WO2007097494A1

WO2007097494A1 - De-differentiation of astrocytes into neural stem cell using bmi-1

Info

Publication number: WO2007097494A1
Application number: PCT/KR2006/001350
Authority: WO
Inventors: Seungkwon You; Jai Hee Moon; Byung Sun Yoon; Ki Dong Kim; Gyuman Park; Eun Kyung Jun; Bona Kim; Seung Jun Yoo; Sung Sik Kwak; Isaac Maeng
Original assignee: Imgen Co., Ltd.
Priority date: 2006-02-27
Filing date: 2006-04-12
Publication date: 2007-08-30
Also published as: KR20070089089A; KR100844971B1; EP1987148A1; EP1987148A4; CN101389761A; JP2009528050A

Abstract

Disclosed are a composition and a method for inducing the de-differentiation of astrocytes into neural stem cells using Bmi-1. The de-differentiated neural stem cells have the ability to differentiate into astrocytes, neurons, and oligodendrocytes.

Description

DE-DIFFERENTIATION OF ASTROCYTES INTO NEURAL STEM CELL

USING BMI-I

Technical Field

Neural stem cells (NSCs) are a subtype of progenitor cells in the nervous system that has the ability to differentiate into astrocytes, oligodendrocytes, and neurons. Originating from the Central Nervous System (CNS) and the Peripheral Nervous System (PNS) , neural stem cells form multicellular neurospheres, which differentiate into glial lineage and neural lineage cells under respective sets of conditions (Sally Temple et al. 2001). These neural stem cells are used in the treatment of incurable diseases, and are being studied as a potential method for cell treatment. Extensive research has been conducted on neural stem cells because they are adult stem cells that entail few ethical problems . Active research on de- differentiation, which has been conducted recently, is increasing the importance of adult stem cells. Adult stem cells are easier to obtain than embryonic stem cells, but there are still many difficulties in the practical application thereof. In addition, immune rejection response can be a problem when using adult stem cells from another person. Therefore, the induction of de-differentiation using a patient's own cells could solve current problems. For this purpose, it is necessary to induce de- differentiation of cells that are already differentiated. Currently, extensive research is underway to induce de- differentiation using methods such as cell fusion and nuclear transfer. In another group using different methods, Alexis J. reported that cells isolated from the skin have the characteristics of neural stem cells when cultured under neural stem cell cultivation conditions (Lancet 2004) . In addition, Toru K. succeeded in de-differentiating oligodendrocyte precursors into neural stem cells (Genes & Development 2004) . This group has been publishing papers on this issue since 2000, and reported in a 2004 paper that gene expression at each stage is relevant to chromatin remodeling and histone modification.

Background Art

In the present invention, a solution to the problems of formerly introduced methods is sought and a novel proper usage method is established. In this invention, one transcription factor which is known to regulate properties of NSCs, is selected and overexpressed to study differentiation into neural stem cells. The selected factor, identified as ^NNBmi-l", is one of the proteins accounting for the regulation of histone modification and cell cycle regulators (Jan W. et al., 1999). A cdkn2a/INK4A locus is reported as a Bmi-1 target. Brtιi-1 is known as being a transcriptional inhibitor of the target gene. Recently, the Bmi-1 gene has been reported to be expressed in neural stem cells and play a role in the self-renewal of neural stem cells (Anna V. M. et al., 2003, 2005, In-Kyoung Park et al., 2004).

Leading to the present invention, intensive and thorough research, conducted by the present inventors with this background, resulted in the finding that the overexpression of Bmi-1 therein induces already differentiated astrocytes to de-differentiate into neural stem cell-like cells capable of self-renewal and differentiation into astrocytes, neurons, and oligodendrocytes .

Disclosure of the Invention

An object of the invention is to provide a composition for inducing the de-differentiation of astrocytes into neural stem cells, comprising a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein.

Another object of the invention is to provide a method of inducing the de-differentiation of astrocytes into neural stem cells, comprising the treatment of astrocytes with a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein.

A further object of the invention is to provide neural stem cells produced using the method.

Still a further object of the invention is to provide a method of differentiating the de-differentiated neural stem cells into astrocytes, oligodendrocytes and neurons.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the overexpression of the Bmi-1 targets pl6^Ink4a and pl9^Arf _f analyzed using immunocytochemistry;

FIG. 2 shows the induction of de-differentiation, in which sphere formation (A) and direct sphere formation (B) are indicated (direct spheres are formed when single cells are cultured under conditions suitable for neural stem cells and spheres are formed when single cells are cultured under conditions suitable for neural stem cells only after being attached under conditions suitable for the culture of astrocytes, and stabilized for 12 hours) ;

FIG. 3 shows the expression of (a) neural stem cell markers as analyzed through immunocytochemistry, in which neural stem cells (A to C) and neural stem cell-like cells (D to F) are differentiated;

FIG. 4 shows the expression of neural stem cell- specific markers as measured through RT-PCR (Ast: astrocytes, NSC: neural stem cell, Ast-Bmi-1: astrocyte+Bmi-1, NSCLC: neural stem cell-like cell) ; and

FIG. 5 shows in vitro differentiation of neural stem cells (A-C) and neural stem cell-like cells (D-G) into astrocytes, oligodendrocytes and neurons.

Best Mode for Carrying Out the Invention

In accordance with an aspect thereof, the present invention relates to a composition capable of inducing the de-differentiation of astrocytes into neural stem cells, which comprises a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein.

As used herein, the term "neural stem cell-like cells" is intended to indicate multipotent stem cells, dedifferentiated from somatic cells capable of differentiating into astrocytes, oligodendrocytes, and neurons. In the present invention, neural stem cell-like cells are also described as neural stem cells.

It is newly disclosed in the present invention that when Bmi-1 is overexpressed, astrocytes, although already differentiated, can de-differentiate into multipotent neural stem cell-like cells.

In the present invention, Brai-1 is provided in the form of a protein or a nucleic acid that codes for the Bmi- 1 protein. As long as it is originated from mammals such as humans, horses, sheep, pigs, goats, camels, antelopes, and dogs, any Bmi-1 may be used in the composition of the present invention. In addition, the Bmi-1 protein of the present invention, used for de-differentiation into neural cells, may be a wild-type or a variant thereof.

The term "Bmi-1 protein variant" is intended to refer to Bmi-1 proteins, occurring naturally or artificially, which are different in amino acid sequence from the wild- type due to the deletion, insertion, non-conservative substitution or conservative substitution of amino acids, or combinations thereof. The variant is a functional equivalent which has the same biological activity as the native protein although its physical and/or chemical properties are modified. Preferably, the variant is increased in structural stability to physical and chemical environments or in physiological activity.

In a preferable embodiment of the present invention, the Bmi-1 is in the form of a nucleic acid material comprising a nucleotide sequence coding for the Bmi-1 protein.

The nucleotide sequence that encodes a Bmi-1 protein is a wild-type or a variant thereof which, naturally occurring or artificially synthesized, is different in amino acid sequence from the wild-type due to the deletion, insertion, non-conservative substitution or conservative substitution of bases, or combinations thereof.

The nucleotide sequence that encodes a Bmi-1 protein can be either a single or a double strand consisting of a DNA molecule (genome, cDNA) or an RNA molecule.

In a preferred embodiment of the present invention, the nucleic acid material containing a nucleotide sequence encoding a Bmi-1 protein is a vector that allows a Bmi-1 protein to be expressed.

The term "vector", as used herein, is intended to refer to a DNA construct containing a DNA sequence which is operably linked to a control sequence capable of effecting the expression of the DNA in a suitable host cell.

The term "operably linked", as used herein, is intended to refer to a functional linkage between a nucleic acid expression control sequence and a second nucleic acid sequence coding for a target protein in such a manner as to enable general functionality. The operable linkage to a recombinant vector may be prepared using a genetic recombinant technique that is well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes that are generally known in the art.

A vector suitable for use in the present invention includes a signal sequence or a leader sequence for membrane targeting or secretion as well as expression regulatory elements, such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal and an enhancer, and can be constructed in various forms depending on the purpose thereof. The promoter of the vector may be constitutive or inducible. In addition, expression vectors include a selectable marker that allows the selection of host cells containing the vector, and replicable expression vectors include a replication origin. The vector may be self-replicable, or may be integrated into the DNA of a host cell.

The vector useful in the present invention may be a plasmid vector, a cosmid vector, or a viral vector, with preference for a viral vector. Examples of the viral vector includes vectors originated from retroviruses such as HIV

(Human Immunodeficiency Virus) , MLV (Murine Leukemia

Virus) , ASLV (Avian Sarcoma/Leukosis Virus) , SNV (Spleen

Necrosis Virus), RSV (Rous Sarcoma Virus), MMTV (Mouse Mammary Tumor Virus), etc., Adeno-associated viruses, and Herpes Simplex virus, but are not limited thereto. In one practical example of the present invention, a pBabe puro vector, derived from a Moloney leukemia virus-based virus vector, carrying a selection marker for puromycin, was used.

The nucleic acid material containing the nucleotide sequence coding for the Bmi-1 protein can be introduced into cells in the form of a vector as naked DNA (Wolff et al. Science, 247:1465-8, 1990: Wolff et al. J. Cell Sci. 103:1249-59, 1992), or with the aid of a liposome or a cationic polymer. For use in gene transfection, a liposome is a phospholipid membrane made of cationic phospholipids such as DOTMA and DOTAP. A cationic liposome, when mixed with a negatively charged nucleotide at a certain ratio, is formed into a nucleic acid-liposome complex. In another preferred embodiment of the present invention, the nucleic acid material containing a nucleotide sequence that encodes the Bmi-1 protein may be a virus which expresses the Bmi-1 protein therein.

The term "virus", as used herein, is intended to refer to a Bmi-1-expressing virus which is prepared by transforming or transfecting a packaging cell with a viral vector carrying a nucleotide sequence coding for the Bmi-1 protein.

Examples of viruses ,useful in the preparation of the Bmi-1-expressing viruses according to the present invention include retroviruses, adenoviruses, adeno-associated viruses, and the Herpes Simplex virus, but are not limited thereto. Preferable are retroviruses. In the following examples, a Bmi-1-expressing virus was prepared by transforming a recombinant pBabe puro vector carrying a nucleotide sequence coding for the Bmi-1 protein (pBabe puro Bmi-1) into PT67 packaging cells.

In accordance with another aspect thereof, the present invention pertains to a method of dedifferentiating astrocytes into neural stem cells, comprising treatment with a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence encoding the Bmi- 1 protein.

In greater detail, the method comprises the steps of (i) culturing astrocytes in a medium; (ii) treating the culture with a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence coding for the Bmi-1 protein; and (iii) inducing the de-differentiation of astrocytes into neural stem cells.

Any conventional culture medium for neural stem cells may be used as a culture medium for astrocytes in step (i) .

Generally, a culture medium contains a carbon source, a nitrogen source, and trace element ingredients. In addition, the culture medium may include antibiotics, such as penicillin, streptomycin, and gentamicin. Preferred is a culture medium containing bFGF.

The Bmi-1 protein or the nucleic acid material containing the nucleotide sequence coding for the Bmi-1 protein with which the cells are treated in step (ii) is as mentioned above.

In accordance with a further aspect thereof, the present invention pertains to neural stem cells prepared in accordance with the aforementioned method.

It has been confirmed that the neural stem cells, prepared through the de-differentiation according to the present invention, express the neural stem cell-specific markers, Nestin, CD133, and Sox2 at the same level, and have the same ability to differentiate as general neural stem cells. The neural stem cells prepared through de- differentiation according to the present invention feature self-renewal, as well.

In accordance with still a further aspect thereof, the present invention pertains to a method of differentiating the neural stem cells, de-differentiated according to the aforementioned method, into astrocytes, oligodendrocytes, and neurons.

When the neural stem cells, de-differentiated using the composition and method of the present invention, are placed under respective differentiation conditions, differentiation into astrocytes, neurons, and oligodendrocytes can be monitored by detecting the expression of markers specific for respective cells.

A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1: Experiment Method

1. Cultivation of mouse astrocytes and mouse neural stem cell

Mouse astrocytes were isolated from the CNS at E13.5 and cultured under suitable conditions. They were treated with trypsin (Gibco 0.05%) before incubation in modified Eagle's medium (DMEM (high glucose, HyClone) ) supplemented with 10% FBS (HyClone) , 1% penicillin/streptomycin, and 1% L-glutamine (Cambrex) . From the next day, the medium was replaced with a fresh one. Cells between passage one and three were used. Neural stem cells were separated from mice (E13.5) and cultured as a control in Dulbecco's modified Eagle's medium/F12 (DMEM/F12, Gibco), supplemented with B27 serum-replacement (Gibco) , human recombinant basic FGF and human recombinant EGF (R&D) , containing insulin (Sigma), apo-transferrin (Sigma) , selenium (Sigma) , progesterone (Sigma) and penicillin/streptomycin (Cambrex) .

2. Retroviral-mediated infection

pBabe puro Bmi-1 (prepared by inserting human Bmi-1 (NCBI accession No.: L13689 NM 024865) into a pBabe puro vector) and pBabe puro were transfected into a PT67 packaging cell line (Clontech) using Lipofectamine

(Invitrogen) and selected in the presence of puromycin (3 μg/ml) (BD science) . After the transformed cell line had grown to 90% or higher confluency, the supernatant was passed through a filter (0.45 μm) (Millipore) to remove cell debris, after which the supernatant was twice allowed to infect the astrocytes, which were separated at intervals of 10 hrs using polybrene (Sigma) . Selection was subsequently conducted in the presence of puromycin (0.5 μg/ml) .

3. Western blot analysis and semi-quantitative PCR

Total cell extracts were isolated from the cells using RIPA buffer, followed by Bradford assay (Bio-rad) to determine the protein concentrations thereof. 50 μg of each cell extract was subjected to 10% or 4~12% precasted SDS- PAGE (Invitrogen) . The proteins thus separated were transferred onto a PVDF membrane (Millipore) and blocked with 3~5% skimmed milk TBST. Incubation with anti-Bmi-1 (Upstate) anti-plβ^INK4A (Santacruz), anti-pl^ (Novous), and α-tubulin (sigma) was done at 4°C overnight with shaking, followed by reaction with HRP conjugated anti-mouse IgG, anti-rabbit IgG (Zymed) at RT. A Supersignal West Pico kit

(Pierce) was used for detection. Total RNA was isolated from each cell line using Trizol (Invitrogen) . cDNA was synthesized from 500 ng of the total RNA in the presence of oligo d(T) 12-18-mer (Invitrogen) using Superscriptase II reverse transcriptase (Invitrogen) . RT-PCR was performed with 1 μl of the cDNA in the presence of 10 pmol of each of suitable primers using a PCR premix (IU Tag DNA polymerase, 250 μM dNTPs, 10 mM Tris-HCl, 40 mM KCl and 1.5 mM MgCl₂ Bioneer, Korea). The primers used in the RT-PCR were adapted to amplify the mouse markers GAPDH, Nestin, and Sox2, and their base sequences are shown as follows.

Primers for mouse GAPDH

Forward: 5'-GATGACATCAAGAAGGTGGTGAAG-SMSEQ ID NO.:1) Reverse: 5'-GTTGCTGTAGCCGTATTCATTGTC-SMSEQ ID NO.^) Primers for mouse nestin

Forward: 5'-GGCATCCCTGAATTACCCAA-S' (SEQ ID NO.: 3) Reverse: 5'-AGCTCATGGGCATCTGTCAA-S' (SEQ ID NO.: 4) Primers for mouse sox2 Forward: 5'-AGTGGTACGTTAGGCGCTTC-S' (SEQ ID NO.: 5)

Reverse: 5'-TGCCTTAAACAAGACCACGA-S' (SEQ ID NO. 6)

4. Induction of de-differentiation

De-differentiation was induced under the same culture condition used for neural stem cells. Cell culture was conducted using two different methods. Cells were plated at a density of IxIO⁵ cells/well in β-well plates and incubated for 12 hrs, after which the medium was replaced with a fresh Dulbecco' s modified Eagle's medium/Fl2 (N2) (DMEM/F12, Gibco) , supplemented with B27 serum-replacement

(Gibco) , human recombinant basic FGF and human recombinant EGF (R&D) , containing insulin (Sigma) , apo-transferrin

(Sigma) , selenium (Sigma) , progesterone (Sigma) and penicillin/streptomycin (Cambrex) . Cells were treated with bFGF every day, and the medium was replaced with a fresh onemedium every other day. Alternatively, cells which had been cultured under proper conditions were trypsinized and seeded at a density of 3^χlO⁵ cells in a 60-mm bacterial culture plate. Dulbecco' s modified Eagle's medium/F12 (N2) (DMEM/F12, Gibco) , supplemented with B27 serum-replacement

(Sigma) , selenium (Sigma) , progesterone (Sigma) and penicillin/streptomycin (Cambrex) , was used as a culture medium.

5. Determination of overexpression and respective markers through immunocytochemistry

After fixation with 4% paraformaldehyde (EMS) at 4⁰C for one hour, neurospheres were incubated overnight at 4⁰C in 20% sucrose with shaking. Then, the neurospheres were subjected to cryopreservation using an OCT compound in an

8-well chamber slide (Nunc) and sectioned 8~10 μm thick before staining. After blocking with PBS containing 10% normal goat serum (Jackson ImmunoResearch) +0.1% BSA

(Sigma) +0.3% Triton X-100 (Sigma), the sections were incubated with anti-nestin (Chemicon) , anti-CD133 (MACS) and anti-Sox2 (Sigma) at 4°C overnight and then with anti- mouse-cy3 (Jackson ImmunoResearch) , anti-rabbit-FITC (Molecular probe) , and anti-goat-cy3 (Zymed) at RT, finally followed by nuclear staining with DAPI (Sigma) . A Zeiss confocal lens (Carl Zeiss) was used for examination after staining. Differentiated cells were stained in the same manner as described above. In this regard, anti-GFAP (Dako), anti-SlOOβ (Zymed), anti-β-tubulin III (Covance) , anti-Map2a (Sigma), anti-TH (Sigma), anti-04 (R&D) , and anti-CNPase (Chemicon) were used as antibodies.

6. In vitro differentiation

After being coated with PLO (poly-L-ornithine) (Sigma) and laminin or fibronectin (Sigma) , cells were cultured under the differentiation conditions given below.

For differentiation into astrocytes, cell culture was conducted for 5-7 days in DMEM (HyClone, high glucose) supplemented with 10% FBS (HyClone) in the presence of human recombinant bFGF and EGF (R&D) or of CNTF (recombinant rat ciliary neurotrophic factor) (Upstate) .

Differentiation into neurons was induced by culturing the cells in a medium containing N2 and B27 serum- replacement, supplemented with human recombinant FGF, for 4 days and then in an FGF-free medium for 8 days. Alternatively, cells were cultured for 7 ~ 14 days in the presence of 1 ~ 10 μM of RA (retinoic acid, Sigma) as a differentiation inducer. As an additional alternative, cells were cultured for 7 ~ 14 days in the co-presence of 1 ~ 10 Mm of VPA (valproic acid, Sigma) and 1 ~ 10 μM of RA (retinoic acid, Sigma) to induce differentiation into neurons .

Differentiation into oligodendrocytes was induced by incubating in an N2 medium supplemented with B27 serum replacement in the presence of PDGF-AA (platelet derived growth factor-AA, R&D), T3 (3, 3, 5-triiodo-L-thyronine, Sigma) , human recombinant basic FGF and EGF (R&D) .

While being cultured under the aforementioned differentiation conditions, the cells were monitored for morphology, and immunocytochemistry using antibodies specific to respective differentiation markers was conducted to examine whether the differentiation proceeded accurately.

EXAMPLE 2: Results The Brai-1 gene was overexpressed using a retroviral vector transduction system in mouse astrocytes, which had been differentiated. The overexpression of the Bmi-1 gene was verified through Western blotting analysis while the Bmi-1 target genes plβ^Ink4a and pl9^Art were not expressed

(FIG. 1) .

Then, de-differentiation was induced using conditions for culturing neural stem cells. The astrocytes were seeded at a density of IxIO⁵ cells/well in β-well plates, and 12 hrs later, the cells were incubated under conditions suitable for neural stem cells. Within 3 - 4 days of incubation, the cells were observed to have neural stem cell-like morphology. When the cells were plated at a density of 3^χlO⁵ cells/plate in 60-mrn bacterial plates and cultured under the same conditions as neural stem cells, they were also observed to have the morphology of neural stem cells. On the contrary, when the cells transfected with the control vector pBabe puro EGFP were cultured, no neurospheres were formed (FIG. 2) . On day 6, the neurospheres were observed to have the same morphology as neural stem cells when they were transferred to and cultured in new plates . In order to examine whether the cells de-differentiated with the Bmi-1 gene ensure self- renewal, a subsphere formation assay was conducted. As a result, spheres were formed in single cells one week later (data not shown) .

In order to find out whether these cells had the same characteristics as neural stem cells, immunocytochemistry was performed using respective markers. In this regard, the markers nestin, CD133 and Sox2, which are most abundantly expressed in neural stem cells, were also observed in the neural stem cell-like cells after staining. Also, the cryo- section of the neurospheres thus formed ensured the expression of nestin, CD133 and Sox2 (FIG. 3) . Also, nestin and sox2, both expressed in neural stem cells, were found to be expressed in the cells dedifferentiated according to the present invention, as analyzed through RT-PCR. Therefore, the cells dedifferentiated according to the present invention were judged to be neural stem cell-like cells (FIG. 4) .

In order to examine whether the neural stem cell-like cells could differentiate like neural stem cells, differentiation into astrocytes, oligodendrocytes and neurons was induced in the same manner as described above. The neural stem cell-like cells were found to differentiate into the three types of cells like neural stem cells as analyzed with antibodies against the respective markers specific therefor. Identification was conducted with the expression of GFAP and SlOO for differentiation into astrocytes, with the expression of β-tubulin III (Tujl) and Map2a for differentiation into neurons and with the expression of 04 and CNPOase for differentiation into oligodendrocytes (FIG. 5) .

Taken together, the data obtained through this study demonstrate that the Bmi-1 gene plays a critical role in the de-differentiation of astrocytes into neural stem cell- like cells, and that these de-differentiated neural stem cell-like cells can differentiate back into astrocytes, oligodendrocytes, and neurons.

Industrial Applicability

As described hitherto, Bmi-1 is useful in inducing the de-differentiation of astrocytes into neural stem cells, and the de-differentiated neural stem cells can be used for the treatment of various diseases.

Bibliography of Prior Art

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Claims

1. A composition for inducing de-differentiation of astrocytes into neural stem cells, comprising a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein.

2. The composition as set forth in claim 1, wherein the nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein is a vector that allows the Bmi- 1 protein to be expressed.

3. The composition as set forth in claim 1, wherein the nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein is a virus that expresses the Bmi-1 protein.

4. The composition as set forth in claim 1, wherein the de-differentiated neural stem cells have ability to differentiate into astrocytes, neurons and oligodendrocytes .

5. A method for inducing the de-differentiation of astrocytes into neural stem cells by treating the astrocytes with a Bmi-1 protein or a nucleic acid material containing a nucleotide sequence coding for a Bmi-1 protein.

6. The method as set forth in claim 5, wherein the de-differentiated neural stem cells have ability to differentiate into astrocytes, neurons and oligodendrocytes .

7. The method as set forth in claim 5, comprising steps of:

(i) culturing astrocytes in a medium; (ii) treating the astrocytes with the Bmi-1 protein or the nucleic acid material; and

(iii) inducing the astrocytes to differentiate into neural stem cells.

8. The medium as set forth in claim 7, wherein the medium of step (i) contains bFGF.

9. A neural stem cell, produced using the method of claim 5.

10. A method of differentiating the neural stem cells produced using the method of claim 5 into astrocytes, neurons, and oligodendrocytes.