KR101614010B1 - Composition for inducing neurogenesis comprising inhibitors of antioxidant enzyme - Google Patents

Abstract

The present invention relates to a composition for inducing differentiation into a neuron, comprising an antioxidant enzyme expression or an activity inhibitor. The present invention also relates to a transformant transformed with an expression or activity inhibitor of peroxiredoxin, a method for inducing differentiation into neural cells using the transformant, a neural cell induced thereby, and a cell therapy agent containing the same. The present invention also relates to a composition for retaining stem cell function comprising peroxiredoxin. The present invention also relates to markers for detecting stem cell function or nerve cells comprising peroxiredoxin, a composition for detection, a kit for detection, and a detection method.
Based on the relationship between the antioxidant enzyme according to the present invention, particularly peroxiredoxin and the differentiation of stem cells into neurons, it is possible to produce effective nerve cells or maintain stem cell function, And can be effectively used for management.

Description

[0001] The present invention relates to a composition for inducing differentiation into neurons, which comprises an antioxidant enzyme expression inhibitor or an activity inhibitor,

The present invention relates to a composition for inducing differentiation into a neuron, comprising an antioxidant enzyme expression or an activity inhibitor.

The present invention also relates to a transformant transformed with an expression or activity inhibitor of peroxiredoxin, a method for inducing differentiation into neural cells using the transformant, a neural cell induced thereby, and a cell therapy agent containing the same.

The present invention also relates to a composition for retaining stem cell function comprising peroxiredoxin.

The present invention also relates to markers for detecting stem cell function or nerve cells comprising peroxiredoxin, a composition for detection, a kit for detection, and a detection method.

Embryonic stem cells (ESCs) have a unique ability to self-renew and differentiate into various cell lines, which impart therapeutic potential to embryonic stem cells. Despite the continuing research into the loss of stemness and the onset of differentiation, the mechanism for this has not yet been elucidated.

Recently, a mechanism has been proposed that states that the redox state is a major regulator of stem cell function and differentiation. Indeed, differentiation experiments involving various stem cells have shown that reactive oxygen species (ROS) play an important role in the loss of stem cell function. In stem cells or progenitor cells, stimulation of differentiation increases the level of ROS, thereby inducing the differentiation of cells into specific cell lines. These observations suggest that the signaling associated with the differentiation of ESCs is very closely related to the balance between ROS production and neutralization.

Although differentiated cells exhibit an elevated level of ROS, antioxidant enzymes are not always indicative of expression according to intracellular ROS levels. For example, transcript levels of superoxide dismutase-2 (SOD2), glutathione peroxidases (Gpxs) and catalase are lower in mouse embryoid bodies compared to non-differentiated ESCs, whereas in spontaneously differentiated human ESCs Expression of Gpx1 was increased. Similar to the antioxidant enzymes mentioned above, peroxiredoxins (Prxs), a novel antioxidant enzyme family that has been implicated in the removal of hydrogen peroxide (H2O2) in cell defense, The difference was confirmed. Suggesting involvement in ROS-mediated differentiation. Therefore, it is necessary to study the role of specific Prx isoforms in cell differentiation.

The involvement of these MAPKs in the signaling and differentiation processes leading to activation of MAPKs (mitogen-activated protein kinases) known as JNK (c-Jun N-terminal kinase), ERK (extracellular signal- Lt; RTI ID = 0.0 > redox signal. ≪ / RTI > In neuronal development, the activity of the ERK1 / 2 and JNK pathways is required for loss of stem cell function and neurogenesis, respectively. In contrast, a high level of neurogenesis is achieved when the pathway of p38 MAPK is suppressed or eliminated. However, mechanisms for regulating MAPK activity in the loss of stem cell function and nerve development have not yet been elucidated. Therefore, there is a need for research into the involvement of certain antioxidants in the regulation of redox signals in ESC-mediated neurogenesis.

The present inventors have demonstrated that the level of ROS increases in the neural differentiation of ESCs and that Prx I - / - or Prx II - / - cells do not decrease ROS levels in both ESCs and differentiated cells. Also, escape of ROS from Prx I- or Prx II-mediated antioxidant monitoring has been shown to destroy ESC stem cell function and induce accelerated neuronal commitment, which is largely dependent on the activity of the JNK signal, The present invention has been completed on the basis thereof.

An aspect of the present invention is to provide a composition for inducing differentiation into a neural cell, comprising an antioxidant enzyme expression or an activity inhibitor.

Also, one aspect of the present invention provides a transformant transformed with an expression or activity inhibitor of peroxiredoxin, a method for inducing differentiation into neurons using the same, a neuron induced thereby, and a cell therapy agent comprising the same will be.

Another aspect of the present invention is to provide a composition for retaining stem cell function comprising peroxiredoxin.

Yet another aspect of the present invention is to provide a marker for detecting stem cell function or nerve cell comprising peroxiredoxin, a composition for detection, a kit for detection, and a detection method.

An aspect of the present invention provides a composition for inducing differentiation into neurons, comprising an antioxidant enzyme expression or an activity inhibitor.

Hereinafter, the present invention will be described in detail.

In the present invention, the antioxidant enzyme means an enzyme which removes or inhibits active oxygen in a cell or a colony. In the present invention, reactive oxygen species (ROS) are chemically reactive molecules including oxygen atoms.

The antioxidant enzyme may preferably be peroxiredoxin. In the present invention, peroxiredoxin is a type of antioxidant enzyme that transfers electrons to hydroperoxide, one of the metabolic intermediates of intracellular oxygen, and reduces it to water. And its relationship is reported in various fields such as proliferation and tumorigenesis. Specifically, peroxiredoxins are scavengers of hydrogen peroxide and alkylhydroperoxides in vivo and are classified in the mammal as peroxiredoxin isotypes of type I to VI and are observed in various parts of the tissue. In particular, in the present invention, inducing differentiation into neurons may be peroxiredoxin I or II.

The antioxidant enzyme expression inhibitor according to the present invention may be used as an antioxidant enzyme for the gene encoding antioxidant enzyme or antisense nucleotide for mRNA, small hairpin RNA, shRNA, small interfering RNA, siRNA and ribozyme ), But the present invention is not limited thereto. In particular, the antioxidant enzyme expression inhibitor may be an inhibitor of peroxiredoxin expression.

The antioxidant enzyme activity inhibitor according to the present invention may be at least one selected from the group consisting of a compound specifically binding to an antioxidant enzyme protein, a peptide, a peptide mimetic, a substrate analog, an aptamer, an antibody or an antagonist, It is not. In particular, the antioxidant enzyme activity inhibitor may be an inhibitor of the activity of peroxiredoxin.

The antisense nucleotide binds (hybridizes) to a complementary base sequence of DNA, immature-mRNA or mature mRNA, as defined in the Watson-Crick base pair, to interfere with the flow of genetic information as a protein in DNA will be.

The siRNA is composed of a sense sequence of 15 to 30 mers selected in the nucleotide sequence of the mRNA encoding the sulfating enzyme protein and an antisense sequence complementarily binding to the sense sequence, The sense sequence is preferably composed of 25 bases, though not particularly limited thereto.

The peptide mimetics inhibit the binding domain of the sulfated enzyme protein and inhibit the activity of the sulfated enzyme protein. Peptide mimetics can be peptides or non-peptides and can be composed of amino acids joined by non-peptide bonds, such as psi bonds. Also included within the scope of the present invention are "conformationally constrained" peptides, cyclic mimetics, at least one exocyclic domain, a binding moiety (binding amino acid) Can be. Peptide mimetics can be structured similar to the secondary structural features of sulfated enzyme proteins and mimic the inhibitory properties of large molecules such as antibodies or water-soluble receptors, and can be novel small molecules that can act as an equivalent of natural antagonists .

The aptamer is a single-chain DNA or RNA molecule, which has high affinity for a specific chemical molecule or biological molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) Can be obtained by separating oligomers that bind with selectivity. Aptamers can specifically bind to a target and modulate the activity of the target, for example, by blocking the function of the target through binding.

The antibody can specifically and directly bind to the sulfated enzyme protein and effectively inhibit the activity of the sulfated enzyme protein. As the antibody specifically binding to the sulfated enzyme protein, it is preferable to use a polyclonal antibody or a monoclonal antibody. The antibody specifically binding to the sulfated enzyme protein may be prepared by a known method known to those skilled in the art, and commercially available sulfated enzyme antibodies can be purchased and used. The antibody may be prepared by injecting an immunogen, p34 protein, into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen may be injected intramuscularly, intraperitoneally or by subcutaneous injection, and may generally be administered with an adjuvant to increase the antigenicity. Blood can be collected periodically from the external host and the antibody formed can be collected by collecting sera showing specificity to the antigen and the titer formed.

Induction of differentiation into neurons of the present invention means induction of differentiation from stem cells into neurons.

In addition, one aspect of the present invention provides a method for inducing differentiation into neural cells and a neural cell derived therefrom, comprising the step of treating the expression or activity inhibitor of peroxiredoxin in stem cells.

In the present invention, a stem cell refers to a cell capable of differentiating into various types of body tissues, that is, undifferentiated cells. The stem cells of the present invention may be embryonic stem cells or adult stem cells, and the adult stem cells may be derived from all tissues and may be derived from bone marrow, cord blood, blood, liver, skin, , Placenta-derived, nerve-derived, adrenal-derived, epithelial-derived or uterine-derived stem cells. The stem cells of the present invention are preferably embryonic stem cells.

In the present invention, "differentiation" generally means a phenomenon in which a relatively simple system is separated into two or more qualitatively different partial systems. Specifically, a phenomenon in which a structure or function is mutually specialized while cells are growing by proliferation It means a phenomenon in which the cells and tissues of a living organism change shape or function in order to carry out a given task. Relatively, "undifferentiated" refers to a state in which the above-mentioned differentiation does not yet occur and contains characteristics as stem cells.

In addition, another aspect of the present invention provides a cell therapy agent comprising the nerve cell induced by the above-described method.

The term "cellular therapeutic agent" of the present invention is a drug (FDA regulation of the United States of America) used for the purpose of treatment, diagnosis and prevention of cells and tissues produced by separation, culture and special manipulation from human, Or to restore the function of the tissue, through a series of actions such as proliferating the living organism, allogeneic, or xenogeneic cells in vitro, or otherwise altering the biological characteristics of the cell, Means drugs used for the purpose.

In addition, the present invention provides a composition for retaining stem cell function comprising peroxiredoxin.

The stemness is a special feature of stem cells, which means the ability to differentiate into various types of body tissues. When the peroxidoredoxin of the present invention is treated, the stem cell ability of stem cells can be maintained for a long time.

The present invention also relates to a composition for detecting stem cell function, comprising a peroxidoredoxin I, a marker capable of measuring the expression level of peroxiredoxin I, a marker for detecting stem cell function, and a stem A kit for detecting cellularity is provided.

In addition, the present invention relates to a composition for detecting neurons, comprising a peroxidoredoxin II-containing marker for detecting the differentiation of neuronal cells, an agent capable of measuring the level of expression of peroxiredoxin II, A kit for detecting neuronal differentiation is provided.

The agent for measuring the expression level of the peroxidoredoxin I or II comprises an antisense oligonucleotide, a primer pair or a probe that specifically binds to the mRNA of the peroxiredoxin I or II.

The agent for measuring the expression of the mRNA is selected from the group consisting of an antisense oligonucleotide specific to the gene, a primer pair, a probe, and a combination thereof. That is, the detection of the nucleic acid can be performed by an amplification reaction using a nucleic acid molecule encoding the gene or one or more oligonucleotide primers hybridized to the nucleic acid molecule.

For example, the detection of mRNA using a primer can be performed by amplifying a gene sequence using an amplification method such as PCR, and then confirming amplification of the gene by a method known in the art.

According to a preferred embodiment of the present invention, the agent for measuring the expression level of the protein comprises an antibody, a peptide or a nucleotide that specifically binds to the protein.

The agent for measuring the expression of the protein refers to an antibody that specifically binds to the protein, and includes both a polyclonal antibody, a monoclonal antibody, a recombinant antibody, and a combination thereof.

Such antibodies include both full forms with polyclonal antibodies, monoclonal antibodies, recombinant antibodies and two full-length light chains and two full-length heavy chains as well as functional fragments of antibody molecules, such as Fab, F (ab ' ), F (ab ') 2, and Fv. Antibody production can be easily performed using techniques well known in the art to which the present invention is applicable, and commercially produced antibodies can be used.

The composition of the present invention can be used not only as a preparation for measuring the expression of the gene described above but also as a label for quantitatively or qualitatively measuring the formation of an antigen-antibody complex, a common tool and reagent used for immunological analysis .

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . Enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, The present invention relates to a method for the treatment and / or prophylaxis of cancer, comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound selected from the group consisting of GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, ≪ RTI ID = 0.0 > Ratama < / RTI > The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, ^{_{hydroquinone, K 4 W (CN) 8}} , [Os (bpy) 3] 2+ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include include ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 13 1I, 186 Re , and without limitation.

Examples of such tools or reagents include, but are not limited to, suitable carriers, solubilizers, cleaning agents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, Acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, other paper, glass, metal, agarose, and combinations thereof.

The kit may include an agent for measuring the expression level of the gene as well as tools, reagents and the like commonly used in the art used for immunological analysis.

Examples of such tools or reagents include, but are not limited to, suitable carriers, markers capable of generating detectable signals, chromophores, solubilizers, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, Acrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

The present invention also provides a method for measuring whether a cell has stem cell potential or differentiation into a neural cell, comprising the step of measuring the level of expression of peroxiredoxin I or II in a biological sample.

If the level of peroxycorticosin I is higher than that of the differentiated cells, it can be judged to have stem cell ability. If the level of peroxiredoxin II is higher than that of stem cells, it is judged to be differentiated into neurons .

Based on the relationship between the antioxidant enzyme according to the present invention, particularly peroxiredoxin and the differentiation of stem cells into neurons, it is possible to produce effective nerve cells or maintain stem cell function, And can be effectively used for management.

1 is a diagram showing that ROS causes loss of stem cell function during embryonic stem cell-mediated nerve development.
FIG. 2 is a graph showing that Prx I and Prx II show opposite expression patterns in the neural differentiation process of embryonic stem cells.
FIG. 3 is a graph demonstrating that deficiency of Prx II inhibits Prx I and Oct4 expression and induces loss of stem cell viability of embryonic stem cells during neuronal development.
FIG. 4 is a graph showing that Prx deficiency induces the production of ROS and accelerates the loss of stem cell function in neuronal development.
Figure 5 shows the accelerated neurogenesis process in Prx I - / - and Prx II - / - embryonic stem cells by regulation of the ROS-dependent signaling pathway.
FIG. 6 is a graph showing that rapid stem cell loss of embryonic stem cells requires JNK activity dominated by Prx / ROS balance.
Figure 7 is an illustration showing that Prx is essential for the maintenance of stem cell function of ESC in developmental teratoma.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Cell culture

(Invitrogen, Grand), 5% FBS (HyClone, Logan, UT), 10% ESC-qualified fetal bovine serum (Grand Island, NY) for the maintenance of J1 ESC from blastocysts and the production of ESCs (Invitrogen, Grand Island, NY) containing 500 U / ml leukemia inhibitory factor (Chemicon, Temecula, CA) and antibiotics (Invitrogen, Grand Island, NY) Cells were cultured on MI-MEFs (mitotically inactivated mouse embryonic fibroblasts). DMEM containing 10% FBS (HyClone, Logan, UT) and antibiotics were used to culture P19 mouse embryonic carcinoma cells and CRL-2073 human ESCs.

Example  2: Prx  I - / - or Prx II - / - Of ESCs  Judo

To establish ESC cell lines, blastocysts were harvested and cultured from wild-type, Prx I - / -, and Prx II - / - 129 / SvJae mice to form ICM (inner cell mass) clumps. This clump was subcultured until the colonies exhibited a typical ESC morphology, such as a large percentage of nuclear / cytoplasmic and glowing colony boundaries. Individual colonies were cloned and expanded to 7 or 8 passages, and stored cold at -196 ° C.

Example  3: ESCs Differentiation and chemical treatment

Exponentially growing mouse ESCs were isolated from MI-MEFs by treatment with trypsin. After centrifugation, ESCs were resuspended in NBM (neurobasal medium; DMEM / F12 [all Invitrogen, Grand Island, NY] containing 20 μl / ml N2 and 10 μl / ml B27 additive) Gelatin-coated culture dish. In some experiments, the effect of ROS on neural differentiation of ESCs was confirmed using B27 (B27-AO; Invitrogen, Grand Island, NY) without antioxidant components. Cells were subcultured using 0.25% trypsin / 5.3 mM EDTA (Invitrogen, Grand Island, NY) and plated on fibronectin (5 μg / ml) -coated culture dishes (Invitrogen, Grand Island, NY) After transfer, they were cultured in NBM at 37 ° C under 5% CO ₂ .

(N-acetyl-1-cysteine, 2.5 mM; Sigma-Aldrich, St. Louis, MO), H ₂ O ₂ (0.05 and 1 mM; Sigma-Aldrich, (Both Calbiochem, San Diego, Calif.) Of p38 MAPK (5 urn SB203580), ERK (10 urn PD98059), and JNK (2.5 or 5 urn SP600125) were used.

Example  4: ROS  Measure

Cells were cultured with 5 mM CM-H2DCF-DA (Invitrogen, Grand Island, NY) for 15 min at 37 ° C and then centrifuged at 200 g for 5 min using phosphate-buffered saline And washed. Cells were resuspended in PBS and ROS levels were measured using a FACSCalibur instrument (BD Biosciences, San Jose, Calif.).

Example  5: Alkaline  Phosphatase ( Alkaline Phosphatase Active essay

All active assay procedures were performed according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, Mo., Leukocyte Alkaline Phosphatase Kit, Cat # 86R).

Briefly, after removing the culture medium, the cells were fixed with citrate-acetone-formaldehyde fixative for 30 seconds at room temperature and then washed with calcium and magnesium PBS (Invitrogen, Grand Island, NY). Ml of Naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO) and 1 mg / ml Fast Red TR salt (Sigma-Aldrich) in 100 mM Tris buffer, St. Louis, MO). The staining reaction was terminated with PBS.

Example  6: Semi - qPCR  And Real - Time RT - qPCR

Total RNA was isolated using Trizol reagent (Invitrogen, Grand Island, NY) according to manufacturer's instructions and quantitated using spectrophotometry. Initial template cDNA was amplified by PCR using 0.75 DN DNase-treated total RNA (Invitrogen, Grand Island, NY) and 7.5 U AMV reverse transcriptase (Promega, Madison, Wis.) At 20 부 volume for 60 min at 37 캜 . PCR was performed for 22-28 cycles at 94 ° C for 30 seconds, 55-60 ° C for 30 seconds, and 72 ° C for 30 seconds, and the final culture was performed at 72 ° C for 10 minutes. PCR primers for amplifying human and mouse cDNA are available from Silico in Primer3 software http://frodo.wi.mit.edu/primer3/input.htm; Designed using Supporting Information.

Example  7: Double-label immunocytochemistry

Cells were fixed with 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) solution for 30 minutes, washed with PBS (PBST) containing 0.02% Tween-20 for 30 minutes and then incubated for 30 minutes with 0.26% ammonium chloride (Sigma-Aldrich, St. Louis, Mo.). Were incubated with 10% normal goat serum (Invitrogen, Grand Island, NY) at room temperature for 1 hour to prevent nonspecific binding. Subsequently, cells were incubated overnight at 4 ° C with polyclonal rabbit antibodies to Prx I (Alexis, San Diego, CA) or Prx II (Proteintech Group, Chicago, IL). After washing with PBST for 1 hour, the cells were incubated with Alexa488- or Alexa598-conjugated goat anti-rabbit antibody (Invitrogen, Grand Island, NY) for 1.5 hours at 37 ° C and excess antibody was incubated with PBST for 1 hour Washed and removed. For double-labeling, cells were seeded at 4 ° C overnight with monoclonal antibodies against SSEA1, Nestin, Tuj1, MAP2, GFAP (all Chemicon, Temecula, CA), and Oct4 (Santa Cruz Biotechnology, Santa Cruz, CA) , Followed by incubation with Alexa 488- or Alexa 598-conjugated goat anti-mouse antibody for 1 hour at 37 ° C. The nuclei were stained with 4 μg / ml of 4 ', 6-diamino-2-phenylindole (Sigma-Aldrich, St. Louis, Mo.) for 30 minutes at room temperature.

Example  8: Western Blot  analysis

Cell protein (30 μg / lane) was separated on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, Bedford, Mass.). The membranes were treated with Prx I, Prx II (all from Abfrontier, Seoul, Korea), Oct4 (Santa Cruz Biotechnology, Santa Cruz, Calif.), Nanog, MAP2 from both Chemicon, Temecula, CA, pJNK, , a-tubulin (Sigma-Aldrich, St. Louis, MO), and GAPDH (Abfrontier, Seoul, Korea) overnight at 4 ° C. The membranes were washed five times with TBS (Tris-buffered salineS; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.2% Tween-20 and incubated with Horsesdesdysperoxidase-conjugated goat anti- rabbit IgG Sigma-Aldrich, St. Louis, Mo.) for 1 hour at RT. Excess antibodies were removed by washing with TBS and specific binding was detected using a chemiluminescence detection system (Amersham, Berkshire, UK) according to the manufacturer's instructions.

Example  9: siRNA -medium mRNA Knockout

The following siRNA (small interfering RNA) target sequence was used for human Oct4, Prx I, and Prx II.

Oct4, 5'-AGC AGC UUG GGC UCG AGA A-3 ';

Prx I, 5'-AAA CUC AAC UGC CAA GGA-3 ';

Prx II, 5'-CGC UUG UCU GAG GAU UAC GUU-3 '.

The following siRNA target sequences were used for mouse Oct4, Prx I, and Prx II.

Oct4, 5'-AGG UGU UCA GCC AGA CCA C-3 ';

Prx I, 5'-GCG CUU CUG UGG AUU CUC AUU-3 ';

Prx II, 5'-AAA UCA AGC UUU CGG ACU AUU-3 '.

The scrambled siRNA sequence, 5'-UUC UUC GAA CGU GUG UCA CGU UU-3 'was used as a negative control. All siRNA oligonucleotide duplexes were purchased from Dharmacon (Lafayette, CO). SiRNAs (5 nM) were transfected into P19 or CRL-2073 ESCs using the Lipofectamine RNAi Max Transfection Reagent kit (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. Prx I, Prx II, and Oct4 mRNA and protein levels were measured via PCR (qPCR; 48 h after transfection) and immunoblotting (72 h after transfection).

Example  10: Teratoma  ( Teratoma ) formation

Exponentially growing ESCs were isolated from MIMEFs, and 0.5-1.0 × 10 ⁷ cells were loaded into a 1-ml syringe equipped with a 26-gauge needle and injected into the epidermis of athymic nude mice. After 3-5 weeks, the tumors were surgically resected and analyzed using semi-qPCR and immunohistochemical staining.

Example  11: Immunohistochemistry

Teratoma was fixed overnight with 10% neutral formalin, trapped in paraffin, and cut into 5 ㎛ thick. For antigen retrieval, the deparaffinized sections were briefly heated for 4 minutes in a pressure cooker containing 10 mM citrate buffer (pH 6.0). The subsequent procedure proceeded at room temperature. The sections were pretreated with 3% H ₂ O ₂ in 0.1 M TBS for 30 min to quench endogenous peroxidases and treated with protein block solution (Dako, Carpinteria, CA) for 20 min. Were incubated with antibodies against Prx I, Prx II, Oct4, or PCNA (proliferating cell nuclear antigen; Santa Cruz Biotechnology, Santa Cruz, Calif.) For 30 min in a humidified chamber.

The sections were washed with TBS containing 0.01% Tween-20 and incubated with EnVision anti-rabbit (Dako, Carpinteria, CA) polymer for 30 min. The peroxidase conjugated to the antibody complex was visualized by treatment with 3,3'-diaminobenzidine chromogen substrate solution (Dako, Carpinteria, CA). The color reaction was observed under a microscope to determine the optimal incubation time and then terminated by multiple washes with TBS. Immunolabeled sections were dehydrated with graded ethanol series, degreased with xylene and fixed.

The sections were examined in bright-field using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and images were obtained on an Olympus DP 70 camera (Olympus, Tokyo, Japan).

Example  12: Statistical analysis

Data were presented by means and SD through three independent experiments (n = 3). Experimental differences were tested for statistical significance using ANOVA. p <.05 were considered to be statistically significant, and they are shown on the graph. p <.01 and <.001 are represented by 2 and 3, respectively.

Example  13: ROS (Nerve development) Neurogenesis ) In progress ESC of Stem cell ability  (Stemness) is verified to adjust the voice

To test the involvement of ROS in neurogenesis, ESCs were cultured and differentiated on differentiation medium supplemented with B27 or B27-AO (respectively, NBM or NBM-AO, respectively), and the ROS indicator CM-H2DCF-DA Cultured together, and flow cytometry was performed.

The results are shown in Fig. The levels of ROS were increased during neurogenesis in both NBM and NBM-AO; NBM-AO.

Next, AP (alkaline phosphatase) activity assay, Western blot, and qPCR analysis were performed to observe the effect of increasing the ROS level on ESC stem cell function regulation.

The results are shown in Figs. 1B to D.

As shown in Figures 1b-d, AP activity and expression levels of Oct4 and Nanog showed a more rapid decrease in cells incubated with NBM-AO than those incubated with NBM, whereas levels of MAP2 were significantly higher in NBM-AO treated group Respectively.

Consistent with these results, supplementation of H ₂ O ₂ (0.05 and 0.1 mM) in NBM caused a sharp decrease in the level of Oct4 and Nanog transcripts in concentration and proportion in the neural differentiation of ESCs (FIG. 1e).

Example  14: Mouse ESC - in the derived neuron Prx  I and Prx II of Intracellular  Distribution

To confirm the involvement of Prxs in ESC-mediated neuronal development, the expression profiles and intracellular distribution of Prx I and Prx II, the isotypes of Prx, were determined.

The results are shown in Fig.

Analysis of semi-qPCR (Fig. 2a) and qPCR (Fig. 2B) analysis showed that Prx II transcript levels progressively increased in time proportionally with neural markers Nestin, NeuroD1, and MAP2.

In contrast, Prx I expression showed a continuous decrease in neurogenesis, as did the stem cell markers Oct4 and Nanog (Fig. 2a, 2b).

Consistent with these results, differential expression kinetics of Prx I and Prx II were detected by western blot analysis (Fig. 2C).

The results of the double-label immunocytochemistry study indicate that the contradictory expression kinetics of Prx I and Prx II result from a unique distribution in each of the undifferentiated ESCs and nerve cell lines.

Strong Prx II immunoreactivity was clearly colocalized with Nestin, Tuj1, and MAP2, while Prx I immunoreactivity was detected only in SSEA1-positive cells (Fig. 2d, 2e). Indicating that Prx I and Prx II may be markers of stem cell function and neurogenesis, respectively.

Example  15: Prx II -lack of ESCs Confirms that it rapidly differentiates into neurons

In order to confirm the role of Prxs in the differentiation process, ESC cell line was established at 3.5 days post-coitum (p.c), and wild type, Prx II - / - 129 / SvJae blastocyst was established and differentiated into neuronal cell line.

Interestingly, Prx II - / - ESCs showed very rapid differentiation into neurons within 7 days of neural differentiation and formed an abundant population of Nestin- and MAP2-positive cells compared to the wild type. (Fig. 3A). This indicates that active nerve development is induced from Prx II - / - ESCs.

In contrast, colonies positive to Prx I and SSEA1 were frequently found in wild-type cells in the neural differentiation process whereas immunostaining of the two antibodies was not detected in the differentiating Prx II - / - ESCs (FIG. 3B). This implies a sudden loss of stem cell function in the neurogenic differentiation of Prx II - / - ESCs cells.

Consistent with these results, Nestin and MAP2 expression levels of neuronal markers were higher in Prx II - / - than in wild type at 7 days after neural differentiation. On the other hand, the expression of Oct4 and Nanog genes related to stem cell function was much lower in Prx II - / - ESCs than in wild type (FIG. 3c). Western blot analysis also confirmed the rapid decrease of Prx I and Oct4 and the increase of MAP2 in Prx II - / - ESCs compared to the wild type in neurogenesis (Fig. 3d).

SiRNAs were used to knock down Oct4 and Prx isotypes in P19 and CRL-2073 EC (embryonic carcinoma) cells, based on Prx I and similar expression patterns in stem cell function-related transcription factors, particularly Oct4. Using western blot and qPCR analysis, it was found that knockdown of Oct4 caused reduced expression of Prx I, but did not affect Prx II (Fig. 3e, 3f). Indicating that Oct4 is an upstream regulator of the Prx I transcript. In contrast, the knockdown of Prx I and Prx II did not affect the expression levels of Oct4 and Nanog (Fig. 3g, 3h).

Prx I - / - ESC cell lines were established (Supporting Information Figs. S2 and S3) and compared with wild-type and Prx II - / - ESCs to examine the effect of Prx isotype on ESC stem cell potency.

The expression level of SSEA1, Oct4, or Nanog was not changed and AP activity was observed in wild-type, Prx I - / -, or Prx II - / - ESCs (Fig. 2i).

Example  16: Prx  I- and Prx II - Null ESCs Is high ROS  Level and by active nerve development Stem cell function  Confirmed a sudden loss

To confirm the effect of Prx deficiency in ROS production during neurogenesis, the levels of intracellular ROS in wild type, Prx I - / -, and Prx II - / - ESCs in neurogenic differentiation using flow cytometry Respectively.

Although neurogenesis causes an increase in ROS levels in all three genotypes, it has been found that ROS continues to occur at higher levels in Prx I - / - or Prx II - / - cells than in wild type cells ( 4A). In addition, the expression of transcription factors associated with high levels of AP activity and stem cell function is maintained in the wild type cells during differentiation, whereas when the antioxidant is greatly reduced from Prx I - / - or Prx II - / - or MBM, (Figs. 4B and 4C). Wild-type ESCs form ESC-like colonies with high cell density during neurogenesis, whereas in Prx I - / - or Prx II - / - cells these colony-like forms collapse and extensive neurite formation is observed (Fig. 4d, brightfield). Double-label immunocytochemistry revealed that Prx I and SSEA1 were expressed at high levels in wild-type ESC-derived colonies, while Prx II and MAP2 were expressed only in the colony peripheries (Fig. 4d, fluorescence field). On the other hand, many Prx-knockout cells were immunostained with anti-MAP2 and / or anti-Prx II antibodies and scattered rather than maintaining colony status (Fig. 4d).

Example  17: ROS The Prx -lack of ESCs Of the brain.

Prx I - / -, and Prx II - / - ESCs were differentiated in NBM in the presence or absence of antioxidant NAC, to confirm whether ROS directly involved ESC-mediated neurogenesis, Their ROS levels and neural gene expression profiles were determined using flow cytometry and semi-qPCR, respectively.

NAC treatment of the differentiating Prx I - / - or Prx II - / - cells effectively relieved the increase in ROS levels (Fig. 5a), resulting in a decrease in neuron-like cell development 5b). Similar to these results, the NAC treatment significantly reduced the expression of the nerve genes Nestin, MAP2, NeuroD1, and Ngn1, while the expression of Oct4 and / or Prn in the differentiating Prx I - / - or Prx II - (Fig. 5C). &Lt; / RTI &gt;

Similar to these results, Western blot analysis showed that a sharp decrease in Oct4 level in differentiating Prx-deficient ESCs was restored to the wild-type level by supplementation of NAC in the neural differentiation process (Fig. 5d).

Example  18: In the process of neurogenesis Prx  I - / - or Prx II - / - ESCs In Stem cell function  Loss is JNK of ROS - Confirms that dependence activity is required

Prx I - / -, and Prx II - / - ESCs were differentiated from NBM in the presence or absence of NAC and phosphorylated ) Western blots were performed using antibodies against ERK1 / 2, p38 MAPK, and JNK.

In the neurogenesis process, the phosphorylation of the three MAPKs was much more rapid (pp38 MAPK) or increased (pERK1 / 2 and pJNK) in Prx I - / - or Prx II - / - ESCs than in the wild type. On the other hand, NAC inhibited or delayed phosphorylation (FIG. 6A).

The presence or absence of signaling inhibitors of JNK (SP600125), ERK1 / 2 (PD98059), and p38 MAPK (SB203580) in order to determine whether the ROS-sensitive MAPK pathway is involved in the loss of ESC stem cell function during neurogenesis Under conditions, Prx I - / - or Prx II - / - ESCs were differentiated from NBM and semi-qPCR and qPCR analyzes were performed to confirm the expression of Oct4 and Nanog.

ERK and p38 MAPK inhibitors did not affect Oct4 or Nanog transcript levels in Prx I - / - or Prx II - / - cell differentiation, whereas treatment of JNK inhibitors resulted in significant recovery (FIG. 6b). Double-label immunocytochemistry also confirmed that the reduction of Oct4- and Prx I-positive cells in differentiating Prx I - / - or Prx II - / - colonies was reversed by NAC treatment and JNK inhibition (FIG.

Example  19: under development In teratoma ESC Stem cell function  Long-term survival confirms that cytoplasmic Prxs are required

Teratomas were prepared in athymic nude mice to confirm the regulation of the ROS / Prx axis in ESC differentiation, which was prepared by culturing wild type, Prx I - / -, or Prx II - / - ESCs on their epidermis. Teratoma was surgically removed and semi-qPCR, qPCR, and histological analysis were performed.

The levels of Prx I, Oct4, and Nanog transcripts were maintained in wild-type teratomas, whereas they were not retained in Prx I - / - or Prx II - / - teratomas (Fig. 7a, 7b). Consistent with these results, according to immunohistochemistry, Oct-4 positive cells could be identified only in wild-type teratomas (FIG. 7c). In addition, the immuno-staining chemistry for successive sections of wild-type teratoma showed strong immunoreactivity of Prx I and Prx II in Oct4-positive cells and nerve tissue, respectively, accounting for the entire nerve developmental spectrum (Figures 7c, 7d ). In addition, most of the immunoreactivity to PCNA was detected in Oct4-positive cells of wild-type teratoma and nerve root of Prx I - / - teratoma, while PCNA immunoreactivity was very small or absent in Prx II - / - teratoma , Except for a small portion of respiratory epithelial cells (Fig. 7C). In particular, it was confirmed that Prx I and Prx II were coming and going from Oct4-positive cells to other types of cells in vivo and in vitro (Fig. 7d).

Claims (19)

A composition for inducing differentiation into a neural cell, comprising an expression or activity inhibitor of peroxiredoxin I. delete delete The method according to claim 1,
Wherein the expression inhibitor is selected from the group consisting of antisense polynucleotide, RNAi, miRNA, siRNA, shRNA and ribozyme for the gene encoding the peroxiredoxin I or mRNA. / RTI &gt; Claim 1
Wherein the activity inhibitor is selected from the group consisting of a compound specifically binding to the peroxiredoxin I, a peptide, a peptide mimetic, a substrate analog, an aptamer, an antibody, and an antagonist. / RTI &gt; A method for inducing differentiation into a neural cell, comprising the step of treating the stem cell with an expression or activity inhibitor of peroxiredoxin I. A neuron derived by the method of claim 6. A cell therapy agent comprising the nerve cell of claim 7. A composition for maintaining stem cell function, comprising peroxiredoxin I. delete A marker for detecting stem cell function, comprising peroxiredoxin I. A composition for detecting stem cell function, comprising an agent capable of measuring the level of expression of peroxiredoxin I. A kit for detecting stem cell function, comprising the composition of claim 12. delete delete delete Measuring the level of expression of peroxiredoxin I in the biological sample. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 18. The method of claim 17,
Determining if the level of peroxidoredoxin I is higher than the differentiated cells, and determining stem cell potency. delete

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