KR100809410B1 - Compositions for Inducing the Differentiation of Stem Cells and Uses thereof - Google Patents

Abstract

The present invention relates to stem cell differentiation, comprising a composition for inducing stem cell differentiation comprising N-acetylcysteine as an active ingredient, a method for inducing stem cell differentiation using the same, and treating neurological diseases including differentiated neurons produced by the same. It relates to a pharmaceutical composition for. According to the present invention, differentiated cells (preferably, neurons, more preferably dopaminergic neurons) can be obtained from stem cells. Neurons produced by the present invention are very effective in treating various neurological diseases caused by nerve injury.

Stem Cells, Differentiation, N-acetylcysteine, Neurons, Dopaminergic Neurons

Description

Compositions for Inducing Stem Cell Differentiation and Uses thereof {Compositions for Inducing the Differentiation of Stem Cells and Uses}

1A is a photograph showing the effect of N-acetylcysteine (NAC) on the formation of an embryonic body (EB) from P19 EC (embryonal carcinoma) cells. Panel A is a picture of retinoic acid (RA) treated, EB formed for 3 days and observed under a phase contrast microscope. Panel B was treated with RA and NAC and formed EB for 3 days. Panel C was treated with α-estradiol and RA and confirmed the formation of EB. For NAC or α-estradiol treatment RA was pretreated for 1 hour. As a result of simultaneous treatment with NAC and RA, more and larger EBs are formed than the results of RA alone.

1B is a graph analyzing the number of EBs formed according to NAC or NAC + RA based on diameter. When NAC and RA were processed, it was confirmed that the number of larger EBs was larger than when RA was only processed.

2A is a photograph showing MAP-2-positive cells differentiated from EB. P19 EC cells were differentiated into neurons, followed by immunocytochemical staining using MAP-2 antibody. Panel A was treated with RA only, Panel B was treated with RA and α-estradiol, Panel C was treated with RA + NAC, and Panel D was treated with RA + NAC + BSO. When RA + NAC treatment, it can be seen that a lot of neuronal differentiation proceeds, whereas the antioxidant α- estradiol did not show an effect. In addition, it was confirmed that the treatment of BSO, a GSH formation inhibitor, suppressed the effect of promoting NAC differentiation by NAC.

FIG. 2B is a graph quantifying the results of FIG. 2A.

3 is a graph showing the change in the amount of intracellular GSH (reduced form of glutathione) according to RA + NAC treatment. When GSH and RA were treated together, it was confirmed that there was no significant difference in the amount of intracellular GSH compared with the treatment with RA alone. However, when treated with RA + NAC, it was confirmed that the intracellular GSH improves significantly after 24 hours, and that the level is maintained for 3 days when EB is formed. When treated with BSO, no increase in intracellular levels of GSH by NAC was found.

4A is a photograph of Western blotting results showing changes in N-cadherin expression and PI3-kinase activity in differentiation of P19 EC cells by RA + NAC treatment. Western blotting confirmed that N-cadherin, an adhesion protein associated with the formation of EB, increased its expression level when treated with RA + NAC. And to determine the degree of PI3-kinase activation associated with neuronal differentiation, we identified the amount of phosphorylated form of Akt downstream of it. When RA + NAC was treated, it was confirmed that the amount of phosphorylated Akt was increased at a faster time than when RA alone was treated, indicating that RA + NAC activates PI3-kinase, a neuronal differentiation regulator.

4B is a graph analyzing the effect of LY294002, an inhibitor of PI3-kinase, on embryogenic development and neural differentiation. The number of EBs was counted in 100 mm Petri dishes. EBs were plated on glass coverslips, differentiated and stained with MAP-2 antibody. In the graph, positive cells represent cells stained with MAP-2 antibody and LY represents LY294002, an inhibitor of PI3-kinase.

FIG. 5A is a photograph showing P19 EC cell-derived GAD (GABA)-and TH (dopamine) -forgikib cells, and GFAP (glia) -positive cells by RA + NAC treatment. Differentiated P19 cells were dual stained with TH- (green) and MAP-2 (red) -antibodies. TH ⁺ / MAP-2 ⁺ cells are yellow in the integrated image of TH and MAP-2 immunostaining.

FIG. 5B is a graph quantifying the results for GFAP (glial cells) -positive cells in FIG. 5A. The ratio of MAP-2 positive and GFAP-positive cells in total cells is shown.

5C is a graph quantifying the results for TH- and GAD-positive cells in the FIG. 5A results. The ratio of TH-positive cells and GAD-positive cells in total cells is shown.

6 is a photograph showing the viability of the transplanted cells (Panel A) and the differentiation efficiency into TH-positive cells (Panel B) after transplanting cells obtained from the isolation of P19 cell-derived EB into Parkinson's disease mouse model. Cell transplantation was performed 2 weeks after injection of 6-OHDA. The viability of P19 EC cells was confirmed by staining with anti-estin (surviving neuronal marker) antibodies, and images were obtained by fluorescence microscopy. At 4 weeks of cell transplantation, brain sections of Parkinson's animal models were double stained with TH- (red) / MAP-2 (green) antibodies. TH ⁺ / MAP-2 ⁺ cells appear yellow in the integrated image.

7 is a graph showing amphetamine-induced rotational response over time. Rotational response to amphetamine was examined at 2, 4 and 6 weeks after transplantation. Data is expressed as change in rotational score relative to pre-graft value. Large changes in rotational scores were observed in mice implanted with RA + NAC-treated P19 cells.

The present invention relates to stem cell differentiation, and more particularly, to a composition for inducing stem cell differentiation, a method for inducing stem cell differentiation, and a pharmaceutical composition for treating neurological diseases including stem cells.

Stem cells are undifferentiated cells of the stage before they are differentiated into each cell constituting the tissue, and differentiation proceeds to specific cells by specific differentiation stimulus (environment). Stem cells, unlike differentiated cells that have ceased cell division, are capable of self-renewal by cell division, which proliferates and expands. It is characterized by having plasticity in differentiation because it can be differentiated into other cells by different environment or differentiation stimulus.

Currently, stem cells are in the spotlight as cell therapy, and many studies have been made as cell therapy of various neurological diseases caused by nerve cell damage. In particular, cerebral nervous system disease is considered to be the most suitable target for cell transplantation than any other disease. Unlike other tissues, cerebral nervous system has almost no immune rejection reaction, so long-term survival of transplanted cells when transplanted from outside Because you can expect. Attempts have been made to apply stem cells to the treatment of diseases such as stroke, Alzheimer's disease, Parkinson's disease, demyelinating disease and spinal cord injury (Isacon O, Deacon T, Trends. Neurosci) . ., 10: 477-482 (1997) ; Studer et al, Nat Neurosci, 1:... 290-295 (1998)).

On the other hand, in order to increase the usefulness of stem cells as cell therapeutics, a technique for efficiently differentiating stem cells into specific cells is required.

WO 2005/003320 discloses a method for inducing differentiation of stem cells into neurons, and more specifically, (a) culturing stem cells with basic fibroblast growth factor; (b) culturing the cells of step (a) with fibroblast growth factor 8 and Sonic Hedgehog; (c) culturing the cells of step (b) with brain-derived neurotrophic factors; And (d) co-culturing the cells of step (c) with glial astrocytic cells. WO 2004/093812 discloses that compounds with specific chemical formulas can act as inducers for the differentiation of embryonic stem cells into neurons.

WO 2004/05308 discloses a method for producing dopaminergic neurons by interfering with TGF-β signaling in stem cells.

However, to date, no efficient technology for differentiating stem cells into specific cells (especially neurons) has been developed.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors screened various substances for efficient differentiation of stem cells, and found that N-acetylcysteine is the most effective substance for differentiating stem cells, and injecting stem cells differentiated by N-acetylcysteine into animals. In this case, the present invention has been completed by confirming that a desired therapeutic effect appears.

Accordingly, it is an object of the present invention to provide a novel stem cell differentiation inducing composition.

Another object of the present invention to provide a method for inducing stem cell differentiation.

Another object of the present invention to provide a pharmaceutical composition for the treatment of neurological diseases, including stem cells.

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

According to one aspect of the invention, the present invention provides a composition for inducing stem cell differentiation comprising N-acetylcysteine as an active ingredient.

According to another aspect of the present invention, the present invention provides a method for inducing stem cell differentiation comprising culturing stem cells in a composition for inducing stem cell differentiation comprising N-acetylcysteine as an active ingredient.

The present inventors screened various substances for efficient differentiation of stem cells, and found that N-acetylcysteine is the most effective substance for differentiating stem cells, and injecting stem cells differentiated by N-acetylcysteine into animals. In addition, it was confirmed that the desired therapeutic effect appeared.

As used herein, the term “induced stem cell differentiation” refers to an embryonic body formed at an intermediate stage prior to complete differentiation from stem cells to specific cells, as well as when completely differentiated from stem cells to specific cells. It also includes formation. That is, the composition for inducing stem cell differentiation of the present invention not only effectively achieves complete differentiation of stem cells into specific cells, but also shows very great efficiency in forming embryonic-like structures in stem cells.

N-acetylcysteine used as a stem cell differentiation inducing agent in the present invention may exist in various stereoisomers, most preferably N-acetyl-L-cysteine.

According to a preferred embodiment of the present invention, the composition of the present invention further comprises other differentiation inducing agents rather than including N-acetylcysteine alone as a stem cell differentiation inducing agent, a synergic effect between these differentiation inducing agents To be generated. Differentiation inducing agent that may be additionally included in the composition of the present invention may be any known as the differentiation inducing agent of stem cells, preferably retinoic acid, ascorbic acid, melatonine, cytokine ( cytokine) or various growth factors (eg, GLDF (glial cell line-derived neurotrophic factor), EGF (epidermal growth factor), NGF (nerve growth factor)), and most preferably additionally include retinoic acid. According to the prior art, retinoic acid induces differentiation of stem cells, such as P19 EC (embryonic carcinoma cell) cells, into neurons. However, in the case of differentiation by retinoic acid alone, the differentiation rate is low because only about 20-30% of the finally differentiated P19 EC cells are positive cells for neuronal markers. On the other hand, N-acetylcysteine first adopted in the present invention shows a synergistic stem cell differentiation rate when used with retinoic acid.

Stem cells that can be differentiated by the present invention is not limited, cells having characteristics of stem cells, that is, undifferentiated, infinite proliferation and differentiation into specific cells are cells to which the present invention can be applied. Stem cells include embryonic stem cells, adult stem cells, embryonic germ cells and embryonic tumor cells, preferably embryonic stem cells and adult stem cells.

According to a preferred embodiment of the present invention, the differentiation of stem cells according to the present invention is particularly useful for differentiation into neurons, more preferably for differentiation into dopaminergic neurons.

Stem cell differentiation induction method of the present invention can be largely carried out in three steps: (a) preparing a stem cell; (b) Embryonic structure by culturing stem cells in a medium containing a composition for inducing stem cell differentiation comprising N-acetylcysteine (preferably, N-acetylcysteine and other stem cell differentiation inducer) as an active ingredient body); And (c) culturing the cells obtained by dissociation from the embryo-like structure to obtain differentiated cells.

In step (b), the treatment of N-acetylcysteine and other stem cell differentiation inducing agents (eg, retinoic acid) is not limited in time to each other. For example, N-acetylcysteine can be treated before, simultaneously or after retinoic acid. The culture dish used in this process is a bacterial grade culture dish without a functional group to which cells can be adhered for efficient formation of an embryo-like structure.

The amount of N-acetylcysteine used in the present invention is generally 1-500 μM, preferably 20-300 μM, more preferably 30-250 μM, even more preferably 60-150 μM, most preferably About 100 μΜ. The amount of other stem cell differentiation inducing agents such as retinoic acid used in the present invention is generally 1-500 nM, preferably 20-300 nM, more preferably 30-250 nM, even more preferably 60-150 nM, Most preferably about 100 nM.

Step (c) may be carried out in the absence or presence of N-acetylcysteine and / or other stem cell differentiation inducing agents, preferably in the absence of N-acetylcysteine and / or other stem cell differentiation inducing agents.

On the other hand, in steps (a) and (c), tissue culture dishes, multi-well plates or poly-L-lysine coating-glass coverslips which are commonly used in animal cell culture and capable of attachment of animal cells may be used.

In step (c), dissociation of individual cells from embryonic analogues, which are cell aggregates, can be carried out according to conventional methods known in the art. For example, it can be carried out by mechanical methods and enzymatic methods, and an enzymatic separation method using an enzyme such as trypsin is preferred.

Step (b) is generally carried out for a sufficient time to form an embryo-like structure, preferably 1-10 days, more preferably 1-8 days, even more preferably 2-5 days, Most preferably about 3 days. Step (c) is generally carried out for a sufficient time to form differentiated cells, preferably 2-20 days, more preferably 3-15 days, even more preferably 5-12 days, most Preferably about 9 days.

As the medium used in the present invention, any one conventionally used for culturing stem cells may be used. For example, α-MEM medium containing serum, a buffer, an antioxidant, and the like may be used.

One specific embodiment of the stem cell differentiation induction process according to the present invention, using P19 EC cells as stem cells and a case where the target differentiation cells are neurons as an example: bicarbonate, antibiotic-antifungal agent, bovine Stem cells (eg, PC EC cells) are inoculated in culture dish suspension with α-MEM medium supplemented with serum, fetal bovine serum, pyruvate and tyrosine solution and cultured undifferentiated.

Subsequently, P19 cells are cultured for 3 days in the presence of N-acetylcysteine and retinoic acid as a cell aggregation inducing agent to obtain an embryonic body. The culture dish used in this process is a bacterial grade culture dish without a functional group to which cells can be adhered for the efficient formation of embryo-like structures.

After generation of embryo-like structures for 3 days, the cell aggregates are separated with trypsin and transferred to tissue culture dishes, multiwell plates or poly-L-lysine coating-glass coverslips. P19 cells are then cultured for 9 days in the absence of N-acetylcysteine and retinoic acid to give differentiated neurons.

N-acetylcysteine used in the present invention improves the formation of cell-like structures in the differentiation process of stem cells, and also improves the rate of differentiation into specific cells (preferably neurons, more preferably dopaminergic neurons).

According to another aspect of the invention, the present invention (a) stem cell-like structure formed by culturing stem cells in the above-described composition for inducing stem cell differentiation of the present invention comprising N-acetylcysteine as an active ingredient (embryonic body Pharmaceutically effective amount of differentiated neurons obtained by culturing stem cells in the cells obtained by dissociation or the composition for inducing stem cell differentiation; And (b) provides a pharmaceutical composition for the treatment or prevention of neurological diseases (neuronal disease) comprising a pharmaceutically acceptable carrier.

According to a preferred embodiment of the present invention, the composition for inducing stem cell differentiation is retinoic acid (retinoic acid), ascorbic acid (ascorbic acid), melatonine (melatonine), cytokines or various growth factors [eg, GDNF (glial cell) line-derived neurotrophic factor (EGF), epidermal growth factor (EGF), and NGF (nerve growth factor)], and most preferably further include retinoic acid.

According to a preferred embodiment of the present invention, cells obtained from the stem cell analogues used in the pharmaceutical composition of the present invention have activated PI3-kinase. PI3-kinase is known as a physiological regulator of neuronal differentiation.

According to a preferred embodiment of the invention, the differentiated neurons used in the pharmaceutical compositions of the invention are double stained with anti-MAP-2 antibodies and anti-TH antibodies. That is, differentiated neurons have MAP-2 and TH. MAP-2 and TH are known as markers of mature neurons and markers of dopaminergic neurons, respectively.

Diseases or conditions that can be treated by the pharmaceutical composition of the present invention include all neurological diseases caused by damage to nerve cells. Preferably, the neurological disease is selected from the group consisting of neurodegenerative diseases and diseases caused by ischemia or reperfusion. More preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis, most preferably Parkinson's disease. According to a preferred embodiment of the invention, the disease caused by ischemia or reperfusion is ischemic stroke.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19 ^th ed., 1995).

The pharmaceutical compositions of the present invention can be administered parenterally, and local injection is the most preferred method of administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 2 x 10 ^5-2 x 10 ⁶ cells once, and is usually administered once or twice.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. The formulations may then be in the form of solutions, suspensions or emulsions in oil or aqueous media, and may further comprise dispersants or stabilizers.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Experimental Materials and Methods

P19  Cell Culture and Differentiation

Subclones of P19 EC (embryonal carcinoma) cells (ATCC), P19 mouse embryonic tumor cell lines were used. P19 EC cells are cells that are widely used in the in vitro model system for neuronal development. P19 cells can be easily maintained and expanded in tissue culture in an undifferentiated state. In addition, P19 EC cells are believed to reflect early stages of in vivo neural development and are widely used as in vitro model systems that are well suited for analyzing regulatory mechanisms of mammalian neural development (Masaharu et al., 2002). .

For subcloning, bicarbonate (Sigma, 2.2 mg / L), 1% antibiotic-antifungal (GIBCO), 7.5% bovine serum (GIBCO), 2.5% fetal bovine serum (GIBCO), pyruvate (Sigma, 100 mM ) And 100 PC EC cells were inoculated in a 100 mm culture dish suspension with 100 ml α-MEM (GIBCO) medium supplemented with 1% tyrosine solution (Sigma). Nerve differentiation proceeded as shown in FIG. 1. P19 EC cells were cultured in undifferentiated cell state in a tissue culture dish (step 1). P19 cells were cultured for 0-3 days in the presence of RA (retinoic acid, Sigma, 1 μM, DMSO 0.1%>) as a coagulation inducer to obtain an embryonic body (step 2). After generation of embryo-like structures for 3 days, cell aggregates were separated with 1% trypsin (GIBCO) and transferred to tissue culture dishes, multiwell plates or poly-L-lysine coated-glass coverslips. Isolated P19 cells were grown in the absence of RA. After 9 days, P19 EC cells differentiated into neurons (step 3). N-acetylcysteine (Sigma) or ascorbic acid (Sigma) was added to the medium so that the final concentration was 100 μM (the most efficient concentration for neural differentiation).

Immunocytochemistry

Cells were immobilized and permeabilized for 5 minutes at room temperature in methanol / acetone (50/50), then washed three times with PBS and treated with 0.1 M PBS-3% H ₂ O ₂ for 5 minutes. Blocked for 1 hour with PBS containing 5% BSA (bovine serum albumin). Then, it was stained according to the DAB / nickel staining method. Dilutions and dilutions used are as follows: TH monoclonal antibody 1: 1000 (Santa Cruz biotechnology, CA, USA), microtubule-associated protein-2 (MAP-2) monoclonal antibody 1: 300 and glial -Fibrillar acid protein (GFAP) monoclonal antibody 1: 1000 (Santa Cruz biotechnology, CA, USA), dopamine transporter (DAT) monoclonal antibody 1: 500 (Santa Cruz biotechnology, CA, USA), yes Stain polyclonal antibody 1:50 and pest 33258. For detection of primary antibodies, biotin-binding secondary antibodies and fluorescent labels (Alexa 488 or Alexa 594, Molecular Probes, Oregon), according to the manufacturer's protocol. , USA) secondary antibody was used. Cells on glass coverslips (coated with poly-L-lysine) were placed in mounting medium and photographed using fluorescence microscopy (Nikon, Tokyo, Japan) and confocal microscopy (LSM 510 meta, Zeiss, Feldbach, Switzerland). .

Immunoblotting

After 3 days embryogenesis from P19 EC cells, cells were washed twice with ice-cold PBS. Lysis buffer (1% TX-100, 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM Na ₃ VO ₄ , 40 mM NaF, 5 mM ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA), 0.2% sodium dodecyl sulfate (SDS), 0.5% sodium deoxy cholate (SDC) and 0.2 mM phenylmethylsulfonyl fluoride (PMSF)) were added to the cells It was. Cell extracts were sonicated and then centrifuged at 15,800 × g for 10 minutes and the resulting supernatant (RIPA lysate) was used for immunoblotting. Protein concentrations of RIPA lysates were determined by the Bradford (Bradford 1976) method using BSA as a standard. Sample buffer was added to the fraction of lysate (50 μg protein), boiled for 3 minutes, and then SDS-PAGE (Laemmli 1970) under reducing conditions. The proteins were then transferred to nitrocellulose (NC) membranes (Amersham Pharmacia Biotech, Little Chalfont, UK) and immunoblotted with UK) phospho-Akt antibody (Santa Cruz, CA, USA). Blots were detected with an ECL detection system (Amersham Pharmacia Biotech). Relative protein amounts were determined using normalized densitometers (Pilber Lourmat, Torcy, France) for p-Akt and actin bands.

In vivo research

25 g of male C57BL / 6 mice were used for the in vivo study. All experiments were conducted as approved by Chung-Ang University. All mice were male to 5-11 week old and were bred at 21 ± 1 ° C., 50 ± 10% humidity and 12 hr / hr light / cancer cycle, and had free access to feed and water. C57BL / 6 mice were purchased from SAMTAKO (Korea, KyungGi-Do). Under pentobarbital anesthesia, 3 μl 6-OHDA (3 μg / L in normal saline) was stereostatically injected into the striatum bundle (0.0 mm to posterior vestibule; 2.0 mm to middleline side; 3.0 mm to dural mask). The cut bar was set at 3.55 mm below zero (intradural line).

Two weeks after 6-OHDA treatment, the experimental animals were examined for amphetamine-induced turning behavior (amphetamine 0.1 mg / kg i.p.). Mice were examined at 1.5 minute intervals, and animals were selected that exhibited net rotational asymmetry of at least 400 full turns per minute away from other injured areas. Amphetamine-ration at 2, 4 and 6 weeks after transplantation was assessed.

On day 3 of embryogenesis, cells were trypsinized and suspended in medium. The animals were anesthetized with pentobarbital and placed in stereotactic frame. Using an 18-gauge needle, 5 μl of cell suspension (4000 cells / μl) was injected into the damaged striatum over 10 minutes (0.0 mm to posterior vestibule; 2.0 mm to the midline side; 3.4 mm to the dural mask).

Two and six weeks after transplantation, animals were anesthetized and 4% paraformaldehyde in PBS was perfused intracardiacally. The brain was removed and soaked in 30% sucrose in PBS overnight, then sectioned with frozen microtome. Free-flowing brain sections (30 μm thick) were immunohistochemically stained with TH-, GAD, and MAP-2- (Santa Cruz biotechnology, CA, USA) as described above, and the images were confocal microscopy (LSM 510). meta, Zeiss, Feldbach, Switzerland).

Axon  Measure of growth

Images of MAP-2 ⁺ cells obtained from randomly selected sites of at least six cultures from three independent experimental sets were obtained. Morphological characteristics were quantified using an Olympus phase contrast microscope equipped with an Olympus digital camera system and an Olympus image analyzer (Olympus, Tokyo, Japan). The length of the primary neurite was defined as the distance from the tip of the longest branch to the soma. The total extent of axons was defined as the total length of all axons per cell. The number of axons per cell was defined as all processes that were longer than two cell diameters in length.

Cell count Counting  And statistical analysis

Eyepiece reads at 40 or 200 magnifications were used to determine the number of immunoreactive or DAPI-stained cells at 6-12 single random selection sites in each well. In each experiment 3-6 culture wells were analyzed. Data are shown as mean ± SEM. ANOVA with Tukey post hoc analysis (SPSS, Chicago, IL, USA) was used for statistical comparisons involving more than two groups.

Experiment result

NAC In the process of neuronal differentiation Embryonic structure  Improves production

Formation of embryonic bodies is an important step in neuronal differentiation. Undifferentiated P19 EC cells were aggregated with retinoic acid (1 μM) and incubated for 3 days in suspension in 100 mm non-adhesive Petri dishes. To assess the effect of N-acetylcysteine (NAC) on the development of embryo-like structures, the embryo-like structures formed after co-treatment of NAC (100 μM) and RA (1 μM) or RA (1 μM) alone The number and size were observed. Development of embryo-like structures by RA alone and co-treatment with NAC and RA was significantly different (FIG. 1A). Embryonic similar structures were classified into 0.05 μm to 0.2 μm in units of 0.05 μm in diameter (FIG. 1B). In the case of co-treatment with NAC together with RA, an embryo-like structure with a much larger diameter than that of RA alone was formed (FIGS. 1A and 1B). The number of embryo-like structures developed by NAC + RA less than 0.10 μm in diameter was less than that developed by RA. However, the number of embryo-like structures developed by NAC + RA was 0.15 μm or more in diameter than those developed by RA. On the other hand, the total number of embryo-like structures in a 100 mm tissue culture dish was investigated, and the results are shown in Table 1 below.

Processing method Number of embryo-like structures RA 1826 ± 70.65 NAC + RA 2242 ± 113.07 α-estradiol + RA 1901 ± 76.66 NAC + RA + BSO 1912 ± 60.23

As shown in Table 1, the number of embryo-like structures formed by co-treatment of NAC and RA is much larger than in the case of RA alone. The number of embryo-like structures formed by co-treatment of NAC and RA increased significantly to 2242 ± 113.07. On the other hand, such an increased pattern was not observed when treated with α-estradiol, a type of antioxidant such as NAC. As a result, it can be seen that NAC is a very effective promoter for P19 cell aggregation by RA. In embryonic analogues formed by co-treatment of NAC and RA, cell-to-cell interactions were increased. In addition, the treatment of BSO (inhibitor of glutathione-reduced GSH, L-buthionine (S, R) -sulfoximine, GIBCO) in the NAC-RA co-treatment group significantly reduced the number of embryo-like structures. NAC increases the concentration of intracellular GSH, increasing the number of embryo-like structures.

NAC Improves the efficiency of neuronal differentiation

In order to confirm whether the improvement of embryogenicity by NAC directly affects neuronal differentiation, the differentiation of P19 EC cells by RA or NAC + RA was examined by immunochemical staining. Differentiated P19 EC cells were trypsinized, then gently resuspended and incubated for 7 days on poly-L-lysine coated glass coverslips (α-MEM with 2% CS). Cells were immobilized with ethanol / acetone (50/50) and then immunocytochemical staining was performed for neuronal specific marker MAP-2 (FIG. 2A, Panels A-D).

As a result of immunocytochemical staining, the expression level of neuronal marker was more than twice that of RA in NAC + RA. However, in the case of RA and RA + α-estradiol, the rate of neuronal differentiation was about the same. In addition, BSO treatment in NAC and RA co-treatment groups resulted in a slightly lower neuronal differentiation rate than RA.

The length and number of initial axons were analyzed for P19 cells stained for MAP-2 (Table 2).

Item RA treated-p19 EC cells (n = 50) RA + NAC treated-p19 EC cells (n = 90) Initial Axon (μM) 19.69 ± 0.89 20.13 ± 0.64 Total Axon Degree (μM) 34.61 ± 1.27 82.32 ± 1.56 Axons Per Cell 2.11 ± 0.12 4.57 ± 0.31

NAC + RA significantly increased the length and number of early axons compared to RA. In particular, the number of early axons in P19 cells differentiated by NAC + RA increased more than two times compared to RA.

Thus, it can be seen that the improvement of embryogenesis by NAC + RA effectively increases the rate of neuronal differentiation from P19 EC cells. In addition, neurons differentiated by NAC + RA had greater maturity compared to neurons differentiated by RA.

NAC On by GSH  Judo P19  EC cells Embryonic development  And affects neural differentiation

In previous experiments, it was confirmed that NAC promoted embryonic development and neural differentiation. N-acetyl-L-cysteine (NAC) is a well known GSH precursor. However, GSH + RA treatment conditions did not promote embryogenesis and neural differentiation compared to RA alone treatment. After treatment with GSH + RA in P19 EC cell development, there was little change in intracellular GSH levels when cells were treated with GSH. Higher and sustained concentrations of intracellular GSH could be achieved when P19 EC cell embryogenesis with NAC + RA. To investigate the effect of NAC-induced GSH on neuronal differentiation of P19 EC cells, P19 EC cells were treated with BSO (100 μM) to lower the concentration of GSH. BSOs are well known as irreversible inhibitors of γ-glutamylcysteine synthetase (Toxicology 152, 2000, 75-84). As a result of treatment with BSO + NAC + RA, no NAC effect was observed in embryogenesis and neuronal differentiation (FIG. 3). BSO reduced the effect of NAC further treatment in neuronal differentiation system by RA. BSO significantly reduced intracellular GSH content (FIG. 3). Intracellular GSH content regulation plays an important role in inducing embryonic development and neural differentiation by NAC. Thus, GSH induction by NAC affects embryonic development and neural differentiation of P19 EC cells.

NAC By + RA joint treatment Embryonic structure  During development PI3 - Kinase  Activity is up-regulated

 It was confirmed that NAC increased the embryogenic and neuronal differentiation rate of P19 EC cells. Adhesion molecules involved in N-cadherin and L-cadherin play an important role in cell-to-cell interaction for neuronal differentiation as well as embryogenicity (Ann NY Acad Sci. 2004 Apr; 1014: 140-54). For NAC + RA treatment, the expression pattern of N-cadherin was examined by Western blotting method to study whether adhesion molecule-mediated signaling promotes embryogenicity. The expression of N-cadherin in NAC + RA neuronal differentiation conditions showed a significantly increased pattern than RA in the early embryogenic stage (24 hr). An increase in early N-cadherin expression in NAC + RA conditions is in close agreement with the results that NAC + RA treatment produces more embryonic analogs initially than RA. However, the expression of N-cadherin in RA conditions was up-regulated from 48 hr so that little difference was observed at 72 hr compared to NAC + RA.

 PI3-kinase is known as a physiological regulator of neuronal differentiation cells. To investigate whether activation of PI3-kinase is involved in the improvement of NAC + RA-induced differentiation, the activity of Akt downstream in the signal transduction of P13-kinase was analyzed by Western blot. On day 3 cell lysates were prepared from aggregated P19 cells and treated with antibodies recognizing phosphorylated Akt. Western blotting showed that NAC + RA-treated P19 cells showed an increased amount of phosphorylated-Akt than RA alone (FIG. 4A). Activated PI3-kinase was maintained during development of embryonic analogues by NAC treatment. Since α-estradiol does not affect embryonic analogues and neural differentiation, it does not affect the increase in the activated form of Akt. LY294002 (GIBCO) was used to inhibit PI3-kinase. Treatment with LY294002 (10 μM) reduced the number of embryo-like structures by NAC + RA co-treatment. Treatment of PI3-kinase inhibitors (10 μM LY294002) with NAC and RA inhibited neuronal differentiation than in NAC and RA co-treated cells (FIG. 4B). These experimental results show that PI3-kinase is a major mediator in neural development by NAC + RA co-treatment.

NAC increases differentiation efficiency into dopaminergic cells

The above experimental results clearly show that NAC + RA treatment increases neuronal differentiation from P19 EC cells. Furthermore, to investigate whether NAC treatment contributes to the differentiation of dopaminergic and GABA-producing neurons, anti-GAD antibodies against GABA-producing neurons, anti-TH antibodies to dopaminergic neurons, and glial cells Dual immunofluorescence staining was performed using anti-GFAP antibody and anti-MAP-2 antibody against mature neurons. Cells were differentiated from embryonic analogues for 9 days, followed by dual immunofluorescence staining.

P19 EC cells differentiated by NAC + RA were stained from antibodies to MAP-2 and TH, while cells differentiated by RA were slightly or slightly stained (FIG. 5A). The average percentage of mergence for TH ⁺ cells and MAP-2 in NAC + RA treated total culture was 48.72 ± 2.12%, but 7.12 ± 9.32% for RA alone. In particular, as in the previous results, MAP-2 positive cells had significantly more NAC + RA cases than RA.

Dual immunofluorescence staining was performed using anti-GAD and anti-MAP-2 antibodies against GABA-producing neurons or anti-GFAP and anti-MAP-2 antibodies against glial cells. The average percentage of mergence for GAD ⁺ cells and MAP-2 in NAC + RA treatment was 21.7 ± 7.5%, but 24.32 ± 0.456% for RA alone. Similarly, GFAP positive cells in P19 cells differentiated by NAC + RA treatment showed a reduced pattern than RA treatment.

The experimental results show that NAC increases the development of dopaminergic neurons but inhibits the development of GABA-producing neurons and glial cells.

NAC In a Parkinson's disease animal model P19  Increased efficiency of cell therapy by cell transplantation Let

In vitro experiments demonstrated that NAC + RA co-treatment significantly increased dopaminergic neuronal differentiation efficiency. Furthermore, in order to confirm whether such NAC effects are reproduced in vivo , cell transplantation was performed in 6-OHDA Parkinson's disease animal model. Prior to transplantation, cells were transplanted from embryonic analogues developed in vitro and tagged with Hoechst 33324. It was analyzed whether the NAC + RA co-treatment culture system used improved neuronal differentiation and whether the NAC + RA-treated cells used for transplantation contained dopaminergic neurons in vivo .

P19 cell transplantation was performed 2 weeks after 6-OHDA injection. The viability and neural differentiation opportunities of the transplanted cells were examined morphologically by double staining with Hoechst 33324 and anti-estin antibodies and then observed by confocal microscopy (FIG. 6A). As can be seen in FIG. 6A, 92.6% of NAC + RA-treated transplanted cells exhibited the mergence of Hoechst 33324 and anti-estin antibodies at 2 weeks post-transplantation, whereas RA-treated transplanted cells and controls were immune. No mules for tissue staining were detected. Four weeks after transplantation, dual immunofluorescence staining was performed with MAP-2 antibody and TH antibody. As can be seen in FIG. 6B, the MAP-2 and TH mutagenesis was stronger in NAC + RA-treated P19 transplanted cells than RA alone. Next, in the 6-OHDA-induced Parkinson's disease mouse model, the functional recovery of amphetamine-induced motor asymmetry was examined. Rotational response to amphetamine was examined at 2, 4 and 6 weeks after transplantation. Animals with cells differentiated by NAC + RA treatment showed recovery from amphetamine-induced turning behavior over time, while animals with differentiated cells by RA did not (FIG. 7). Decrease in rotational scores was gradual, and animals with NAC + RA-treated differentiated P19 EC cells significantly reduced rotational scores at 2, 4 and 6 weeks after transplantation. However, animals transplanted with cells by the RA-differentiation system showed no difference compared to the control (sham) at 2, 4 and 6 weeks after transplantation (FIG. 7).

As described above in detail, the present invention provides a novel composition for inducing stem cell differentiation and a method for inducing stem cell differentiation. The present invention also provides a pharmaceutical composition for the treatment of neurological diseases, including stem cells. According to the present invention, differentiated cells (preferably, neurons, more preferably dopaminergic neurons) can be obtained from stem cells. Neurons produced by the present invention are very effective in treating various neurological diseases caused by nerve injury.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (22)

A composition for inducing stem cell differentiation, comprising N-acetylcysteine as an active ingredient and comprising retinoic acid as a differentiation inducing agent. delete delete The composition for inducing stem cell differentiation according to claim 1, wherein the stem cells are embryonic stem cells or adult stem cells. The composition for inducing stem cell differentiation according to claim 1, wherein the stem cell differentiation is differentiation into neurons. The composition for inducing stem cell differentiation according to claim 5, wherein the neuron is a dopaminergic neuron. A method of inducing stem cell differentiation comprising culturing stem cells in a composition for inducing stem cell differentiation comprising N-acetylcysteine as an active ingredient and comprising retinoic acid as a differentiation inducing agent. delete delete 8. The method of claim 7, wherein the stem cells are embryonic stem cells or adult stem cells. 8. The method of claim 7, wherein the differentiation of stem cells is differentiation into neurons. 12. The method of claim 11, wherein the neurons are dopaminergic neurons. delete delete delete delete delete delete delete delete delete delete

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