentiation into neural stem cell-like cells in vitro.

FIG. 2A shows a process for inducing differentiation. In FIG. 2A, ac is a astrosite (control) in which only a vector (Vector) is introduced, and df is a result of culturing astrosite overexpressed with Bmi-1 gene in neural stem cell culture. . g is a neural stem cell, h is a cell overexpressing the Bmi-1 gene.

FIG. 2B shows the results of experiments to determine whether the reverse differentiation of Bmi-1 overexpressed astrosite is growth factor dependent (N2: N2 culture only, N2 + EGF: EGF addition to N2 culture) , N2 + bFGF: bFGF added to N2 culture, N2 + bFGF + EGF: bFGF and EGF added to N2 culture solution).

Figure 2c shows whether the maintenance of self-renewal capacity of neural stem cell-like cells (NSCLCs) that have re-differentiated Astrosite overexpressing the Bmi-1 gene is dependent on bFGF. The result is.

FIG. 2D shows various inhibitors in neural stem cell culture by making Bmi-1 overexpressed cells into single cells to determine whether the FGF signaling pathway is involved in the process of dedifferentiating Bmi-1 overexpressed astrosite. ) Is the result of confirming whether reverse differentiation is induced. Inhibition of some of the factors involved in the FGF signaling pathway resulted in the confirmation of the formation of neurospheres.

FIG. 2E shows the result of confirming that the neural stem cell-like cells which dedifferentiated Astrosite overexpressing Bmi-1 have the same characteristics as the neural stem cells by immunocytochemistry. Nestin, Sox2, and CD133 are astrosite specific markers, Ki-67 is a proliferation marker, ad of FIG. 2E represents neural stem cells (NSCs), and eh represents neural stem cell-like cells (NSCLCs).

FIG. 2F is a result of confirming the marker identified in FIG. 2E through the RT-PCR method. In neural stem cell-like cells, Nestin and Sox2 are expressed like neural stem cells, but not in astrocytes incorporating Vector or overexpressing Bmi-1 gene (vector: astrocyte-pBabe puro, neural stem cell, Bmi) -1: astrocyte-pBabe puro Bmi-1, Bmi-1 sphere: NSCLCs: neural stem cell-like cells).

FIG. 3 shows the results of confirming whether neural stem cell-like cells (NSCLCs) have multipotency like neural stem cells (NSCs) through in vitro differentiation experiments.

Figure 3a is a result of differentiation of neural stem cells (NSCs) and neural stem cell-like cells (NSCLCs) to astrocyte (astrocyte), and confirmed by immunocytochemistry (Immunocytochemistry). In Figure 3a, a, c is a neural stem cell, b, d is a neural stem cell-like cell, it shows the expression of GFAP and S100β, astrosite-specific markers.

Figure 3b is after the differentiation of neural stem cells (NSCs) and neural stem cell-like cells (NSCLCs) into neurons, specific markers (Tuj1, Map2, TH) was confirmed by immunocytochemistry (Immunocytochemistry). Figure 3b, a, ce is a neural stem cells, b, fh is a result confirmed after differentiating cells such as neural stem cells.

Figure 3c is the result of differentiation of neural stem cells and neural stem cell-like cells (NSCLCs) into oligodendocytes (oligodendrocyte), the specific markers (O1, O4, CNPase) was confirmed by immunocytochemistry. Ac and gi of Figure 3c, the neural stem cells, df, jl is a result confirmed after differentiating neural stem cells-like cells.

4 shows the results of experiments to determine whether the reverse differentiation process is dependent on the Bmi-1 gene.

4A shows the results of confirming the expression of Bmi-1 in astrosite infected with the inducible vector pBI-Bmi-1-EGFP and pBabe puro rTTA. 1 μg / ml of Doxycycline can induce the expression of the Bmi-1 gene, and Western blot analysis confirms the difference in the expression of Bmi-1 with and without doxycycline (DOX). The result is.

Figure 4b is a result of comparing with and without doxycycline when switched to the culture of neural stem cells.

Figure 4c is a result of confirming that the cells of the same shape as the neural stem cells are made, and GFP is expressed when switched to the culture medium of neural stem cells, and doxycycline is added. Figure 4c- a is a photograph of a cell of the same shape as the neural stem cells, Figure 4c-b is a result of confirming the GFP expression of these cells, In addition, 4c-c is confirmed by the immunochemical staining method to express the markers of neural stem cells to be.

4d is a result confirming whether the cells identified in FIG. 4c have differentiation ability. In Figure 4d, a, b is astrosite, ce is a neuron, f, g is the result of inducing differentiation into oligodendrosite, and then the respective specific markers by immunochemical staining.

Figure 4e is a result confirming the differentiation ability in vivo (in vivo). Subcutaneous injection of neural stem cell-like cells cultured in a culture medium of neural stem cells to which doxycycline was added to nude mice of 6 weeks old was fixed, and the tissues were fixed two weeks later, and each differentiation marker was fixed. Confirmed using.

5 is a result confirming whether the action by the Bmi-1 gene is inhibited by the HDAC inhibitor (inhibitor).

Figures 5a-f is the result of confirming the growth rate and reverse differentiation of Astrosite overexpressed Bmi-1 gene while treating TSA (Trichostatin A).

Figure 5a shows that the growth rate is reduced when TSA is treated with the astrosite overexpressed Bmi-1 gene, Figure 5b is a cell picture after the treatment for 3 days, the rightmost picture of 5B is 100ng / ml treatment Afterwards, SA-β-galactosidase assay (SA-β-galactosidase assay) is a cell photo showing the results of senescence. Figure 5c shows the difference in protein expression at each concentration, Figure 5d is a result of confirming whether the neurospheres are formed when subjected to reverse differentiation process. 5E shows that the expression of nestin and sox2, which are markers of neural stem cells, decreases at the RNA level extracted from the sample identified in FIG. 5D. In addition, FIG. 5F shows 100 cells incubated for 2 weeks while treating TSAs in concentrations such as neural stem cell culture conditions for each concentration, and confirming whether neurospheres are formed.

Figure 5g-j is the result of treatment of VPA (Valproic acid) to the astrosite overexpressed Bmi-1 gene. Figure 5g shows the difference in the growth rate of astrosite when treated with VPA concentration, Figure 5h shows the shape of the cells after treatment for 3 days in the culture of neural stem cells. 5i shows that the expression of nestin and sox2, which are markers of neural stem cells, is reduced in RNA extracted from the sample identified in FIG. 5h . In addition, in FIG. 5J , 100 cells of Bmi-1 overexpressed astrosite were cultured for 2 weeks in a culture solution of neural stem cells and a culture solution treated with VPA at different concentrations.

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Neural stem cells (NSCs) are subtypes of progenitor cells in the nervous system that are capable of differentiating into astrocytes, oligogodendrocytes, and neurons. Have Multicellular neurospheres can be formed from the central nervous system (CNS) and the peripheral nervous system (PNS), which can produce glial lineage and neural lines under different conditions. lineage) (Sally Temple et al. 2001). The neural stem cells have been applied to the treatment of intractable diseases and are currently being studied as a method of cell therapy. These neural stem cells are one of the adult stem cells, so the situation is being studied a lot because there is little ethical problem. The importance of adult stem cells has been emphasized more recently in the context of research on dedifferentiation. Although adult stem cells are easier to obtain than embryonic stem cells, there are still many difficult parts to apply. In addition, when using adult stem cells of another person, an immune rejection reaction may be raised. Therefore, inducing dedifferentiation using own cells can solve the problems that are being raised. For this purpose, it is important to induce reverse differentiation using cells that have already undergone differentiation. Currently, research is being conducted to induce differentiation using methods such as cell fusion and nuclear transfer, and another method is called Alexis J (Lancet 2004). Neural stem cells were reported to have characteristics of neural stem cells when cultured in culture conditions. Toru K. (Genes & Development 2004) also reported that oligoendrocyte precursors dedifferentiated into neural stem cells. The group has studied and published papers since 2000, and recently reported in 2004 that gene expression is related to chromatin remodeling and histone modification at each stage of differentiation.

In the present invention, to solve the problems of various existing methods, to establish a more suitable method for use. One of the transcription factors controlling the properties of NSCs was selected and overexpressed to study differentiation into neural stem cells. The Bmi-1 gene of the polycomb group selected by the inventors is a gene encoding one of the proteins that regulate histone modification and cell cycle regulators (Jan W. et al. 1999). The cdkn2a / INK4A locus is known to be a target of the Bmi-1 gene, and the Bmi-1 gene is known to act to inhibit this target gene during transcription. Recently, it has been reported that the Bmi-1 gene is expressed in neural stem cells and acts to maintain self-renewal of neural stem cells (Anna VM et al. 2003). , 2005, In-Kyung Park et al. 2004).

Against this background, the inventors overexpressed the Bmi-1 gene in already differentiated mouse astrosites, which have neural stem cell-like regeneration ability and differentiation ability to asstroocytes, neurons, and oligoendrocytes. It was confirmed that the cells (neural stem cell-like cell) can be differentiated and completed the present invention.

One object of the present invention is to provide a composition for inducing the differentiation of astrocytes (astrocytes) comprising a Bmi-1 protein or a nucleotide sequence encoding a Bmi-1 protein into neural stem cells.

Still another object of the present invention is to provide a method for inducing differentiation of astrosite into neural stem cells, comprising the step of processing a Bmi-1 protein or a nucleic acid that is a nucleotide sequence encoding a Bmi-1 protein.

Another object of the present invention is to provide a neural stem cell produced by the above method.

Still another object of the present invention is to provide neural stem cells which are produced by the above method and have differentiation ability into astrosites, neurons or oligodendrosites.

In one aspect, the present invention relates to a composition for inducing reverse differentiation of an astrocyte (astrocyte) comprising a Bmi-1 protein or a nucleotide sequence encoding a Bmi-1 protein into neural stem cells.

As used herein, the term "neural stem cell-like cells" refers to a multipotent stem cell capable of differentiating into cells such as neurons, astrosites, oligodendrosites, and the like. It is a neural stem cell originated by dedifferentiation of somatic cells. Neural stem cell-like cells in the present invention may be described as neural stem cells.

The inventors have found that when overexpressing Bmi-1 in astrosite, de-differentiation of the already differentiated cell type astrosite into multipotent neural stem-like cells. Found.

Bmi-1 of the present invention is provided in the form of a protein or a nucleic acid encoding the protein thereof.

Bmi-1 used in the compositions of the present invention includes all Bmi-1 derived from humans and animals such as horses, sheep, pigs, goats, camels, antelopes, dogs, and preferably human Bmi-1. In addition, the Bmi-1 protein of the present invention, which is used for reverse differentiation into neurons, includes variants of the Bmi-1 protein as well as proteins having their wild type amino acid sequences.

A variant of Bmi-1 protein refers to a protein in which the natural amino acid sequence of Bmi-1 and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. The variant may be a functional equivalent exhibiting the same biological activity as the native protein or a variant in which the physicochemical properties of the protein are modified as necessary. Variants with increased structural stability or increased physiological activity to the physical and chemical environment

Preferably, it is provided in the form of a nucleic acid having a nucleotide sequence encoding a Bmi-1 protein.

The nucleotide sequence encoding the Bmi-1 protein is a nucleotide sequence encoding the Bmi-1 protein in wild-type or variant form as described above, wherein one or more bases can be mutated by substitution, deletion, insertion, or a combination thereof, It can be isolated from nature or prepared using chemical synthesis.

Nucleic acid having a nucleotide sequence encoding the above-described Bmi-1 protein may be single- or double-stranded, and may be a DNA molecule (genomic, cDNA) or RNA molecule.

In one preferred embodiment, the composition for inducing reverse differentiation of astrosite into neural stem cells in the present invention comprises a vector expressing a Bmi-1 protein comprising a nucleic acid that is a nucleotide encoding a Bmi-1 protein.

As used herein, the term "vector" refers to a gene construct, which is an expression vector capable of expressing a protein of interest in an appropriate host cell, and which contains essential regulatory elements operably linked to express the gene insert.

As used herein, the term “operably linked” refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. The operative linkage of can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

Vectors of the present invention include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers and can be prepared in various ways depending on the purpose. . The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA.

Vectors include plasmid vectors, cosmid vectors, viral vectors, and the like. Preferably, it is a viral vector. Viral vectors are retroviruses such as Human immunodeficiency virus HIV (Murine leukemia virus) Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV) and Mouse mammary (MMTV). tumor viruses, and the like, but are not limited to vectors derived from Adenovirus, Adeno-associated virus, Herpes simplex virus, and the like. In a specific embodiment of the present invention, a pBabe puro vector containing a selection marker for puromycin and a pBI-EGFP vector including a GFP marker were used as a MLV-based vector.

Nucleic acid having a nucleotide sequence encoding a Bmi-1 protein can be delivered intracellularly by methods known in the art, such as naked DNA in vector form (Wolff et al. Science, 1990: Wolffet al. J Cell Sci. 103). : 1249-59, 1992), liposomes, cationic polymers and the like can be delivered intracellularly. Liposomes are phospholipid membranes prepared by mixing cationic phospholipids such as DOTMA or DOTAP for gene transfer. Nucleic acid-liposomal complexes are formed when cationic liposomes and anionic nucleic acids are mixed in a proportion.

In another preferred embodiment, the composition for inducing differentiation of astrosite into neural stem cells in the present invention comprises a virus expressing a Bmi-1 protein comprising a nucleic acid that is a nucleotide encoding a Bmi-1 protein.

As used herein, the term “virus” refers to a virus that expresses Bmi-1 produced by transforming and infecting a viral vector comprising a nucleic acid encoding a Bmi-1 protein with packaging cells.

Viruses that can be used to make viruses expressing the Bmi-1 protein of the invention include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and the like. Preferably, it is a retrovirus. In a specific embodiment of the present invention, a packaging cell for generating a high titer virus capable of infecting a wide range of mammalian host cells with a vector (pBabe puro Bmi-1) prepared by inserting a nucleotide sequence encoding a Bmi-1 protein into a pBabe puro vector PT67 cells were transformed to prepare a virus expressing the Bmi-1 protein to infect the astrosite.

In another aspect, the present invention relates to a method for inducing reverse differentiation of an astrosite into neural stem cells comprising treating the astrosite with a nucleic acid that is a nucleotide sequence encoding a Bmi-1 protein or a Bmi-1 protein.

More specifically, (i) culturing the astrosite in the medium; (ii) infecting the vector with the transfected packaging cells with a vector inserted with a Bmi-1 protein or a nucleic acid that is a nucleotide sequence encoding the Bmi-1 protein; And (iii) inducing the reverse differentiation into neural stem cells by culturing the infected astrosite under the same culture conditions as neural stem cell culture conditions.

The medium for culturing the astrosite in step (i) of the method includes all of the medium conventionally used for culturing neural stem cells in the art. The medium used for culturing generally contains a carbon source, a nitrogen source and a trace element component. In addition, the medium may include antibiotics such as penicillin, streptomycin, gentamicin, and the like. Preferably it is a medium containing bFGF (Basic Fibroblast Growth Factor). In a specific embodiment of the present invention, the astrosine isolated from the brain of the mouse is trypsinized, Dulbecco's modified Eagle's medium containing fetal bovine serum (FBS), penicillin / streptomycin, L-glutamine medium, DMEM).

The nucleic acid, the nucleotide sequence encoding the Bmi-1 protein or Bmi-1 protein processed in step (ii) of the method, is a human and all Bmi-derived animals, such as horses, sheep, pigs, goats, camels, antelopes, and dogs. May be 1 and include wild type and variant. In a specific embodiment of the present invention, human Bmi-1 (NCBI accession No. L13689) was used.

Neural stem cell culture conditions of step (iii) of the method includes all of the medium commonly used in the culture of neural stem cells in the art. In a specific embodiment of the present invention, two methods were used. Plate the cells in one way and after 12 hours, serum replacement B27 and human recombinant BFGF (Basic Fibroblast Growth) in DMEM / F12 medium (N2) containing insulin, apo-transferrin, selenium, progesterone, penicillin / streptomycin Factor) was added to the medium, bFGF treatment daily, and the medium was incubated with a change every other day. Another method is trypinization of cells cultured in Growth medium DMEM containing 10% FBS, 1% Penicillin or Streptomycin, and 1% L-glutamine. After seeding in bacterial culture plates and cultured in the same medium to induce differentiation into neural stem cells.

In another embodiment, the present invention relates to neural stem cells produced by the above method.

Neural stem cells prepared by the reverse differentiation method of the present invention, it was confirmed that Nestin, CD133, Sox2, which is a specific marker of neural stem cells, express the same level as neural stem cells and have the same multipotency as general neural stem cells. In addition, the neural stem cells produced by the reverse differentiation method of the present invention has the characteristics of self-renewal.

In still another aspect, the present invention is to provide a method for differentiating neural stem cells produced by the above method into astrosites, neurons or oligodendrosites.

When the neural stem cells prepared according to the compositions and methods of the present invention induce differentiation under conventional astrosite, neuron or oligodendrosite differentiation conditions known in the art, it is indicated that each cell-specific marker induces differentiation to each cell. It was confirmed by the expression of.

To induce differentiation into astrosite, human recombinant BFGF (Basic Fibroblast Growth Factor) and EGF (Epidermal Growth Facor) or rat recombinant CNTF (recombinant rat ciliaryneurotrophic factor) can be added to DMEM containing FBS. As specific markers, GFAP, S100β and the like can be used.

Differentiation into neurons can be induced by treatment with human recombinant FGF in N2 medium containing B27, a serum replacement, followed by culturing in a medium without FGF for several days, and alternatively by adding retinoic acid (RA). Or by incubating with addition of VPA (valproic acid) with RA to induce differentiation. As a neuron specific marker, Tuj1, Map2, TH, etc. can be used.

Differentiation to oligodendroseite was performed by platelet derived growth factor-AA (PDGF-AA), T3 (3,3,5-triiodo-L-thyronine) and human recombinant bFGF in B27-supplemented N2 cultures with serum replacement. It can be added together to incubate differentiation. Oligodendrosite specific markers such as 01, 04, CNPase and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Mouse Astrocytes and Mouse Neural Stem Cells neural stem cell Culture

Mouse astrocytes were isolated from the brains of neonatal mice (1-5 days) and cultured in suitable culture conditions. The isolated cells were treated with trypsin (Gibco 0.05%) and then made into single cells, 10% FBS (HyClone), 1% penicillin / streptomycin, 1% L-glutamine (L-glutamine). (Dulbecco's modified Eagle's medium; DMEM; high glucose, Hyclone) containing (cambrex). After incubation, the medium was changed from the next day, and cells between 1-3 passages were used. GFAP (Dako), a marker specifically expressed in astrosite, was confirmed by immunocytochemistry ( FIG. 1A left ).

 Example 2 Retroviral-mediated Infection

pBabe puro Bmi-1 (from Dr. GPDimri, Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine, Northwestern University, Evanston, IL) prepared by inserting the human Bmi-1 (NCBI accession No. L13689) gene into a pBabe puro vector 60201, USA) and pBabe puro (control) were transfected with PT67 packaging cell line (Clontech) using lipofectamine (Invitrogen), followed by puromycine (puromycine, 3 μg / ml). (BD science). The PT67 packaging cell line produces a high titer virus that can infect a wide range of mammalian host cells.

When the cell line thus formed is 90% or more, a supernatant is taken and filtered with 0.45 μm (Millipore) to remove cell debris and then polybrene (6 μg / ml) (sigma) is removed. The mice were infected twice with 10 hr intervals and were selected by treatment with puromycine at 0.5 μg / ml for 3-5 days.

Example 3 Western blot analysis and semi-quantitative PCR

Western blot analysis was used to confirm that the Bmi-1 gene was overexpressed in the cells selected in Example 2. It was confirmed that Bmi-1 was expressed, and it was confirmed that expression of p16 ^INK4a and p19 ^Arf , which are the target genes, was reduced ( FIG. 1B ). In addition, immunocytochemistry was used to confirm that the cells thus identified maintain the character of astrosite. It was confirmed that S100β, a specific marker of astrosite, was expressed ( right side of FIG. 1A ), and its target gene, p16 ^INK4A. And it was confirmed that the expression of p19 ^Arf is reduced ( FIG. 1B ).

The cells selected in Example 1 were resuspended in ice-cold cell lysis buffer, RIPA buffer (containing proteinase inhibitor cocktail and phosphatase inhibitor), and then resuspended in ice. Vortexing was performed while incubating for minutes. After centrifugation at 14000 rpm for 30 minutes, the supernatant was obtained, the protein concentration was measured by Bradford assay reagents (Bio-rad), and each 50 μg-100 μg was prepared by 10% or 4-12%. Running on a precasted SDS-PAGE NuPAGE gel (Invitrogen). This was transferred to a PVDF membrane (Millipore) and then blocked with Trist buffered saline with 0.1% Tween 20 containing 3-5% skimmed milk. Wherein -Bmi-1 (Upstate Biotechnology), wherein -p16 ^INK4A (Santa Cruz Biotechnology), anti -p19 ^ARF (Novous Biologies), anti-acetyl-H3 (anti-acetyl H3, Cell signalling), wherein -p21 ^WAF1 (Neomarker) , horseradish peroxidase-conjugated anti-secondary IgG, 1 hour at room temperature after shaking overnight at 4 ° C using α-tubulin (Sigma) Invitrogen) and detected with a Super Signal West Pico Chemiluminescent Substrate kit (Pierce).

After isolation of RNA using Trizol (Invitrogen) in each cell, reverse-transcription (RT) was performed at 500 ng of total RNA, oligo d (T) _12-18 primer (Invitrogen). CDNA was generated using superscriptase II reverse transcriptase (Invitrogen). RT-PCR contained 1 μl of cDNA, 10 pmol of each primer, and PCR premix (1U Tag DNA polymerase, 250 μM dNTPs, 10 mM Tris-HCl, 40 mM KCl and 1.5 mM MgCl ₂ , Bioneer, Korea). It was carried out using. Mouse GAPDH, Nestin, Sox2 was used as a control and each marker, and PCR amplification according to the respective primer conditions. Primer sequences used are as shown in SEQ ID NOs: 1-6.

 Example 4 Measurement of Growth Rate of Astrosite Overexpressed with Bmi-1

Growth curve analysis was performed to see the difference in the growth rate of the cells identified as overexpression. 0.01% crystal violet after plating 2.5-5.0 × 10 ⁴ cell / 6 well plates to determine growth rate of vector (control) and Bmi-1 overexpressed astrosite violet) was used every day for 5 days and stained for analysis. The extract of the crystal violet stain was measured on a spectrophotometer (600 nm) using 10% acetic acid and compared relative growth rates. In addition, the SA-β-galactosidase assay was used to confirm the senescence when the growth rate was decreased when the histone deacerylase inhibitor was treated. . Neural stem cells used as controls were isolated from E13.5-day-old mice and used insulin (Insulin, sigma), apo-transferrin (sigma), selenium (sigma), and progesteron (progesteron). sigma), serum-replacement in Dulbecco's modified Eagle's medium / F12, DMEM / F12, Gibco medium (N2) containing penicillin / streptomycin (cambrex). ) Were cultured with the addition of B27 (Gibco), human recombinant basic fibroblast growth factor (bFGF), and human recombinant epidermal growth factor (R & D). The culture was carried out by pipetting (Pipetting), and the sphere growth was also measured through a subsphere assay. 100 cells were seeded in 12 well plates, and 14 days later, the number of spheres and the total number of cells were measured.

Figure 1c is a result of comparing the growth rate of the vector and Bmi-1 overexpressed astrosite, it was found that the growth rate of the astrosite overexpressed Bmi-1 gene is increased compared to the vector.

Example 5 De-differentiation Induction

Reverse differentiation was induced by culturing cells overexpressing the Bmi-1 gene and cells infected with the corresponding vector under the same conditions as the neural stem cell culture conditions. Cultures of neural stem cells include insulin (Insulin, Sigma), apo-transferrin (Sigma), selenium (Selenium, Sigma), progesteron (Sigma), penicillin / streptomycin (penicillin / streptomycin, cambrex) Serum-replacement B27 (Gibco) and human recombinant basic Fibroblast Growth in Dulbecco's modified Eagle's medium / F12, DMEM / F12, Gibco (N2) Medium with Factor, R & D systems) was used. bFGF treatment was performed daily, and cells overexpressing the Bmi-1 gene and its control vector only were plated in 6 well plates by 2500-5000 cells / cm ₂ . After 12 hours of plating, the cells were changed to neural stem cell culture, and cell shape changes were observed.

Two methods were used, with the same culture conditions as neural stem cells, one of which was plated with 1 × 10 ⁵ cells in a 6 well plate and 12 hours later. After switching to the culture, bFGF is treated every day, and the medium is incubated with a change every other day. The results of the process of inducing differentiation are shown in FIG. 2A . In FIG. 2A, ac is a vector and df is a result of culturing astrocytes overexpressed with Bmi-1 gene in neural stem cell culture. After attaching the same number of cells, the cells were cultured under neural stem cell culture conditions when stabilized after 12 hours, wherein a and d were 12 hours after attachment; b, e is after 3 days; c and f are the cells after 6 days. On the third day of incubation in the culture of neural stem cells, neurospheres were formed in cells overexpressing the Bmi-1 gene, and on the 6th day, the neurospheres were formed, which was significantly different from the vector. On the other hand, cells infected only with vectors rarely grew and did not form neurospheres. The cells of the vector show senescence, but the cells overexpressing the Bmi-1 gene are similar to neural stem cells. In FIG. 2A , g is a neural stem cell, and h is a cell overexpressing the Bmi-1 gene, and the two cells showed similar results.

In the same culture conditions as the neural stem cells culture, another method is cultured in a growth medium containing 10% FBS, 1% penicillin or streptomycin, and 1% L-glutamine After trypinization of the cells, the cells were seeded in 60 mm bacterial culture plates (BD Bioscience) by 3 × 10 ⁵ each to insulin (sigma), apo-transferrin (sigma), selenium (sigma), progesterone (sigma), Serum-substituted B27 (Gibco) and human recombinant basic fibroblast growth factor (R & D) in Dulbecco's modified Eagle's medium / F12 (DMEM / F12, Gibco) (N2) containing penicillin / streptomycin (cambrex) ) Is cultured in the added medium. Since cells such as neural stem cells were made, the cells were cultured in 60 mm bacterial plates by treating bFGF and EGF in neural stem cell culture.

This is to see whether bFGF used during the dedifferentiation process was confirmed by different conditions as shown in Figure 2b . Neurospheres were formed only with bFGF alone and with bFGF and EGF.

Secondary sphere formation by culturing 100 single cells in culture of neural stem cells to determine whether self-renewal ability is maintained in dedifferentiated neural stem cell-like cells Even in the case of attempting, spheres were formed only when bFGF or bFGF and EGF were treated together as in FIG. 2c . When N2 culture and EGF were added, spheres were not formed, but when bFGF or bFGF and EGF were treated together, 12.3 ± 4.5 and 15.6 ± 1.5 neurospheres were formed. These results indicate that bFGF is required for the basal culture of neural stem cells.

In addition, in order to compare the proliferative capacity of neural stem cells and dedifferentiated neural stem cell-like cells, the total cell count of secondary spheres formed by incubating 10000 single cells for 14 days in a 60 mm plate. As a result of performing the subsphere forming assay by calculating the cell number, it was confirmed that the total number of cells formed in the neurosphere was further increased to 180,000 ± 30,550 (NSCLCs) and 160,667 ± 75,000 (NSCs).

< Example  6> Immunocytochemistry  ( Immunocytochemistry )and RT - PCR As each Marker  Confirm

In order to check whether the neurospheres prepared above have the characteristics of neural stem cells, the neurospheres were fixed with 4% paraformaldehyde (EMS) at 4 ° C. for 1 hour, and then 20% sucrose was added thereto. Shaking overnight at &lt; RTI ID = 0.0 &gt; Thereafter, cryopreservation was performed on an 8-well chamber slide (Nunc) with an OCT compound (Tissue Tek, Sacura). Sections were stained after 8-10 μm. In addition, the neurospheres were plated on a plate coated with PLO (poly-L-orthinine, sigma) and fibronectin (sigma), and then treated with 4% paraformaldehyde (EMS) for 20 minutes. Afterwards, the cells were washed with PBS (containing Ca and Mg) containing 0.1% BSA (bovine serum albumine, sigma) and stained. Cells immobilized by two methods were blocked, permeabilized and antibody incubated in the following manner.

After blocking with PBS containing 10% Normal donkgey serum, Jackson Immunoresearch, 0.1% BSA (Sigma) and 0.3% Triton X-100 (Sigma), anti-nestin (Chemicon), anti-CD133 ( MACS) (CD133 is a surface marker, limiting the permeabilization time to about 10 minutes) and anti-mouse-cy3 (Jackson Immunoresearch at room temperature after overnight reaction at 4 ° C using anti-Sox2 (Sigma). ), And then reacted with anti-rabbit-FITC (Molecular probe) and finally nuclear stain using DAPI (sigma). After staining, it was confirmed by Zeiss confocal (Carl Zeiss). Differentiated cells were stained in the same manner as above, and the antibodies used were anti-GFAP (Dako), anti-S100β (Sigma), anti-β-tubulin III (Covance), anti-Map2a (Sigma), Anti-TH (Chemicon), anti-NeuN (Chemicon), anti-O4, anti-O1, anti-A2B5 (R & D).

Through immunocytochemistry, make a specific marker of neural stem cells results, Nestin, Sox2, CD133 is the same expression, Nestin- positive cells was expressed with the proliferation marker Ki-67 also (Fig. 2e, in Figure 2 e ad; neural stem cells, eh of FIG. 2 e; neural stem cells-like cells) .

Results confirm the markers identified in the through RT-PCR method is shown in Fig. 2f. Nestin and Sox2 were expressed in neural stem cell-like cells (Bmi1-sphere) as in neural stem cells (NSC), but not in astrocytes (Bmi1) in which the vector or Bmi-1 gene was overexpressed.

< Example  7> Differentiate into inbeats ( In in vitro differentiation )

In order to determine whether neural stem cell-like cells have multipotency like neural stem cells, the cultured neurospheres were treated with PLO (poly-L-ornithinte, Sigma), laminin, or fibronectin (sigma). ) Was cultured under the following differentiation conditions in a plate coated with ().

Differentiation into astrocytes was achieved by adding human recombinant bFGF and EGF (R & D systems) to DMEM (Hyclone, high glucose) containing 10% FBS (Hyclone), or by recombinant rat ciliaryneurotrophic factor (CNTF) (upstate). Differentiation was induced by incubation for 7 days. GFAP and S100β were used as markers to confirm differentiation with astrosites ( a, c in FIGS. 3a and 3a; neural stem cells, b and d in FIG. 3a; neural stem cells-like cells ).

Differentiation into neurons was treated with human recombinant FGF for 4 days in N2 culture medium containing B27 (Gibco), a serum-substituted product, and then cultured in medium without FGF for 8 days. Alternatively, RA (retinoic acid, sigma) was added at 1-10 μM and cultured for 7-14 days. Another method was used to induce differentiation by adding VPA (valproic acid, sigma) at 1-10 mM concentration with RA (retinoic acid, sigma) at 1-10 μM concentration for 7-14 days. ΒIII-tubulin (Tuj1), Map2a, and TH were used as markers for differentiation into neurons ( a, ce of FIGS. 3b and 3b; neural stem cells and b, fh of neural stem cell-like cells ) of FIG. 3b ).

Differentiation into oligodendrocytes is characterized by platelet derived growth factor-AA (R & D) and T3 (3,3,5-triiodo-L-thyronine, sigma), N2 cultures with serum replacement B27. Human recombinant bFGF and EGF (R & D) were added together and cultured, followed by incubation for 20 days while exchanging the added components one by one to induce differentiation. 04, CNPase, 01 was used as a marker to confirm the differentiation into oligodendrosite ( ac, gi of Figure 3c, Figure 3c; neural stem cells, df, jl of Figure 3c; neural stem cells-like cells ).

After culturing under the above-mentioned conditions, observing the shape of each differentiated cell, and using the antibody used as the differentiation marker, it was confirmed by immunocytochemistry. Differentiation was found to be the same in neural stem cell-like cells (NSCLCs) ( FIG. 3 ).

< Example  8> Bmi -1 gene In reverse differentiation  Check impact

pBI-Bmi-1-EGFP and pBabe puro rTTA were transfected together and cells in which Bmi-1 gene expression was induced with doxycycline were cultured in neural stem cell culture.

The Doxycycline-inducible pBI-Bmi-1-EGFP vector digests the pBI-EGFP (BD Bioscience) vector with pvuII and then ligations the human Bmi-1 gene. Cloning was performed. This vector and pBabe puro rTTA were transfected together with astrosite, and treated with 1 μg / ml of doxycycline (Dox) while screening with 0.5 μg / ml of puromycine and confirming expression with GFP. ( FIG. 4A ). Doxycycline can induce the expression of the Bmi-1 gene (Dox +), and when there is no doxycycline (Dox-) it can be seen that the Bmi-1 gene is not expressed. When switched to the culture medium of neural stem cells, it was confirmed that when there is doxycycline, neurospheres are formed, and when there is no neurospheres ( FIG. 4B) . This means that the Bmi-1 gene plays a role in inducing the differentiation of astrosite.

When cells in which Bmi-1 gene expression was induced were cultured in a culture medium of neural stem cells to which doxycycline was added, cells similar to neural stem cells were formed, GFP was expressed ( FIG. 4C ), and markers of neural stem cells were expressed. ( C in FIG. 4C )

After inducing differentiation of neural stem cell-like cells cultured in the culture medium of doxycycline-added neural stem cells, each specific marker was identified by immunochemical staining . Asstrosite ( a and b in FIG. 4d ) and neurons ( FIG. 4d ce ), oligodendrosite ( f, g of Figure 4d ) was confirmed that differentiation was induced.

< Example  9> in vivo differentiation ( In vivo differentiation )

Neural stem cell-like cells cultured in a culture medium of doxycycline-added neural stem cells were injected into subcutaneous tissues of 6-week-old nude mice, and the tissues were excised two weeks later to be differentiated into different markers. It was confirmed. Differentiation markers were stained by immunocytochemistry and merged with GFP to confirm whether they were differentiated by the injected cells. As a result, the cells were found to have differentiation ability in vivo ( FIG. 4e ).

< Example  10> biochemical inhibitors ( Biochemical inhibitors Processing Bmi -1 gene overexpressed Astrosite  Growth and In reverse differentiation  Impact Analysis

Among the factors involved in Valproic acid (Sigma), TSA (Trichostatin A, Sigma) and FGF signal pathways belonging to the histone deacetylase (HDAC) inhibitor Several concentrations of NL-71-101 (Sigma), PD98059 (Calbiochem), Wortmannin (Sigma), and U0126 (Promega), which are substances that inhibit the key molecule, are overexpressed, resulting in the overexpression of the Bmi-1 gene. When biochemical inhibitor treatment was performed on the growth media (Growth media, GM) of the grown astrosites and the neural stem cell cultures for dedifferentiation into neural stem cell-like cells, the growth and neuronal stem cells of the Bmi-1 gene were overexpressed. The effect on the reverse differentiation of was analyzed.

When the growth medium was treated, the cells were plated with an appropriate amount, and then treated for 3 days, and then stained with 0.01% crystal violet (crystal violet) every day and extracted with 10% acetic acid to measure relative growth rates. In addition, when treated in the neural stem cell culture solution, it was confirmed that reverse differentiation was induced during the treatment after plating the appropriate amount of cells. Alternatively, the above mentioned inhibitors were treated by plating 100 cells / well of cells in 12 well plates with neural stem cell culture. It was observed 14 days after treatment whether neurospheres were formed.

As a result, it was confirmed that the action of Bmi-1 was inhibited by the HDAC inhibitor, and no reverse differentiation into neural stem cells was performed (FIG. 5). Treatment with VPA (Valproic acid) and TSA (Trichostatin A) decreased the growth rate of cells induced by overexpression of the Bmi-1 gene, and observed that incubation under neural stem cell culture conditions did not induce differentiation. Could. As a result of treatment with TSA in the range of 20 ng / ml to 100 ng / ml, it was confirmed that the growth rate was remarkably decreased as the concentration was increased ( FIG. 5a, FIG. 5b) . When the SA-β-galactosidase assay was performed, it was observed that there were cells showing positive ( arrow in FIG. 5B ). In addition, since TSA acts as a histone deacetylase inhibitor, it was confirmed that histone H3 was acetylated by TSA treatment. At this time, expression of p16 and p21 also increased, and TSA concentration was increased. The higher the higher the expression of the protein ( Fig. 5c ). As a result of induction of differentiation into neural stem cells, such treatment did not form neurospheres ( FIG. 5D ), and the expression of nestin and sox2 was reduced compared to the control ( FIG. 5D ) . 5e ). In addition, TSA was added to 100 Astrosite cells induced with overexpression of the Bmi-1 gene, and cultured for 2 weeks under the neural stem cell culture conditions, and the formation of neurospheres was observed. It was confirmed that it is inhibited ( FIG. 5F ).

In addition, even when treated with 1mM, 5mM, and 10mM VPA to the astrocytes overexpressed Bmi-1 gene, the growth rate of cells induced by overexpression of Bmi-1 gene was significantly decreased as the treatment concentration increased ( FIG. 5G). ), Even when cultured with a culture of neural stem cells for reverse differentiation, the higher the VPA treatment concentration, the more likely the formation of neurospheres ( FIG. 5J ). Figure 5h shows the appearance of the cells after treatment with VPA for 3 days in the culture of neural stem cells Astrosite overexpressed Bmi-1 gene. In addition, it was confirmed that the expression of nestin and sox2 also decreased compared to the control in the VPA treatment ( Fig. 5i ). In addition, VPA was added to 100 Astrosite cells induced with overexpression of the Bmi-1 gene, and cultured for 2 weeks under culture conditions of neural stem cells, and the formation of neurospheres was observed. It was confirmed that the inhibition ( FIG. 5J ).

In addition, it was confirmed that bFGF induced dedifferentiation into neural stem cell-like cells and experimented with their related pathways. We examined whether the P13K-AKT and MEK1 / MAPK pathways, which are activated by bFGF, are involved in the mechanism of retrodifferentiation into neural stem cells. P13K-inhibitor (wortmannin), AKT-inhibitor (NL-71-101), EPK-inhibitor (PD98059), and MEK1-inhibitor (U0126) are neuronal stem cell culture media for inducing differentiation of astrosite overexpressed with Bmi-1 gene When treated at 1μM, 10μM, 1μM was not significantly different when compared with the control group, but at 10μM it could be seen that almost no neurospheres formed. However, when PD98059 was treated, it was confirmed that the number of neurosphere formation was not significantly different from that of the control group ( FIG. 2D ).

Therefore, it can be seen that the HDAC inhibitor acts to reduce the proliferation, dedifferentiation, and maintenance of neural stem cell-like cells.

As described above, Bmi-1 may be used to induce differentiation of astrosite into neural stem cells, and the dedifferentiated neural stem cells may be used for treating various diseases.

<110> Imgen Co., Ltd <120> a composition for de-differentiating astrocytes into neural stem          cell comprising human Bmi-1 protein or a nucleic acid encoding          the same and a process of de-differentiation using said          composition <130> PA9702-0146 / KR <150> KR10-2006-19018 <151> 2006-02-27 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer specific for GAPDH <400> 1 gatgacatca agaaggtggt gaag 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer specific for GAPDH <400> 2 gttgctgtag ccgtattcat tgtc 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer specific for nestin <400> 3 ggcatccctg aattacccaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer specific for nestin <400> 4 agctcatggg catctgtcaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer specific for sox2 <400> 5 agtggtacgt taggcgcttc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer specific for sox2 <400> 6 tgccttaaac aagaccacga 20  

Claims (17)

A composition for inducing the differentiation of an astrocyte (astrocyte) comprising a human Bmi-1 protein or a nucleotide sequence encoding a human Bmi-1 protein into neural stem cells. The composition of claim 1, wherein the composition comprises a Bmi-1 protein expression vector inserted with a nucleic acid that is a nucleotide sequence encoding human Bmi-1 protein. The composition of claim 2, wherein the vector is prepared by inserting a nucleotide sequence encoding a human Bmi-1 protein into a pBabe puro vector. The composition of claim 1, wherein the composition comprises a virus in which a nucleic acid, which is a nucleotide sequence encoding human Bmi-1 protein, is introduced to express the Bmi-1 protein. The method of claim 4, wherein the virus is produced by transfection of a Bmi-1 protein expression vector containing a nucleic acid, a nucleotide sequence encoding human Bmi-1 protein, into a PT67 packaging cell line. Characterized in that the composition. The composition of claim 1, wherein the de-differentiated neural stem cells are neural stem cells having multipotency to an astrocyte, a neuron, or an oligoendrocyte. (i) culturing the astrosite in the medium, (ii) infecting the cultured astrosite with packaging cells transfected with the vector into which the human Bmi-1 gene is inserted, and (iii) the infected astrosite A method of inducing dedifferentiation into neural stem cells, comprising the step of culturing in neural stem cell culture conditions. 8. The method of claim 7, wherein step (i) separates the astrosite from the brain of the mouse to make a single cell after trypsin treatment, fetal bovine serum (FBS), penicillin or streptomycin (streptomycin) And culturing in Dulbecco's modified Eagle's medium (DMEM) containing L-glutamine. The method of claim 7, wherein the vector of step (ii) is prepared by inserting a nucleic acid, which is a nucleotide sequence encoding human Bmi-1 protein, into a pBabe puro vector. 8. The method of claim 7, wherein said packaging cells of step (ii) are PT67 packaging cells. According to claim 7, Neural stem cell culture conditions of step (iii) 12 hours after culturing the cells plated, insulin (Insulin), apo-transferrin, selenium (selenium), progesteron (progesteron) ), And Dulbecco's modified Eagle's medium / F12 containing penicillin or streptomycin added to a medium in which serum-replacement B27 and basic Fibroblast Growth Factor (bFGF) were added. After giving, bFGF treatment every day and the culture method of changing the medium every two days. The method according to claim 7, wherein the neural stem cell culture conditions of step (iii) is a tripine to cells cultured in a growth medium containing 10% FBS, 1% penicillin or streptomycin, and 1% L- glutamine After trypinization, seeded in bacterial culture plates and in serum-alternate B27 and bFGF added to Dulbecco's modified Eagle's medium / F12 containing insulin, apo-transferrin, selenium, progesterone, and penicillin or streptomycin Culturing. Neural stem cells produced by the method of any one of claims 7 to 12 having a differentiation ability to astrosite, neurons or oligodendrosite. The method of claim 13, wherein the differentiation of the astrosite cultured neural stem cells by adding a basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) or CiliaryNeuroTrophic Factor (CNTF) to the Dulbecco modified eagle medium containing FBS Neural stem cells that are made by. The method of claim 13, wherein the differentiation of the neurons into the neuronal stem cells N-culture medium containing serum-substituted B27 (insulin, apo-transferrin, selenium, progesterone, and DMEM / F12 medium containing penicillin or streptomycin) 4 days after the FGF treatment, and for 8 days neural stem cells that are made by culturing in a medium without FGF. The method of claim 13, wherein the differentiation of the neurons to the neuronal stem cells in the N2 culture medium containing serum-substituted B27 is added 1-10μM retinoic acid (RA) or 1-10mM VPA (valproic acid) Neural stem cells that are made by incubating for 7 to 14 days by adding with 1-10 μM RA (retinoic acid). The method according to claim 13, wherein the differentiation into oligodendrosite is the neural stem cells platelet derived growth factor-AA (PDGF-AA), T3 (3,3,5-triiodo) in N2 culture medium containing serum-substituted B27 -L-thyronine), the neural stem cells that are made by culturing for 20 days while adding and bFGF and EGF cultured by replacing the added components one by one.

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