KR102184341B1 - Composition for inducing dedifferentiation for induced pluripotent stem cells - Google Patents

Abstract

The present invention relates to a composition for inducing dedifferentiated pluripotent induced cells, a cell therapy agent comprising dedifferentiated pluripotent induced cells produced by the composition, and a method of producing the dedifferentiated pluripotent induced cells. The composition for inducing dedifferentiated pluripotent induced cells of the present invention can effectively introduce a single strand of unstable mRNA into a cell, and the introduced mRNA is naturally degraded in the cell after a certain period of time and is integrated into the DNA of the host cell. It can be avoided and stably generates dedifferentiated pluripotent induced cells. In addition, cells isolated from patients as well as normal have the effect of generating effective dedifferentiation pluripotent induced cells, and thus can be usefully used in related industries.

Description

Composition for inducing dedifferentiation for induced pluripotent stem cells}

The present invention relates to a composition for inducing dedifferentiated pluripotent induced cells, a cell therapy agent comprising dedifferentiated pluripotent induced cells produced by the composition, and a method of producing the dedifferentiated pluripotent induced cells.

Dedifferentiation pluripotent induced cell technology is currently regarded as a promising technology for regenerative medicine for the treatment of various incurable diseases. Dedifferentiation pluripotent induced cells can solve many ethical problems caused by the use of embryonic stem cells and many problems when used as a therapeutic agent. Due to these advantages, dedifferentiated pluripotent induced cells can be applied to patients with various stem cell therapies, and the immune rejection reaction that occurs at this time can be avoided, so the utility is high. These dedifferentiated pluripotent induced cells are derived from stem cell-induced transcription factors (Oct4, Sox2, etc.) through non-viral methods (episomal vectors, Sendai virus, PiggyBac transposon, small molecules, etc.) and viral methods (lentivirus, retrovirus, adenovirus, etc.). Myc, Nanog, Klf4, Lin28, etc.) have been reported to be produced through reprogramming of cells by transducing cells ((Abdal Dayem, Nanomaterials 2018, 8, 761.), (Zulliger, R., Journal of Controlled Release). 2015, 219, 471-487.), (Okita, K., Science 2008, 322, 949-953.), (Lee, CH, Biomaterials 2011, 32, 6683-6691.), (Takahashi, K, cell 2007 , 131, 861-872.)]. However, conventional methods have various problems such as inducing mutagenesis in a host gene or low transformation efficiency according to a method of introducing stem cell-derived transcription factors, and thus a method of generating dedifferentiated pluripotent induced cells is required.

Diabetes is a chronic metabolic disease characterized by insulin deficiency or high blood sugar due to insulin resistance, and is largely classified into type 1 diabetes (T1D) and type 2 diabetes (T2D). T1D is mainly caused by attacking insulin-producing beta cells due to an abnormal functioning of the immune system due to immune-mediated damage of pancreatic beta cells, and occurs in children and adults, and T2D is caused by decreased insulin secretion and insulin resistance. These two factors Depending on the degree of involvement of the drug, it is divided into diabetes with an insufficient insulin secretion advantage and diabetes with an insulin resistance superiority [Choi, HY, Journal of Controlled Release 2016, 235, 222-235]. Currently, 170 million people worldwide have diabetes, and the number is expected to double by 2030. As described above, because insulin cannot be produced or the generated insulin does not function normally, blood sugar control cannot be performed. Therefore, for treatment, periodic blood sugar measurement and insulin injection are the only treatments for patients. However, since these treatment methods are accompanied by various side effects, the development of other effective treatments is urgent.

Accordingly, the present inventors prepared a complex formed of graphene oxide (GO), polyethyleneimine (polyethylenimine: PEI), and RNA encoding a target gene in order to prepare an efficient dedifferentiated pluripotent induced cell capable of solving the above problem. Developed. The complex has high transformation efficiency and high efficiency of generating dedifferentiated pluripotent induced cells, and confirmed that cells isolated from patients as well as normal can be effectively generated as dedifferentiated pluripotent induced cells, thereby completing the present invention.

An object of the present invention can provide a composition for inducing pluripotent induced pluripotent cells (induced Pluripotent Stem cells, iPSCs).

In addition, an object of the present invention can provide a cell therapy agent.

In addition, it is an object of the present invention to provide a method for producing pluripotent inducible cells.

In order to achieve the above object, the present invention comprises a complex formed by combining graphene oxide (GO), polyethyleneimine (polyethylenimine: PEI), and RNA encoding a target gene, dedifferentiation pluripotent induced cells (induced Pluripotent Stem cells, iPSCs) provides a composition for induction.

In addition, the present invention provides a cell therapy product comprising dedifferentiated pluripotent induced cells produced by a composition for inducing dedifferentiated pluripotent induced cells.

In addition, the present invention comprises the steps of: (a) adding a polyethyleneimine (PEI) solution to a graphene oxide (GO) solution to form a GO-PEI complex; (b) separating total RNA from cells in which one or more genes selected from the group consisting of Oct3, Nanog, Sox2, Oct4, c-Myc, and Klf4 are respectively expressed; (c) forming a GO-PEI-RNA complex by adding the total RNA of step (b) to the GO-PEI complex of step (a); And (d) treating the GO-PEI-RNA complex of step (c) on cells other than human cells.

The composition for inducing dedifferentiated pluripotent induced cells of the present invention can effectively introduce a single strand of unstable mRNA into a cell, and the introduced mRNA is naturally degraded in the cell after a certain period of time and is integrated into the DNA of the host cell. It can be avoided and stably generates dedifferentiated pluripotent induced cells. In addition, cells isolated from patients as well as normal have the effect of generating effective dedifferentiation pluripotent induced cells, and thus can be usefully used in related industries.

1 is a diagram showing a method of separating peripheral blood mononuclear cells extracted from patient blood.
2 is a diagram showing a method of separating patient-derived skin fibroblasts.
3 is a diagram showing a method for islet-derived mesenchymal stem cells derived from a patient.
4 is a diagram schematically illustrating a method of producing dedifferentiated pluripotent induced cells using the GO-PEI/mRNA complex of the present invention.
5 is a diagram schematically illustrating a method for producing dedifferentiated pluripotent induced cells using Sendai virus.
6 is a dedifferentiation produced from patient-derived adult pancreatic stem cells (Islet-MSC) (A) and patient-derived skin fibroblast cells (HDFs) (B) using the GO-PEI/mRNA complex of the present invention and Sendai virus. It is a diagram showing the results of confirming the alkaline phosphatase activity and colony morphology of pluripotent induced cells.
7 is a dedifferentiation produced from patient-derived adult pancreatic stem cells (Islet-MSC) (A) and patient-derived skin fibroblast cells (HDFs) (B) using the GO-PEI/mRNA complex of the present invention and Sendai virus. This is a diagram confirming the expression level of stem cell markers of pluripotent induced cells.
8 is a pluripotent induction of dedifferentiation from patient-derived pancreatic adult stem cells (Islet-MSC) (A) and patient-derived skin fibroblast cells (HDFs) (B) using the GO-PEI/mRNA complex of the present invention and Sendai virus. This is a diagram confirming the results of immunofluorescence staining of stem cell markers of cells.

The present invention induces pluripotent induced pluripotent cells (iPSCs), including a complex formed by combining graphene oxide (GO), polyethylenimine (PEI), and RNA encoding a target gene It provides a composition for use.

The target gene is a pluripotency-related gene capable of inducing dedifferentiated pluripotent inducing cells, and is preferably any one or more selected from the group consisting of Oct3, Nanog, Sox2, Oct4, c-Myc and Klf4, More preferably, Oct3, Nanog and Sox2, but are not limited thereto.

The composition can introduce the target gene into the cell, but does not insert the target gene into the intracellular genome, which means that the introduced RNA is naturally degraded in the cell after a certain period of time and is integrated into the DNA of the host cell (integration ) Can be avoided to stably generate dedifferentiated pluripotent induced cells.

The composition may be treated on one or more cells selected from the group consisting of pancreatic beta cells, skin cells, cardiomyocytes, brain cells, nerve cells, adipocytes, bone cells, chondrocytes, muscle cells, hepatocytes, vascular cells, and lung cells. However, preferably, they are pancreatic beta cells and skin cells, and the cells may be cells isolated from a patient as well as normal.

The graphene oxide and polyethyleneimine may be combined with RNA at an N/P ratio of 1 to 40, and preferably an N/P ratio of 25 to 35, but is not limited thereto. The polyethyleneimine is 1 to 40 kDa, preferably 20 to 30 kDa, but is not limited thereto.

In the present invention, the term "induced pluripotent stem cell" includes induced pluripotent stem cells (iPS cells) produced by reprogramming somatic cells by expression or induction of expression of a reprogramming factor. Dedifferentiated pluripotent induced cells can be generated using fetal, postnatal, neonatal, juvenile or adult somatic cells. Dedifferentiation pluripotent induced cells can be newly generated (by methods known in the art) before differentiation into RPE cells or other cell types begins. Dedifferentiated pluripotent inducers can be specifically generated using material from a specific patient or matched donor for the purpose of generating tissue-matched RPE cells. Dedifferentiated pluripotent inducible cells can be prepared from cells that are not substantially immunogenic in the intended recipient, such as autologous cells or cells prepared from cells that are cytostatically compatible with the intended recipient. As a further example, dedifferentiating pluripotent induced cells can be generated by reprogramming somatic cells or other cells by contacting the cells with one or more reprogramming factors. For example, reprogramming factors can be expressed by cells, for example, from exogenous nucleic acids added to cells, or from endogenous genes that respond to factors that promote or induce expression of the gene, such as small molecules (Suh and Blelloch , Development 138, 1653-1661 (2011); Miyosh et al., ell Stem Cell (2011), doi:10.1016/j.stem. 2011.05.001; Sancho-Martinez et al., Journal of Molecular Cell Biology (2011) 1-3; Anokye-Danso et al., Cell Stem Cell 8, 376-388, April 8, 2011; Orkin and Hochedlinger, Cell 145, 835-850, June 10, 2011) Reprogramming factors are exogenous sources, e.g. For example, it can be provided by adding it to the culture medium and introducing into the cell by a method known in the art such as a cell-transducing peptide, protein or nucleic acid transfection agent, coupling with lipofectin, electroporation, gene gun, microinjection, etc. .

In addition, the present invention provides a cell therapy product comprising dedifferentiated pluripotent induced cells produced by a composition for inducing dedifferentiated pluripotent induced cells.

The cell therapy agent regenerates one or more cells selected from the group consisting of pancreatic beta cells, skin cells, cardiomyocytes, brain cells, nerve cells, adipocytes, bone cells, chondrocytes, muscle cells, hepatocytes, vascular cells, and lung cells. Transplantation or transplantation, preferably pancreatic beta cells, skin cells, the cells may be cells isolated from the patient as well as normal.

In the term of the present invention, "cell therapy" refers to the biological characteristics of cells by proliferating and selecting in vitro or other methods of living autologous, allogenic, and xenogenic cells to restore the function of cells and tissues. It refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the disease. The US has been managing cell therapy products as pharmaceuticals since 1993 and Korea since 2002. These cell therapy products can be broadly classified into two fields. The first is stem cell therapy for tissue regeneration or organ function recovery, and the second is immunity for regulating immune responses, such as suppressing the immune response in vivo or enhancing the immune response. It can be classified as a cell therapy.

The cell therapeutic agent of the present invention may be used as a pharmaceutical composition, and the pharmaceutical composition may further include an adjuvant. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, in the active ingredient. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to humans may occur.

In addition, the present invention comprises the steps of: (a) adding a polyethyleneimine (PEI) solution to a graphene oxide (GO) solution to form a GO-PEI complex; (b) separating total RNA from cells in which one or more genes selected from the group consisting of Oct3, Nanog, Sox2, Oct4, c-Myc, and Klf4 are respectively expressed; (c) forming a GO-PEI-RNA complex by adding the total RNA of step (b) to the GO-PEI complex of step (a); And (d) treating the GO-PEI-RNA complex of step (c) on cells other than human cells.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

<Example 1> Cell preparation and culture

1-1. Induced pluripotent cell culture

Dedifferentiated pluripotent induced cells are mTeSR?? supplemented with 10 μM Y27632 (ROCK inhibitor, Tocris Bioscience) in feeder-free system conditions. 1 medium (StemCell Technologies, Vancouver, BC Canada) was used and cultured in a 37°C, 5% CO ₂ incubator. In addition, the 409B2 hiPSCs line provided by Kyoto University through the RIKEN BioResource Center Cell Bank (HPS0076) was used as a positive control for the verification of the dedifferentiated pluripotent induced cells of the present invention. In addition, cells treated with Accutase (Stem cell Technologies) for subculture of hiPSC were cultured on a culture plate coated with Matrigel (Matrigel, BD Biosciences, SanJose, CA, USA) for 24 hours and then used with a feeder-free system. Replaced.

1-2. Isolation of patient-derived peripheral blood mononuclear cells (PBMC)

As shown in FIG. 1, in order to separate the patient-derived peripheral blood mononuclear cells, specifically, the provided patient-derived blood was separated using a density gradient centrifugation method through Ficoll-Plaque PLUS (GE Healthcare). After 10 ml of blood was mixed with 25 ml of phosphate buffered saline (PBS), 15 ml of Ficoll-Plaque was added, followed by centrifugation at 400 g for 30 minutes. After centrifugation, the PBMC centrifuged with the density gradient layer was transferred to a new tube, washed twice with 50 ml PBS, and centrifuged at 300 g for 10 minutes. The cells were prepared in RPMI1640 (GIBCO-BRL) in an incubator at 37° C., 5% CO ₂ using interleukin 2 (10 U/mL) and 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Cultured.

1-3. Isolation of patient-derived dermal fibroblasts (HDFs)

As shown in FIG. 2, in order to separate patient-derived skin fibroblasts, specifically, for cell separation, the patient's skin was cut 2.5 cm x 1 cm and washed three times with PBS. After that, chop it with a scalpel and add 1% penicillin/streptomycin (P/S, 100 U/ml, Gibco-BRL) and 5% FBS to Dulbecco's modified Eagle's (DMEM, Gibco-BRL). The added medium was treated with 1,000 U/ml Collagenase I (Invitrogen) and reacted in an incubator at 37° C. and 5% CO ₂ for 1 hour and 30 minutes. Thereafter, centrifugation was performed at 2,000 rpm for 15 minutes, followed by washing three times with PBS. The isolated fibroblasts were cultured in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin (P/S) in a 5% CO2 incubator at 37°C.

1-4. Isolation of patient-derived mesenchymal stem cells (MSCs)

As shown in FIG. 3, in order to isolate adult pancreatic stem cells in patient d, the tissue used for isolation of adult pancreatic stem cells was used by separating only normal pancreatic tissue of the patient with the consent of the patient undergoing pancreatic resection. Collagenase P (Invitrogen) was directly injected into the pancreatic duct into the separated pancreatic tissue, and the solution was put into a Ricordi Chamber to react while circulating the injected Collagenase P solution at 37°C. The isolated islet cells were isolated while the Collagenase P solution was circulating. At this time, islet cells were separated after rapidly staining beta cells with dithizone (DTZ) to prevent cell damage. The isolated islet cell fluid was divided into a cell layer according to a concentration gradient using a COBE 2991 apparatus, and pure islet cells stained with DTZ were checked, and the number and amount of cells were recorded, and the cells were separated. The isolated islet beta cells (pancreatic adult stem cells) were cultured in a DMEM medium containing 10% FBS and 1% P/S in a 5% CO2 incubator at 37°C.

<Example 2> Generation of reverse differentiation pluripotent induced cells

2-1. Production of GO-PEI/mRNA complexes and dedifferentiated pluripotent induced cells of the present invention

As shown in FIG. 4, dedifferentiated pluripotent induced cells were produced using the GO-PEI/mRNA complex.

first, The GO-PEI complex was fabricated. Specifically, 25kDa PEI solution (1 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA) was slowly added to GO solution (0.1 mg/ml) (Graphene Supermarket, Calverton, NY, USA) for 10 minutes. . After about 10 minutes of sonication, the mixture was stirred overnight, washed 5 times with deionized water using centrifugation, and re-dispersed in deionized water.

After that, a GO-PEI/mRNA complex was prepared. Specifically, a gel retardation assay was performed to establish conditions for complex formation of GO-PEI and RNA (0.5 ug each of three total RNAs of Oct3, Nanog, and Sox2). To identify the complex formation of GO-PEI and RNA, different N/P ratios (0, 7.5, 15, 30, and 45) of GO-PEI and 2 ug of total RNA complex were analyzed by agarose gel electrophoresis assay. . After incubating the GO-PEI and RNA mixture at room temperature for 20 minutes, the samples were heated at 65° C. for 10 minutes. The GO-PEI RNA complex could be confirmed by electrophoresis on an EtBr stained 1% (w/v) agarose gel. The gel was read using the gel documentation system (GL 2200 PRO Imaging system, CARESTREAM, Rochester, NY, USA). As a result, it was confirmed that there was a significant delay in the complex with the GO-PEI 30 N/P ratio, so a 2 ug RNA complex with GO-PEI of 30 N/P was used. The GO-PEI/mRNA complex of the present invention was put in a serum-free DMEM medium and reacted at 4° C. for 30 minutes, and then patient-derived peripheral blood mononuclear cells (PBMC), patient-derived skin fibroblast cells ( HDFs) or patient-derived pancreatic adult stem cells (MSCs). The efficiency of generation of dedifferentiated pluripotent induced cells was calculated by reflecting the number of colonies generated and the number of initially transformed cells.

2-2. Sendai virus and generation of pluripotent induced cells using the same

As shown in FIG. 5, as a control comparable to the GO-PEI/mRNA complex of the present invention, a method using Sendai virus, a method of transforming RNA, was tested and analyzed for comparison.

Specifically, peripheral blood monocytes were transformed through CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Life Technologies), and the Sendai virus kit was Oct4, Sox2, Klf4, c-Myc, that is, a reprogramming vector encoding Yamanaka factor was used as a virus. It is contained inside. Cells are cultured in a 12-well culture plate for 4 days prior to transformation at 5x10 ⁵ for each well, and the medium is 20% knockout serum replacement (KOSR) and cytokines (SCF, FLT-3, IL-). 3, IL-6) was added IMDM. Afterwards, Sendai virus was treated for 24 hours according to the appropriate MOI. Transformed cells are transferred to a 6-well plate surface-treated with Matrigel, and a medium for dedifferentiation pluripotent induction cells (20% KOSR and 1 mM non-essential amino acid, 1 mM GlutaMax, and 1% penicillin/streptomycin-added KO/F12 medium) Cultured. After 8 days, until colonies were formed (Fig. 5A), the medium was changed to a medium exclusively for dedifferentiated pluripotent induced cells to which bFGF was added. As shown in Fig. 5B, human skin cells and islet-derived mesenchymal stem cells were similarly reprogrammed through Sendai virus.

<Example 3> Alkaline phosphatase (AP) activity analysis and staining

In order to confirm the generation of dedifferentiated pluripotent induced cells using the GO-PEI/mRNA complex of the present invention, Sendai virus introduced-retrodifferentiated pluripotent induced cells and Yamanaka-dedifferentiated pluripotent induced cells were used as controls.

Specifically, Alkaline phosphatase staining kit (Sigma-Aldrich) was used. Specifically, cell fixation and permeability treatment was performed with acetone, formalin, and citric acid fixative, followed by staining with naphthol and fast red alkaline solution. AP activity analysis was performed using a fluorometer, and stained cells were measured with a phase contrast microscope (Olympus).

As shown in Fig. 6A, dedifferentiated pluripotent induced cells generated from patient-derived pancreatic adult stem cells (Islet-MSC) into which the GO-PEI/mRNA complex of the present invention has been introduced are (hR-iPSC-L1, hR- iPSC-L2) alkaline phosphatase activity and colony morphology were confirmed to be similar to embryonic stem cells. In addition, it was confirmed that it was similar compared to the dedifferentiated pluripotent induced cells (hR-SeV-L1, hR-SeV-L2) into which the positive control Sendai virus was introduced.

In addition, as shown in Fig. 6B, the dedifferentiated pluripotent induced cells generated from patient-derived skin fibroblast cells (HDFs) into which the GO-PEI/mRNA complex of the present invention has been introduced are (hR-iPSC-L1, hR- iPSC-L2) alkaline phosphatase activity and colony morphology were confirmed to be similar to embryonic stem cells. In addition, it was confirmed that alkaline phosphatase activity and colony morphology were more excellent compared to the dedifferentiated pluripotent induced cells (hR-SeV-L1, hR-SeV-L2) into which the positive control Sendai virus was introduced.

<Example 4> Confirmation of the efficiency of generating dedifferentiated pluripotent induced cells

Of dedifferentiated pluripotent induced cells produced by introducing the GO-PEI/RNA complex and Sendai virus of the present invention into patient-derived skin fibroblast cells (HDFs) and patient-derived adult pancreatic stem cells (beta cells) (Islet-MSC). The efficiency was compared. Specifically, the number of colonies, the number of dedifferentiated pluripotent induced cells, and efficiency (%) were confirmed, and the results are shown in Table 1 below.

As shown in Table 1, the GO-PEI/RNA complex of the present invention shows 2-3 times more colonies, the number of pluripotent induced cells, and the efficiency (%) compared to the Sendai virus introduction method, which is a positive control. Confirmed.

<Example 5> Quantitative real-time reverse transcription polymerase chain reaction analysis

RNA was extracted using Trizol (Invitrogen), and reverse transcription and cDNA were synthesized with 2 ug RNA using Super Script II (Invitrogen) kit. The DNA primers used in this example were designed using Primer3 software. Using Fast SYBR Green Master Mix (Applied Biosystems, Stockholm, Sweden), a quantitative real-time reverse orthogonal polymerase chain reaction was performed, and the data were standardized based on the expression level of the housekeeping gene GAPDH. Primers for Oct4, Nanog and Sox2 genes are shown in Table 2 below. In addition, exogenous and endogenous levels were confirmed, and primers for Sendai virus were used to confirm the exogenous level.

Gene name Forward Reverse Oct4 CGC AAG CCC TCA TTT CAC CAT CAC CTC CAC CAC CTG Nanog TCC TGA ACC TCA GCT ACA AAC GCG TCA CAC CAT TGC TAT TC Sox2 CAT CAC CCA CAG CAA ATG AC GAA GTC CAG GAT CTC TCT CAT AAA SeV GGA TCA CTA GGT GAT ATC GAG C AAC AGA CAA GAG TTT AAG AGA TAT GTA TC GAPDH AAT CCC ATC ACC ATC TTC CAG ATG ACC CTT TTG GCT CCC

As shown in Fig. 7A, in dedifferentiated pluripotent induced cells generated from patient-derived pancreatic adult stem cells (Islet-MSC) into which the GO-PEI/mRNA complex of the present invention has been introduced (hR-iPSC-L1, hR- iPSC-L2) It was confirmed that Oct4, Nanog and Sox2 expression were high. In addition, as shown in B of FIG. 7, in dedifferentiated pluripotent induced cells generated from patient-derived skin fibroblast cells (HDFs) into which the GO-PEI/mRNA complex of the present invention has been introduced (hR-iPSC-L1, hR- iPSC-L2) It was confirmed that Oct4, Nanog and Sox2 expression were high.

In addition, as a result of confirming the expression of three genes (Oct4, Sox2, Nanog), both endogenous and exogenous, only the expression of the (endogenous) gene expressed in the dedifferentiated pluripotent inducer itself was confirmed, and the expression of three genes introduced from the outside (exogenous). It was confirmed that did not appear.

<Example 6> Immunocytochemistry (ICC) analysis

In order to confirm the generation of dedifferentiated pluripotent induced cells using the GO-PEI/mRNA complex of the present invention, Sendai virus introduced-retrodifferentiated pluripotent induced cells and Yamanaka-dedifferentiated pluripotent induced cells were used as controls.

For immunocytochemical staining, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes at room temperature, and then washed three times with PBS. After washing, the fixed cells were permeable for 15 minutes with 0.1% Triton X-100 and washed three times with PBS. After washing, the cells were blocked with a blocking solution (10% normal goat serum) at room temperature for 1 hour. To confirm the expression of Oct4 and Nanog, which are embryonic stem cell-specific markers, OCT4 (polyclonal, 1: 300, Santa Cruz Biotechnology, Dallas, TX, USA) and NANOG (polyclonal, 1: 300, Santa Cruz Biotechnology, Dallas, TX, USA) after staining with a primary antibody, and a Fluorescently labeled (Alexa Fluor 488 or 568, Molecular Probes, Eugene, OR, USA) secondary antibody was used to detect the primary antibody. Cells were analyzed with a fluorescence microscope (Olympus).

As shown in Fig. 8A, in dedifferentiated pluripotent induced cells generated from patient-derived pancreatic adult stem cells (Islet-MSC) into which the GO-PEI/mRNA complex of the present invention has been introduced (hR-iPSC-L1) embryonic stem It was confirmed that the cell-specific markers Oct4 and Nanog were clearly stained.

In addition, as shown in FIG. 8B, in the dedifferentiated pluripotent induced cells produced from patient-derived skin fibroblast cells (HDFs) into which the GO-PEI/mRNA complex of the present invention was introduced (hR-iPSC-L1) embryonic stem It was confirmed that the cell-specific markers Oct4 and Nanog were clearly stained.

When compared with the dedifferentiated pluripotent induced cells generated from HDFs and MSCs into which Sendai virus or Yamanaka was introduced as a positive control, the dedifferentiated pluripotent induced cells of the present invention had a uniform degree of staining luminescence compared to the positive control, and colony It was confirmed that the number was high.

<110> Konkuk University Industrial Cooperation Corp THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Composition for inducing dedifferentiation for induced pluripotent stem cells <130> PN1810-424 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Oct4 <400> 1 cgcaagccct catttcac 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Oct4 <400> 2 catcacctcc accacctg 18 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Nanog <400> 3 tcctgaacct cagctacaaa c 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Nanog <400> 4 gcgtcacacc attgctattc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Sox2 <400> 5 catcacccac agcaaatgac 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Sox2 <400> 6 gaagtccagg atctctctca taaa 24 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for SeV <400> 7 ggatcactag gtgatatcga gc 22 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for SeV <400> 8 aacagacaag agtttaagag atatgtatc 29 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 9 aatcccatca ccatcttcca g 21 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 10 atgacccttt tggctccc 18

Claims (10)

As a composition for inducing pluripotent induced pluripotent cells (iPSCs), comprising a complex formed by combining graphene oxide (GO), polyethylenimine (PEI), and RNA encoding a target gene The composition is to induce dedifferentiation pluripotent induction cells by treatment with patient-derived skin fibroblasts (HDFs) or patient-derived pancreatic adult stem cells (MSC), and the target gene is to induce dedifferentiation pluripotent inducing cells. Nanog gene as a pluripotency-related gene, the graphene oxide and polyethyleneimine are combined with RNA at a ratio of 30 N/P, the polyethyleneimine is 25 kDa, and the composition contains the target gene A composition characterized in that it can be introduced into the cell, but the target gene is not inserted into the genome of the cell. delete delete delete delete delete delete A cell therapy product comprising dedifferentiated pluripotent induced cells produced by the composition for inducing dedifferentiated pluripotent induced cells of claim 1. delete (a) adding a polyethylenimine (PEI) solution to a graphene oxide (GO) solution to form a GO-PEI complex;
(b) separating total RNA from cells in which the Nanog gene is expressed;
(c) forming a GO-PEI-RNA complex by adding the total RNA of step (b) to the GO-PEI complex of step (a); And
(d) treating the GO-PEI-RNA complex of step (c) on cells excluding human cells; a method for producing dedifferentiated pluripotent induced cells, comprising: Skin fibroblasts (HDFs) or patient-derived pancreatic adult stem cells (mesenchymal stem cells, MSCs) to induce dedifferentiated pluripotent inducing cells, a method for producing dedifferentiated pluripotent inducing cells.

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