KR20130143345A - Stem cell expressing nerve growth factor and its use - Google Patents

Abstract

The present invention relates to a stem cell expressing a nerve growth factor by introducing a nerve growth factor (NGF) and a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising the stem cell as an active ingredient. Stem cells expressing nerve growth factors of the present invention not only have activity to protect neurons from cytotoxic substances, but also when transplanted directly into the brain of cognitive impaired animal models that damage brain neurons, It exhibits neuronal cell damage therapeutic activity that restores memory impairment to levels similar to normal animals.

Description

Stem Cells Expressing Nerve Growth Factor and Their Uses {Stem Cell Expressing Nerve Growth Factor and Its Use}

The present invention relates to stem cells expressing Nerve Growth Factor (NGF) and therapeutic use of neurodegenerative diseases thereof.

Alzheimer's disease (AD) is a disease in which cognitive decline is caused by the degeneration or loss of neurons and synapses in the general area of the brain (42). In AD patients, impairment of the choline activating system due to decreased activity of choline acetyltransferase (CAT), which synthesizes acetylcholine (ACh), is the leading cause of cognitive decline (6, 43). 48). To date, only five drugs have been approved for treating AD patients, and AD drug treatment has been shown to increase the concentration of ACh by inhibiting acetyl cholinesterase (AChE), which is mostly ACh-degrading enzyme. It is based on small molecule drugs (31, 43). However, since these drug therapies are only temporary prescriptions, there is a need for more fundamental and effective treatments for AD patients. It is anticipated that AD-targeted stem cell-based cell therapies will meet these needs.

It is well known that nerve growth factor (NGF) improves the function and survival of cholinergic neurons in the basal brain of rats and primates (7, 10, 22, 20, 21, 44-46). . In previous studies, NGF was injected into the ventricle, but caused side effects of NGF such as abnormal sympathetic and sensory neurogenesis and weight loss (33, 50, 49). Direct implantation of cells genetically modified to produce and secrete NGF into the brain will allow direct and targeted introduction of NGF into damaged neurons. In a similar manner, cell-based NGF treatment in primate brains using fibroblasts encoding the NGF gene has been reported to improve the degeneration of basal cholinergic neurons (46). Based on the results of this preclinical study, a phase 1 clinical trial was conducted to examine the effectiveness of ex vivo NGF gene therapy in AD patients. Cutaneous fibroblasts isolated from AD patients were transformed to produce NGF and implanted stereotically into various sites inside the forebrain of AD patients. After 22 months, the degree of cognitive decline was improved and no surgical side effects were observed (47). Successful introduction of genes into the CNS in vivo depends on finding and using cells that can be used as carriers for delivery therapeutic genes and at the same time effective expression of the therapeutic genes and secretion of expressions.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop stem cell therapeutics loaded with genes that can promote the growth and differentiation of stem cells. Thus, the present inventors have produced stem cells stably expressing nerve growth factor (NGF). When transplanted into the brain of the animal model of the brain neurological disease, the present invention was completed by confirming that not only successfully differentiated into normal neurons in vivo, but also repairing damaged neurons to improve learning and memory.

Accordingly, an object of the present invention is to provide a stem cell expressing a nerve growth factor (NGF).

Another object of the present invention to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases, including as an active ingredient stem cells expressing nerve growth factor (NGF).

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a stem cell transformed with an expression vector comprising a nucleic acid molecule encoding a nerve growth factor (NGF) to express a nerve growth factor (NGF).

As used herein, the term “nerve growth factor (NGF)” refers to a signaling protein that plays an important role in the growth, maintenance, and survival of nerve cells.

According to a preferred embodiment of the present invention, the nerve growth factor (NGF) protein is a human-derived nerve growth factor. More preferably, it is a human-derived nerve growth factor consisting of the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "stem cell" refers to a cell having the ability to continue proliferation, that is, self-renewal, and has a differentiation capacity capable of differentiating into various cell types.

According to another preferred embodiment of the present invention, the stem cells of the present invention may be primary cultured neural stem cells isolated and cultured from human tissue, or tumor genes (eg, , v-myc gene) can be an immortalized stem cell line established by introducing a vector containing a form capable of expressing.

According to a preferred embodiment of the present invention, the stem cells into which the nerve growth factor is introduced are neural stem cells or fat stem cells.

As used herein, the term " neural stem cell " refers to a stem cell capable of differentiating into a neural cell, astrocyte, oligodendrocyte, etc. constituting the central nervous system ≪ / RTI >

A specific method for separating neural stem cells is disclosed in US Pat. No. 5,654,183, which is incorporated herein by reference. Neural stem cells can be cultured by adding basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or fibroblast growth factor (FGF) growth factors to the medium in a suitable concentration range, for example, 5-100 ng / .

According to a more preferred embodiment of the present invention, the neural stem cells used in the present invention is immortalization established by introducing a retroviral vector comprising a form capable of expressing the v-myc tumor gene in the neural stem cells isolated from the brain of the human fetus Human neural stem cell line.

In the present invention, the neural stem cells are selected from the group consisting of nestin, neurofilament low-molecular weight protein (NF-L), neurofilament high-molecular weight protein (NF-H) and glial fibrillary acidic protein Can be identified by confirming the existence of the above-mentioned neural stem cell marker protein.

As used herein, “adipose derived stem cells (ASC)” are multipotent stem cells isolated from adipose tissue, and can differentiate into most mesenchymal cells such as adipocytes, osteoblasts, chondrocytes, and myofibroblasts. It means a stem cell having a characteristic.

Methods for isolating, amplifying and differentiating adipose stem cells from adipose tissue are described in Methods. 2008 Jun; 45 (2): 115-20 and Methods Volume 45, Issue 2, June 2008, 115120, Methods in stem cell research, which are incorporated herein by reference.

Stem cells of the present invention are genetically modified stem cells transformed with an expression vector containing a nucleic acid molecule encoding a nerve growth factor (NGF).

In the present invention, a vector used to introduce a neuronal growth factor (NGF) gene into stem cells may be preferably the following vectors, but is not limited thereto: (i) adenovirus vectors; (Ii) retroviral vectors; (Iii) adeno-associated viral vectors; (Iv) a herpes simplex virus vector; (v) SV40 vector; (Vi) a polyoma virus vector; (Ⅶ) papilloma virus vector; (Vii) picornovirus vectors; (Vii) Vexinia virus vector; (X) helper-dependent adenovirus vector.

The origin of replication contained in the vector of the present invention includes, but is not limited to, f1 replication origin, SV40 replication origin, pMB1 replication origin, Adeno replication origin, AAV replication origin, and BBV replication origin.

Promoters that may be used in the vectors of the invention include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter , The SV40 promoter, the cytomegalovirus (CMV) promoter and the tk promoter of HSV) can be used, and the polyadenylation sequence is included as the transcription termination sequence, for example, the SV40 polA sequence and the BGH polA sequence.

The vector used in the present invention is a selection marker and includes an antibiotic resistance gene commonly used in the art and includes, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There are resistance genes for mycine, puromycin and tetracycline.

According to a specific embodiment of the present invention, the configuration of the expression vector containing the nerve growth factor (NGF) of the present invention is disclosed in panel A of FIG.

Methods for transfection of the vector of the present invention into a stem cell can be carried out by a known transfection method, for example, microinjection (Capecchi, MR, Cell 22, 479 (1980)), calcium phosphate precipitation EMBO J. 1, 841 (1982)), liposome-mediated transfection (Wong, TK et al., EMBO J., (Gopal, Mol. Cell Biol. 5, 1188-1190 (1985)) and the gene bend buddhite (Yang et al., Proc. Natl. Acad. Sci. USA 87, 9568-9572 (1990)), but are not limited thereto.

The step of selecting the transformed stem cells by the introduction of the vector can be easily carried out by using a phenotype expressed by the above-described selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the stem cells described above; And (b) provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising a pharmaceutically acceptable carrier.

Stem cells transformed to express the nerve growth factor gene of the present invention can be used for the treatment of neurodegenerative diseases.

Preferably the neurodegenerative disease is a neurodegenerative disease, more preferably Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, dementia, senile dementia, subcortical dementia, atherosclerotic dementia, ischemic brain injury, or trauma. Brain damage.

As demonstrated in one specific embodiment of the present invention, stem cells expressing the nerve growth factor (NGF) of the present invention is hydrogen peroxide (H ₂ O ₂ ), amyloid beta (Aβ _1-42 ) or in vitro culture Not only does it protect neurons from cytotoxic substances such as ibothenic acid, but when implanted directly into the brain of a cognitive impaired animal model established by damaging brain neurons, it impairs the learning and memory of animals. Neuronal damage therapeutic activity is restored to levels similar to

The pharmaceutical compositions of the present invention may be prepared in the form of injections, typically in the form of a suspension comprising cells. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions which are ready for the preparation of solution or dispersion. In all cases, the pharmaceutical agent in the form of an injectable solution should be sterilized, and it is preferable that the agent is fluid enough to facilitate injection.

The pharmaceutical composition of the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means that it does not cause an allergic reaction or a similar adverse reaction when administered to a human. Such carriers include certain solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and preparations in pharmaceutically active materials is well known in the art. The carrier of the pharmaceutical composition may be, for example, a solvent or dispersion medium containing water, saline, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils . Fluidity can be maintained by the use of a coating agent such as lecithin. To prevent microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be included, and may include an appearance system such as sugar or sodium chloride. In addition, agents for delaying absorption in the composition for prolonging the absorption action upon administration in vivo, for example, aluminum monostearate and gelatin may be included. Sterile injectable solutions are prepared by mixing the required amount of the active compound in a suitable solvent with the various other ingredients mentioned above, as required, followed by filtered sterilization.

The pharmaceutical composition of the present invention may preferably be administered by parenteral, intraperitoneal, intradermal, intramuscular, intravenous routes, and more preferably by direct infusion into the brain region, which is the lesion site.

The pharmaceutical compositions of this invention are administered in therapeutically effective amounts in a manner compatible with the formulation. The dosage can also be adjusted according to the condition or condition of the subject to be treated. For parenteral administration with aqueous injection solutions, the solution should be suitably buffered as needed, first making the liquid diluent isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intradermal and intraperitoneal administration. In this regard, the contents of carriers, formulations, and media that can be used in the present compositions are well known in the art (see Remington's Pharmaceutical Sciences & quot ;, 1995, 15th Edition).

The present invention relates to a stem cell expressing a nerve growth factor by introducing a nerve growth factor (NGF) and a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising the stem cell as an active ingredient. Stem cells expressing nerve growth factors of the present invention not only have activity to protect neurons from cytotoxic substances, but also when transplanted directly into the brain of cognitive impaired animal models that damage brain neurons, It exhibits neuronal cell damage therapeutic activity that restores memory impairment to levels similar to normal animals.

1 shows the results of analyzing the characteristics of human neural stem cell (NSC) strain. Panel A shows the structure of the retroviral vector (pLPCX.NGF) encoding the NGF used for the construction of the F3.NGF human NSC cell line. Panels B and C show the results of phase contrast microscopy on the F3 and F3.NGF human NSC cell lines. Panel D shows the results confirmed by RT-PCR gene expression of cell type specific markers in F3 cells and F3.NGF NSC cells. Both F3 cells and F3.NGF cells are nestin (neural stem cell markers), NF-L and NF-H (neuron markers), GFAP (astrocytic markers), MBP (dinal precursor cell markers) and humans Cell type specific markers of NGF were shown. Scale bars represent 50 μm.
Figure 2 shows the results of detection of immunoreactive human NGF and quantitative analysis of NGF levels in vitro. Panel A shows the results of ELISA analysis for human NGF in the culture medium of F3 cells and F3.NGF human NSC cells. NGF secretion of F3.NGF cells was increased by 10-fold compared to the parental F3 cells (*: p <0.05). Panels B and C show results confirming NGF immunoreactivity in F3 cells and F3.NGF cells by immunohistochemistry. All F3.NGF cells showed an NGF-positive response, but only some of the F3 cells expressed NGF. Scale bars represent 50 μm.
Figure 3 shows the results of measuring the cell viability of F3 cells and F3.NGF human NSC cells after treatment with H ₂ O ₂ , Aβ or ibothenic acid. After 12 hours of treatment with the cytotoxic H ₂ O ₂ , Aβ or Ibothenic acid, the cell viability of F3.NGF cells was much higher than that of parental F3 cells.
4 is 100 μM H ₂ O ₂ (Panel A and Panel B), 5 μM Aβ (Panel C and Panel D) or 25 μg / mL Ibothenic Acid (Panel E and Panel F) for 12 hours followed by phase contrast microscopy of F3 cells and F3.NGF human NSC cells. Show the picture you observed. F3.NGF cells had better survival than parental F3 cells. Bars represent 50 μm.
FIG. 5 shows the results of Western blot analysis of protein levels of caspase-3 and phopho-Akt1 enzymes in F3 and F3.NGF human NSCs after treatment with H ₂ O ₂ , Aβ or ibothenic acid.
FIG. 6 shows results of improved learning ability / memory ability after transplanting F3 cells or F3.NGF human NSC cells to mice with damaged learning ability through a Morris water maze test. In the underwater maze experiment, the F3.NGF-transplanted group showed a similar level of learning / memory ability to the same level as the normal control group. Improved capacity lasted up to 4 weeks after implantation (* p <0.05). ●: control group, ○: Ibothenic acid + PBS, ▼: Ibothenic acid + F3, Δ: Ibothenic acid + F3.NGF.
Figure 7 shows the distribution of hNuMA (human nuclear matrix antigen) immunostained positive F3.NGF NSC at 4 weeks after cell transplantation into the mouse brain injected with ibothenic acid. Panel A is a schematic showing the location of hNuMA-positive F3.NGF cells. Panels B and C show the location of hNuMA-positive F3.NFG cells in the Ibothenic acid-injected mouse brain shown in Panel A. Panel DF shows F3.NGF cells producing NGF protein in mouse brain. Panel GI was observed by the NF response in F3. NGF cells show differentiation into neurons. Panel JL was observed by the GFAP response, F3. NGF cells show differentiation into astrocytes. The scale bar represents 50 μm.
8 shows that NGF protein is overexpressed in ADSC.NGF cells using anti-NGF monoclonal antibody.
9 shows that NGF mRNA and protein are overexpressed in ADSC.NGF cells in greater amounts compared to ADSC cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One: NGF  Cell therapy using gene-derived neural stem cells

Materials and Methods

1. Preparation of F3 human neural stem cell line

As reported in the prior art, a stable clonal human NSC cell line, HB1.F3 (F3) was prepared by introducing an avian v-myc cell cycle control gene into a primary cultured human fetal neural stem cell (NSC) using a retroviral vector. (17, 23). F3 human NSCs expressed phenotypes specific for NSCs such as ABCG2, Musashi1 and nestin. F3 NSC is a DMEM containing 10 μg / mL insulin, 10 μg / mL transferrin, 30 nM sodium selenate, 50 nM hydrocortisone, 0.3 nM triiodothyronine and 20 μg / mL gentamicin and containing high concentrations of glucose Cultured using the constructed serum free medium (DM4) (19). Recombinant human bFGF (10 ng / mL; PeproTech, Rocky Hill, NJ) DM4 was usually supplemented during medium exchange. All compounds except bFGF used Sigma (St Louis, MO).

2. Introduction of Human NGF Gene into F3 NSC

PG13 mouse packaging cell lines were transfected with pLPCX.NGF vector (FIG. 1A) using LipofectAMINE (Invitrogen, Carlsbad, Calif.) And stable PG13 cells were selected for 3 days using 5 μg / ml furomycin. A nonreplicating retroviral vector recovered from PG13.NGF cells was obtained and used to transfect F3 NSC. Puromycin-resistant clones were isolated, screened, amplified and used for transplantation. Expression of NGF in the F3.NGF cell line was analyzed by RT-PCR, ELISA, and immunofluorescence microscopy.

3. RT-PCR Measurement Method

Total RNA was extracted from F3 and F3.NGF NSC cultures using TRIzol (BRL, Gaithersburg, MD). CDNA templates were synthesized by PCR 30 cycles using 400 U Moloney Murine Leukemia virus (MMLV) reverse transcriptase (Promega, Madison, Wis.) On oligoT primers and 1 mg total RNA in each sample. The RT-PCR product was then separated by electrophoresis on 1.2% agar gel containing ethidium bromide and observed under UV light. NGF, nestin, neurofilament low-molecular-weight protein (NF-L), neurofilament high-molecularweight protein (NF-H), glial fibrillary acidic protein (GFAP), human myelin basic protein (MBP) and glycerin Primers for RT-PCR for aldehyde-3-phosphate dehydrogenase (glyceraldehyde 3-phosphate dehydrogenase) are summarized in Table 1.

4. Immunohistochemistry

F3. NGF Immunohistochemistry was performed to confirm NGF protein expression in human NSCs. F3.NGF cells were plated on poly-L-lysine-coated Aclar plastic cover slip (9 mm in diameter) and incubated in DM4 serum-free medium with bFGF for 3-5 days, then washed in PBS, Fixed in cold acidic alcohol (5% glacial acetic acid in 95% ethanol) at −20 ° C. for 10 minutes. The immobilized cultured cells were incubated for 30 minutes at room temperature in a blocking solution composed of PBS containing 10% normal goat serum, followed by primary antibodies specific for human NGF (1: 200, mouse monoclonal antibody, Chemicon, Temecula, CA), incubated for 1 hour at room temperature, incubated with Alexa Fluor 594-anti-mouse, rabbit IgG (Molecular Probes, Eugene, OR) for 1 hour at room temperature, washed in PBS and mounted on slides with Gelvatol. .

5. Exposure of Cytotoxic Substances to NSC Cells

F3 and F3.NGF cells were plated in 96 well-plates (Falcon; Becton Dickinson, Franklin lakes, NJ) with DMEM containing 5% FBS at a concentration of 1 × 10 ⁴ cells per well, followed by various concentrations of H ₂ O ₂ (0-500 μM, Sigma-Aldrich, St Louis, MO), amyloid beta _1-42 (Aβ _1-42 , 0-50 μM, BioSource, Camarillo, Calif.) And ibotenic acid (0 Incubated for 12 hours in a medium containing 100 μg / mL, Sigma-Aldrich). Untreated controls were cultured in media containing only normal media components. The cells were then subjected to viability assay and western blotting assay. Cell viability was assessed using CCK-8 kit (Dojindo Laboratries, Kumamoto, Japan) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a micro plate reader. F3 and F3.NGF cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH8.0) and centrifuged for 5 minutes at 14,000 rpm. . The supernatant was recovered and 20-30 μg of protein was subjected to SDS-PAGE, and the gel protein was electrically transferred onto the Immobilon-P membrane, followed by caspase 3 (1: 500, Chemicon, Temecula, CA) phsopho-Akt (1 Western blotting analysis was performed using primary antibodies against: 500, Cell Signaling Technology, Danvers, Mass.) And Akt1 (1: 500, Chemicon). Finally, immunological detection was performed using secondary antibodies (Amersham, Chicago, IL) and chemiluminescence kit (Amersham).

6. Mouse Cognitive Impairment Model

A mouse cognitive dysfunction model (CDM) was prepared by administering ibotenic acid into the corpus striatum of the brain by stereotactic methods according to the methods described in the prior art (41). After intraperitoneal injection of 1% ketamine (30 mg / kg) and xylazine hydrochloride (4 mg / kg), ICR mice (male, 25-30 g, n = 32) were subjected to stereotactic positioning (Kopf Instruments, Tujunga, CA). ). A burr hole is made, and a 30-gauge needle is inserted through a burhol into a freshly prepared ibothenic acid solution (1 μL saline containing 0.5 μg of ibothenic acid, Sigma), where the junction of sagittal and coronal sutures is bregma. Were injected over 5 minutes into the left and right CA3 hippocampal sites on anterior ± 1.7 mm, positioning ± 2.1 mm, and abdominal-2.5 mm coordinates. The needle was removed after another 5 minutes.

7. Brain Transplantation

F3 and F3.NGF NSCs cultured in culture flasks were isolated into single cells by simple trypsin treatment, suspended in PBS at a concentration of 4 × 10 ⁷ cells / 0.1 ml and stored until use for transplantation. One week after CDM surgery, F3. 2 μL of PBS containing 2 × 10 ⁵ cells (n = 9) or F3.NGF cells (n = 9) were injected. In the simulated control group, 2 μL of PBS without NSCs were injected into CDM mice (n = 7). PBS, including PBS or NSC, was slowly injected into the cerebral cortex (anterior ± 1.7 mm, location ± 2.1 mm, and abdominal-2.0 mm coordinates) from the junction of the hippocampus and coronary sutures. The needle was removed after 5 additional minutes. No immunosuppressant was used.

8. Learning and memory test

To evaluate learning ability and memory, mice were subjected to a Morris water maze test. The underwater labyrinth experiment was conducted in a 27 cm deep 180 cm diameter tank filled with water with a water temperature maintained at 22 ± 2 ° C. The tank was divided into four quadrants, with a hidden escape platform (10 cm in diameter and 25 cm in height) submerged 2 cm below the water in the center of one quadrant. Mice were trained to find hidden escape platforms based on several signals outside the maze, and did not change the location of the signal during the experiment. Mice were trained three times, five minutes each, for four consecutive days, followed by a fifth training one week later. One week later mice were administered ivostenic acid into the striatum of the brain by stereotactic methods to prepare a cognitive impairment model (CDM). Each mouse was given up to 90 seconds to find the hidden platform and 30 seconds more to stay on the platform. The average time taken to escape through the platform during the training, that is, the average escape time, was calculated and compared with the time measured in the experiment. If the mice did not escape from the tank in 90 seconds, they were guided to the platform and allowed another 30 seconds to stay on the platform.

9. Immunohistochemistry in Brain Sections

After conducting a behavioral test, each animal was anesthetized and 4% paraformaldehyde in 0.1 M phosphate buffer was perfused through the heart, and the brain was post-fixed for 24 hours in the same fixative, followed by 24 in 30% sucrose. After a cold-free process for a time, a coronal section of the brain was prepared on a cryostat. Continuous brain slices of 30 μm thickness were made to pass through the anterior 1.0 mm and posterior 1.0 mm positions with respect to the needle insertion position and the plane visible from the brain surface. Six or seven sections were made in this manner. For the prepared brain sections, human NGF (1: 200, mouse monoclonal antibody, Chemicon, Temecula, CA), GFAP (1: 1000, rabbit polyclonal antibody, Chemicon) or NF (1: 200, rabbit poly Double immunostaining of human NGF markers and cell type-specific markers was performed using antibodies specific for the clonal antibody, Chemicon). To confirm that transplanted F3 and F3.NGF cells continue to proliferate in vivo, antibodies to hNuMA (human nuclear matrix antigen) (1: 200, mouse monoclonal antibodies, Abcam, Cambridge, MA) for brain sections ) Double immunofluorescence staining was performed. Brain sections were incubated with primary antibodies overnight at 4 ° C. with free suspension and then incubated with Alexa Fluor 594-anti-mouse, rabbit IgG (Molecular Probes, Eugene, OR) for 1 hour at room temperature. Negative control sections were prepared in the same manner except that the primary antibody was omitted. Stained sections were observed under an Olympus laser confocal fluorescence microscope.

10. Statistical Analysis

Stem cell transplantation was analyzed using two-way ANOVA and post-hoc Tukey test. Data are expressed as mean ± SE.

Experiment result

1. Human NGF Stable human nerve expressing Stem cell line  Establish

F3 human NSCs were infected with a retroviral vector encoding human NGF gene (Panel A of FIG. 1), followed by selection and amplification of clones resistant to puromycin. One of the clones was selected and used for the experiment. The morphology of the selected F3.NGF cells had a bipolar or multipolar morphology and did not differ from the parental F3 NSC cells (Panel B, and Panel C in FIG. 1). RT-PCR analysis of mRNA isolated from F3 and F3.NGF cells is shown in panel D of FIG. Nestin (NSC specific marker), NF-L and NF-H (neurofilament triplet proteins) (neuronal cell specific marker), glial fibrillary acidic protein (GFAP) (astrocytic specific marker) and transcripts of NGF were F3 and F3. All expressed well in NGF cells. However, transcript expression for cell specific markers for MBP, structural proteins and oligodendrocytes was not detected. In addition, transcript expression levels of the human NGF gene were much higher in F3.NGF cells compared to maternal F3 cells. The result of measuring the expression level of NGF in the culture medium of the F3 cell line and the F3.NGF cell line is shown in Panel A of FIG. ELISA analysis revealed that the amount of human NGF secreted in the culture medium of F3.NGF NSC cells was 10-fold higher than that of control F3 maternal NSC cells [1203.0 ± 107.3 ng / 10 ⁶ cells / day vs 120.9 ± 36.3 ng / 10 ⁶ cells / day (mean ± SEM, p <0.001)]. While all F3.NGF cells showed an NGF-positive immune response, only a few of the mother F3 cells expressed NGF (Panel B, and Panel C of FIG. 2).

2. NGF Expressing NSC  In the cell On cytotoxic substances  Cytoprotective effect

Cytoprotective effects of NGF against cell death induced by these cytotoxic substances by exposure to various concentrations of toxins, H ₂ O ₂ , Aβ _1-42 or ibothenic acid in culture with F3 cells and F3.NGF NSC cells The effect was measured. F3.NGF cells showed much higher cell viability against cell death induced by H ₂ O ₂ , Aβ _1-42 or ibothenic acid as compared to F3 control cells (see FIGS. 3 and 4).

The survival rate of F3 parental cells exposed to 100 μM of H ₂ O ₂ for 12 hours was 15%, whereas the survival rate of F3.NGF cells treated under the same conditions was 85%. Similarly, the survival rate of F3 cells treated with 5 μM Aβ _1-42 was 40%, whereas the survival rate of F3.NGF cells treated with the same conditions was 85%.

The cell viability of F3 cells exposed to 25 μg / mL of ibothenic acid for 12 hours was 47%, while the viability of F3.NGF cells treated under the same conditions increased to 70% (see FIG. 3).

We investigated whether the cytotoxic H ₂ O ₂ , Aβ _1-42 or Ibothenic acid induced changes in Caspase-3 cleavage and Akt1 phosphorylation in F3 cells and F3.NGF cells. When F3 cells and F3.NGF cells were exposed to 100 μM H ₂ O ₂ , 5 μM Aβ _1-42 and 25 μg / mL ibothenic acid for 12 hours, active fragments of caspase 3 were expressed in F3 cells (20 kDa). ) Increased, whereas the increase in the active fragment of caspase 3 was decreased in F3.NGF cells (FIG. 5). After treatment with these cytotoxic substances, the phosphorylated form of Akt1 in F3.NGF cells increased, but F3 cells showed the opposite expression pattern (see FIG. 5). Since Akt1 is the most important mediator of growth factor-induced neuronal survival, it is thought to be the cause of high survival rate in F3.NGF cells after phosphorylated form of Akt1 is exposed to cytotoxic substances. These results suggest that the increase in Akt1 phosphorylated morphology and cell viability is due to the production and secretion of NGF in F3.NGF cells.

3. F3 NSC After transplantation CDM  Learning / memory measurement in mouse

Learning ability / memory of CDM mice infused with PBS, F3 or F3.NGF NSC was measured by a Morris water-maze (FIG. 6). CDM mice injected with F3.NGF cells showed improved learning ability / memory in water maze experiments compared to mice injected with PBS or F3 cells. ) Up to (Figure 6). Underwater maze testing for 1-4 weeks after transplantation revealed significant differences between the F3.NGF and F3 cell populations (P <0.05).

4. Transplanted F3. NGF NSC Were differentiated into neurons and astrocytes

After implantation into the cortical site covering the hippocampal lesions of mice, hNuMA + human NSCs were selectively migrated to the hippocampal site and located at the border of the lesion, located farther than the injected site (FIG. 7 panel AC). . A large number of transplanted hNuMA + F3.NGF cells differentiated into neurofilament + (panel GI of FIG. 7; 35-45%) neurons at CA1, CA3 hippocampal sites. Only a small number of transplanted hNuMA + F3.NGF cells were GFAP + astrocytes (Panel JL in FIG. 7; 3-5%), and hNuMA + / GFAP + dual-positive cells were found along the border of CA3. These results suggest that most of the transplanted F3.NGF NSCs differentiate into either neurons or astrocytes in response to signals from the local microenvironment provided by the receptor hippocampal lesion site.

Example  2: NGF  Cell therapy using gene-induced fat stem cells

Materials and Methods

1. Human Fat stem cell line  Produce

Adipose tissue was obtained by liposuction from a healthy adult patient. Adipose tissue obtained by liposuction was washed with Hank's Balanced Salt Solution (HBSS, Gibco, USA) with 2% bovine serum albumin (BSA, Gibco, USA) and crushed into small pieces. The ground tissue was treated with 0.1% collagenase type I (Sigma-Aldrich, USA) for 30 minutes at 37 ° C., followed by 10% FBS (fetal bovine serum), 0.2% Fungizone, 1% penicillin and streptomycin (Gibco, The enzyme reaction was stopped with Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) containing USA, and passed through a 100 μm nylon filter paper (BD bioscience, USA) to remove unnecessary tissue. The cell layer suspended in the upper layer was separated by centrifugation at 1000 rpm for 10 minutes. Erythrocytes and debris were removed from the separated cell layers and cultured in a culture dish. Culture was performed using DMEM: F12 with bFGF (10 ng / ml), 50 mM streptomycin, and 50 U / mL penicillin. Subculture was subcultured at a ratio of 1: 3 when the culture dish was 80% or more.

2. Human Into fat stem cells  human NGF  Introduction of gene

Human adipose stem cells were transfected with pLPCX.NGF vector (FIG. 1A) using LipofectAMINE (Invitrogen, Carlsbad, Calif.), And stable human adipose stem cells were selected for 7 days using 1 μg / ml puromycin. Puromycin-resistant human adipose stem cells were isolated, screened, amplified and used. Expression of NGF in human adipose stem cells was analyzed by RT-PCR, ELISA, and immunofluorescence microscopy.

Nerve growth factor introduced into the pLPCX.NGF vector (FIG. 1A) was transfected into ADSC cells using Lipofectamine 2000 (invitrogen) and replaced with a culture medium without antibiotics the day before transfection. In the presence of about 90-95% of the cells in the culture dish, transfection was performed. In order to transfect with cells of a 60 mm culture dish, 7 μg of DNA and 60 μl of Lipofectamine 2000 were diluted in 500 μl of the culture medium, and then allowed to stand at room temperature for 5 minutes. Thereafter, the diluted DNA and Lipofectamine 2000 were mixed and allowed to stand for 20 minutes, and then added to the culture solution, and the cells were cultured for 24 hours. Stable ADSC cells were cultured for 3 days and selected for 6 days using 1 μg / ml puromycin to culture only puromycin-resistant ADSC.NGF cells. After isolation, amplification and extraction of RNA and protein from puromycin-resistant ADSC.NGF, NGF expression was analyzed by RT-PCR, ELISA, and immunofluorescence microscopy.

3. RT - PCR  How to measure

Total RNA was extracted from human adipose stem cells using TRIzol (BRL, Gaithersburg, MD). CDNA templates were synthesized by PCR 30 cycles using 400 U MMLV (Moloney Murine Leukemia virus) reverse transcriptase (Promega, Madison, Wis.) On oligo T primer and 1 mg total RNA in each sample. The RT-PCR product was then separated by electrophoresis on 1.2% agar gel containing ethidium bromide, and the expression of NGF, glyceraldehyde-3-phosphate dehydrogenase was observed under UV light. Primers for RT-PCR for glyceraldehyde-3-phosphate dehydrogenase are listed in Table 1.

4. Immunohistochemistry and ELISA

Immunohistochemistry and ELISA were performed to confirm NGF protein expression in ADSC.NGF. ADSC.NGF was plated on poly-L-lysine-coated Aclar plastic cover slip (9 mm in diameter) and in DMEM: F12 serum medium (10%, Caisson Labs, North Logan, UT) to which FGF-2 was added After incubation for 3 days, the cells were washed in PBS and fixed in cold acidic alcohol (5% glacial acetic acid in 95% ethanol) at −20 ° C. for 10 minutes. The immobilized cultured cells were incubated for 30 minutes at room temperature in a blocking solution composed of PBS containing 10% normal goat serum, followed by primary antibodies specific for human NGF (1: 200, mouse monoclonal antibody, Chemicon, Incubate with Temecula, CA) for 1 hour at room temperature, incubate with Alexa Fluor 594-anti rabbit IgG (Molecular Probes, Eugene, OR) for 1 hour at room temperature, wash in PBS, slide onto Gelvatol and fluoresce The presence or absence of expression was observed under a microscope.

To quantify the neuronal growth factors secreted from ADSC.NGF, 1 x 10 ⁶ hADSCs were incubated in 3% FBS + DMEM, a conditioned medium of ADSC and ADSC. Filtration was performed for quantitative analysis according to the protocol in the NGF ELISA Kit (R & D systems, Minneaplis, MN). Detected nerve growth factor concentration was determined by calculating the absorbance (450 nm) measured by Microplate Absorbance Reader (Biotek Instruments, Winooski, VT).

Experiment result

1. Human NGF  In human fat stem cells NGF  Expression of protein

In order to confirm whether the selected NGF-induced NGF overexpressing adipose stem cells (ADSC.NGF) express NGF, immunohistochemistry was performed on cells using anti-NGF monoclonal antibodies. As can be seen in Figure 8, it was confirmed that the NGF protein is overexpressed in ADSC.NGF cells.

2. Human NGF  In human fat stem cells NGF mRNA  Expression and Protein Secretion Measurement

RT-PCR and ELISA detection was performed to confirm the secretion of NGF mRNA and protein in selected NGF-introduced NGF overexpressing adipocytes (ADSC.NGF). As can be seen in Figure 9, it was confirmed that NGF mRNA and protein are overexpressed in ADSC.NGF cells in a greater amount compared to ADSC cells. In other words, mRNA expression was increased in ADSC.NGF cells rather than ADSC, and ADSC cells were 302.2 pg / ml in protein secretion results, whereas ADSC.NGF cells incorporating NGF genes secreted 1003.6 pg / ml in ADSC.NGF cells. It was found to secrete 3.3 times more NGF.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (9)

Stem cells that are transformed with an expression vector containing a nucleic acid molecule encoding a nerve growth factor (NGF) to express a nerve growth factor (NGF).
The stem cell of claim 1, wherein the stem cell is a primary cultured stem cell or a genetically modified immortalized stem cell thereof.
According to claim 1, wherein the stem cells are stem cells, characterized in that the neural stem cells or fat stem cells.
The group of claim 3, wherein the neural stem cells are composed of nestin (nestin), neurofilament low-molecular-weight protein (NF-L), neurofilament high-molecularweight protein (NF-H), and glial fibrillary acidic protein (GFAP). Stem cells, characterized in that for expressing at least one neural stem cell marker protein selected from.
The method of claim 1, wherein the expression vector is an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, an SV 40 vector, a polyoma virus vector, a papilloma virus vector, a picarnovirus vector, a Bexi. Stem cells, characterized in that the nia virus vector or a helper dependent adenovirus vector.
The stem cell according to claim 1, wherein the nerve growth factor (NGF) comprises an amino acid sequence of SEQ ID NO: 1 sequence.
The stem cell of claim 5, wherein the expression vector has a vector structure disclosed in panel A of FIG. 1.
(a) a pharmaceutically effective amount of the stem cells of any one of claims 1-7; And (b) a pharmaceutical composition for preventing or treating neurodegenerative diseases, including a pharmaceutically acceptable carrier.
9. The neurodegenerative disease of claim 8, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, dementia, senile dementia, subcortical dementia, arteriosclerosis, ischemic brain injury, or brain injury due to trauma. Pharmaceutical composition.

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