KR20220145043A - Preparation of stem cells improved tissue regeneration - Google Patents

Abstract

The present invention relates to a stem cell medicine that enhances tissue regeneration performance and is manipulated with a miRNA inhibitor. According to the present invention, antagomirs for miR 203a-3p, miR124-3p, miR181-5p, 128-3p, miR145-5p, miR-128-3p or miR 34a-5P or combinations thereof enhance an EMT gradient in an MSC to significantly improve a tissue regeneration promotion performance compared to conventional MSCs, so as to promote myocardial stem cell, recover and improve a heart function, protect myocardial cell, and promote myocardial regeneration to be used for treatment of cardiovascular diseases.

Description

Manufacturing method of stem cells with improved tissue regeneration ability {PREPARATION OF STEM CELLS IMPROVED TISSUE REGENERATION}

The present invention relates to stem cells with improved tissue regeneration ability, and to a stem cell therapeutic agent engineered with miRNA inhibitors to enhance tissue regeneration ability.

BACKGROUND ART Myocardial infarction is a fatal disease in which blood flow to the coronary artery supplying the myocardium is blocked, resulting in necrosis of the myocardium and decreased cardiac function. Currently, there are 16 million cases of myocardial infarction each year worldwide as of 2015, and 1 million cases of myocardial infarction occur in the United States alone each year (2016). Based on the US in 2011, myocardial infarction was found to be one of the five diseases with the highest medical cost during hospital stay, and it was found that it cost 612,000 days of hospitalization and 11 billion dollars of medical expenses per year (National inpatient Hospital Cost, 2011 report) ( Statistical Brief #160 (ahrq.gov) ).

Various types of thrombolytic agents are used for the treatment of myocardial infarction, and percultaneous coronary intervention (PCI) is used. It is also used for treatment (O'Connor et al., 2010). However, despite aggressive treatment such as anticoagulant therapy and coronary artery patency, the mortality rate is 10-12%, so a new approach to treat myocardial infarction is needed (Gershlick and More, 1998; Pollard, 2000; Williams). and Morton, 1995).

On the other hand, mesenchymal stromal cells (MSCs) exhibit multi-lineage differentiation and play a role in promoting the regeneration of various tissues through paracrine effects (Bazoobandi et al., 2020). ; Ryu et al., 2020). While they mainly inhibit cell necrosis and fibrosis in damaged tissues, they are known to promote regeneration of damaged tissues by secreting substances that promote angiogenesis and cell regeneration. In particular, it has been reported that MSCs have the potential to differentiate into vascular endothelial cells or cardiomyocytes in cardiovascular diseases, and that they play a role in regulating the inflammatory response in myocardial tissue due to myocardial infarction (Hatzistergos et al., 2010). ; Hume and Chong, 2020; Shafei et al., 2017). Accordingly, various clinical trials have been conducted for the treatment of myocardial infarction using MSC (Nakamura and Murry, 2019; Terashvili and Bosnjak, 2019).

However, clinical trials and efficacy evaluations using mesenchymal stem cells conducted at various medical institutions showed border line effects (Khodayari et al., 2019). Also, it was reported that MSCs do not directly contribute to the differentiation into cardiomyocytes in the results of confirming the effects of MSCs in animals by pulse-chasing experiments (Wu et al., 2011). Due to such technical limitations, the need for a new technology that can bring a more pronounced clinical effect for the treatment of cardiovascular diseases by MSC is being raised.

An object of the present invention is to provide a composition for improving the regeneration promoting ability of stem cells.

In addition, it is an object of the present invention to provide a method for producing stem cells with improved regeneration promoting ability.

Another object of the present invention is to provide a pharmaceutical composition for tissue regeneration.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular diseases.

Another object of the present invention is to provide a cell therapy agent for the prevention or treatment of cardiovascular diseases.

In addition, it is an object of the present invention to provide a method for promoting regeneration of stem cells.

In order to solve the above problems, the present invention provides a composition for promoting regeneration of stem cells comprising a microRNA inhibitor.

In addition, the present invention provides a method for producing stem cells with improved regeneration promoting ability.

In addition, the present invention provides a pharmaceutical composition for tissue regeneration containing the stem cells prepared by the method of the present invention as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases containing the stem cells prepared by the method of the present invention as an active ingredient.

In addition, the present invention is a composition for enhancing the regeneration promoting ability of stem cells of the present invention; And it provides a cell therapy agent for the prevention or treatment of cardiovascular diseases, including stem cells or cells differentiated from the stem cells.

In addition, the present invention provides a method for improving the regeneration promoting ability of stem cells.

According to the present invention, EMT in MSCs by antagomirs to miR 203a-3p, miR124-3p, miR181-5p, 128-3p, miR145-5p, miR-128-3p or miR 34a-5P, or a combination thereof By improving the gradient, the tissue regeneration promoting ability is significantly improved compared to the conventional MSC, and through this, it has the effect of promoting myocardial stem cell proliferation, restoring and improving cardiac function, protecting myocardial cells, and promoting myocardial regeneration. It can be used for therapeutic purposes.

1 is a diagram showing the results of screening miRNA groups capable of regulating the EMT gradient and pluripotency in human bone marrow-derived MSCs.
FIG. 2 is a grouping of miRNA groups targeting cellular EMT gradients and pluripotency-related genes.
3 is a diagram confirming the EMT gradient and pluripotency-related gene expression patterns of cells for each miRNA combination group.
4 is a diagram confirming the myocardial stem cell proliferation stimulating effect of each EMT-enhancing MSC (EMT-MSC) prepared by transfecting BM-MSC with antagomir for a miRNA combination group.
FIG. 5 is a diagram confirming the therapeutic effect of EMT-MSC (EMT-enhanced MSC prepared by transfection with antagomir for miR124-3p and antagomir for miR-145-5p) in myocardial infarction cell treatment in a permanent ligation animal model:
A: Cardiac function data; and
B: Echocardiography findings.
6 is a diagram confirming the myocardial protective effect of EMT-MSC (EMT-enhanced MSC prepared by transfecting antagomir for miR124-3p and antagomir for miR-145-5p together) in a permanent ligation animal model:
CM: Cardiomyocyte.
7 is a schematic diagram showing an experimental schedule in which EMT-MSC is injected into an IR animal model and the effect thereof is confirmed.
8 is a diagram confirming the therapeutic effect of EMT-MSC (EMT-enhanced MSC prepared by transfection with antagomir for miR124-3p and antagomir for miR-145-5p) in an IR animal model for treating myocardial infarction cells:
A: Cardiac function data; and
B: Echocardiography findings.
9 shows the cardiac function improvement effect of EMT-MSC (EMT-enhanced MSC prepared by transfection with antagomir for miR124-3p and antagomir for miR-145-5p) in an IR animal model through cardiac pressure-volume volume curve. It is confirmed
10 is a diagram confirming the myocardial protective effect of EMT-MSC (EMT-enhancing MSC prepared by transfecting antagomir for miR124-3p and antagomir for miR-145-5p together) in an IR animal model.
11 is a diagram confirming the direct myocardial regeneration effect of EMT-MSC (EMT-enhanced MSC prepared by transfection with antagomir for miR124-3p and antagomir for miR-145-5p) in an animal model of myocardial infarction.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples for the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express a preferred embodiment of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In one aspect, the present invention provides one or more micro RNA inhibitors selected from the group consisting of miR 203a-3p, miR124-3p, miR181-5p, 128-3p, miR145-5p, miR-128-3p and miR 34a-5P. It relates to a composition for improving the regeneration promoting ability of stem cells, including.

In one embodiment, the micro RNA inhibitor may be a nucleic acid molecule that specifically binds to the micro RNA, and the "nucleic acid" includes polynucleotides, oligonucleotides, DNA, RNA, and analogs thereof, and derivatives thereof. and include, for example, peptide nucleic acids (PNA) or mixtures thereof. Nucleic acids may also be single or double-stranded.

In one embodiment, the nucleic acid molecule is RNA (Ribonucleic acid), DNA (deoxyribonucleic acid), ribozyme, aptamer, antagomir, siRNA (small interfering RNA), miRNA, shRNA ( short hairpin RNA), PNA (peptide nucleic acid), and LNA (locked nucleic acid) may be at least one selected from the group consisting of, and more preferably antagomir (antagomir).

The term "antagomir" used in the present invention is a single-stranded chemically modified oligonucleotide, used to block endogenous miRNA, and has a sequence complementary to a target sequence. In the present invention, the complementary RNA sequence of miRNA is modified with hydroxyprolinol-linked cholesterol solid support and 2'-OMe phosphoramidites so that the antagonist can effectively bind to and inhibit miRNA (Kru tzfeldt, J., Rajewsky, N., Braich). , R., Rajeev, K.G., Tuschl, T., Manoharan, M., and Stoffel, M. (2005). Silencing of microRNAs in vivo with “antagomirs.” Nature 438, 685-689), but limited thereto. it's not going to be

As used herein, the term "complementary" means that the antisense oligonucleotide is sufficiently complementary to selectively hybridize to a miRNA target under predetermined hybridization or annealing conditions, preferably physiological conditions, and partially or partially substantially It has a meaning encompassing both substantially complementary and perfectly complementary. Substantially complementary, although not completely complementary, refers to complementarity to the extent that it binds to a target sequence and produces an effect according to the present invention, ie, sufficient to interfere with the expression of the miRNA.

In one embodiment, the microRNA inhibitor may include a nucleic acid encoding and expressing a nucleic acid molecule that specifically binds to the microRNA, and may be a plasmid comprising the same, and may be in the form of a plasmid used in the art. , vector form, antisense form, shRNA form, oligo form, etc. may be administered.

In one embodiment, the composition comprises miR145 antagomir and miR124 antagomir; miR145 antagomir and miR203a antagomir; miR145 antagomir, miR124 antagomir and miR34a antagomir; or a combination of miR145 antagonist, miR124 antagonist, and miR106b antagonist.

In one embodiment, the composition may induce the expression of an epithelial-mesenchymal transition (EMT) gradient-related gene and/or pluripotency-related gene in stem cells.

In one embodiment, the stem cell may be a mesenchymal stromal cell (MSC), and the mesenchymal stem cell is an umbilical cord mesenchymal stem cell, adipose-derived mesenchymal stem cell (adipose). derived mesenchymal stem cell) or bone marrow-derived mesenchymal stem cell (BM-MSC).

As used herein, the term “stem cell” refers to a cell having the ability to differentiate into two or more cells while having self-replicating ability, and “adult stem cell” refers to each organ of the embryo as the development process progresses. It refers to stem cells that appear in the stage of formation or in the adult stage.

The term "mesenchymal stem cell" used in the present invention is an undifferentiated stem cell isolated from human or mammalian tissue, and can be derived from various tissues, and the technology for isolating stem cells from each tissue is already in the art. is known.

In one embodiment, the composition may enhance/increase the tissue regeneration promoting ability of stem cells by enhancing the EMT gradient in stem cells.

As used herein, the term “miR” refers to “micro RNA” and may be used interchangeably.

Small RNA (hereinafter referred to as “sRNA”) refers to a ribonucleic acid having a length of about 17 to 25 nucleotides (nucleotide: hereinafter referred to as “nt”) that regulates gene expression in vivo. sRNA is largely classified into microRNA (microRNA, miR: hereinafter referred to as “miRNA”) and small interfering RNA (hereinafter referred to as “siRNA”) according to the method in which it is produced. miRNA is produced from partially double-stranded hairpin RNA, and siRNA is derived from long double-stranded RNA (hereinafter referred to as "dsRNA"). In general, it plays an important role in various regulatory processes in vivo. sRNA is miRNA, and miRNA is a single-stranded RNA (hereinafter referred to as “ssRNA”) molecule with a length of 19-25 nt and an endogenous hairpin-shaped transcript (Bartel, D.P., Cell 116:281-297, 2004; Kim, V.N., Mol. Cells. 19:1-15, 2005). miRNA acts as a post-transcriptional gene suppressor by complementarily binding to the 3' untranslated regions (UTRs) of the target mRNA, and suppresses the target gene by inducing translational repression and mRNA destabilization. miRNAs play important roles in various processes such as development, differentiation, proliferation, apoptosis and metabolism.

In addition, miRNA biosynthesis is initiated via transcription by RNA polymerase II (Cai. X., et al., RNA 10:1957-1966, 2004; Kim, V.N. Nat. Rev. Mol. Cell. Biol. 6: 376-385, 2005; Lee, Y., et al., EMBO J 21:4663-4670,2002; Lee, Y., et al., EMBO J 23:4051-4060, 2004). The primary miRNA (primary microRNA: hereinafter referred to as "pri-miRNA") usually exceeds a length of several Kb, and includes a 5' cap and poly A tail. pri-miRNA is ribocuclease III, Drosha (Lee, Y., et al., Nature 425:415-419, 2003) and its sub-element DGCR8 (Gregory, R.I. et al., Nature 432:235-240) , 2004; Han, J., et al., Genes Dev. 18:3016-3027, 2004; Landthaler, M., et al., Curr. Biol. 14:2162-2167, 2004). ) is first cleaved by a complex called ) and separated into about 65 nt hairpin-structure precursors (pre-miRNA). The pre-miRNA process is a central step in miRNA biosynthesis, and is defined as the process of generating one end of a molecule containing an mRNA sequence in a long pre-miRNA. The pre-miRNA generated after the initiation process is transported to the cytoplasm by the nuclear transport factor Exp5 (exportin-5) (Bohnsack, M. T., et al., RNA 10:185-191, 2004; Lund, E., et al.) al., Science 303:95-98, 2004; Yi, R., et al., Genes Dev. 17:3011-3016, 2003). In the cytoplasm, Dicer, an intracytoplasmic RNase III type protein, cuts the delivered pre-miRNA to generate a 22 nt miRNA double strand. One strand of the Dicer product is a mature miRNA that exists in the cytoplasm and is assembled with an effector complex called miRNP (microribonucleoprotein) or miRISC (miRNA-induced silencing complex) (Khvorova, A., et al. , Cell 115:209-216, 2003; Schwarz, D.S., et al., Cell 115:199-208, 2003). The RNAi phenomenon, which is a gene silencing mechanism by sRNA, is being used as an effective gene research tool in mammalian systems. Effective and stable gene knockdown occurs as small hairpin RNA (hereinafter referred to as “shRNA”) is expressed and processed into small interfering RNA (siRNA). Recent breakthroughs in RNAi technology have been achieved by constructing shRNA expression cassettes that mimic natural miRNA genes (Dickins, R.A., et al., Nat. Genet. 37:1289-1295, 2005; Silva, J.M. et al., Nat Genet. 37:1281-1288, 2005: Zeng, Y., et al., Mol. Cell 9:1327-1333. 2002). The miRNA-based shRNA regulated by the RNA polymerase II promoter effectively and stably induces gene silencing regulation in cultured cells such as animal models.

In one aspect, the present invention relates to a method for producing stem cells with improved regeneration promoting ability, comprising the step of treating the stem cells with the composition of the present invention.

In one embodiment, the method comprises at least one microRNA selected from the group consisting of miR 203a-3p, miR124-3p, miR181-5p, 128-3p, miR145-5p, miR-128-3p and miR 34a-5P. It may include the step of transfecting antagomir for stem cells.

In one embodiment of the present invention, antagomir for miR124-3p and antagomir for miR145-5p were transfected into mesenchymal stem cells to prepare EMT-enhanced MSCs inhibiting miR124-3p and miR-145-5p. , all these cells were named "EMT-MSC".

In one aspect, the present invention relates to a pharmaceutical composition for tissue regeneration containing the stem cells prepared by the method of the present invention as an active ingredient.

In one embodiment, the tissue may be cardiac tissue.

In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cardiovascular diseases containing the mesenchymal stem cells prepared by the method of the present invention as an active ingredient.

In one embodiment, the cardiovascular disease may be any one selected from the group consisting of arteriosclerosis, heart failure, myocardial infarction, hypertension, thrombosis, precoagulant state and atherosclerosis, more preferably myocardial infarction. .

In one embodiment, the pharmaceutical composition of the present invention can promote proliferation of myocardial stem cells, restore or improve cardiac function, protect myocardial cells, and promote myocardial regeneration.

As used herein, the term "treatment", unless otherwise stated, refers to a disease or condition to which the term applies, or one or more symptoms of the disease or condition to reverse, alleviate, inhibit the progression, or to prevent.

A composition is indicated to be "pharmaceutically or physiologically acceptable" if the recipient animal can tolerate administration of the composition, or if administration of the composition to that animal is suitable. When the amount administered is physiologically important, the agent can be said to have been administered in a "therapeutically effective amount". An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, administration method, target site, patient's condition, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical composition of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. have.

As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal or topical administration according to a desired method) ) or oral administration, and the dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the subject's age, weight, sex, physical condition, etc. is selected taking into account. It is self-evident that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 μg/ml to 50 mg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 50 mg/ml, it may be toxic to the human body.

The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg, silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, coloring, flavoring and sweetening agents. The formulation may be prepared by conventional mixing, granulating or coating methods. In addition, a representative formulation for parenteral administration is an injection formulation, and examples of the solvent for the injection formulation include water, Ringer's solution, isotonic saline or suspension. The sterile, fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil including mono- and di-glycerides can be used for this purpose. In addition, the injection preparation may use a fatty acid such as oleic acid.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of cardiovascular disease by administration of the pharmaceutical composition according to the present invention.

In one aspect, the present invention provides a composition for promoting regeneration of stem cells of the present invention; And it relates to a cell therapy agent for the prevention or treatment of cardiovascular diseases, including stem cells or cells differentiated from the stem cells.

As used in the present invention, the term "cell therapy agent" refers to cells and tissues manufactured through separation, culture, and special manipulation from an individual, and is a drug used for the purpose of treatment, diagnosis and prevention, in order to restore the function of cells or tissues. It refers to a drug in which living autologous, allogeneic or xenogeneic cells are used for the purpose of treatment, diagnosis and prevention of diseases through a series of actions such as in vitro proliferation and selection or changing the biological properties of cells in other ways.

The cell therapy composition can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil.

When formulating the cell therapy composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are mixed with at least one or more excipients such as cotton, starch, calcium carbonate, sucrose or lactose, gelatin, etc. is prepared In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. For oral administration of the pharmaceutical composition, liquid formulations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, Sweetening agents, flavoring agents, preservatives, and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerol gelatin, etc. may be used.

In the present invention, the preferred dosage of the cell therapy composition varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition is preferably administered at 0.01 mg/kg to 10 g/kg per day, preferably 1 mg/kg to 1 g/kg. Administration may be administered once a day, or may be administered in several divided doses.

The cell therapy composition may be administered to mammals such as mice, mice, livestock, and humans by various routes. All methods of administration may be by a conventional method, for example, may be administered through oral and rectal or intravenous methods.

In one aspect, the present invention relates to a method for enhancing the regeneration promoting ability of stem cells, comprising the step of treating the stem cells with the composition of the present invention.

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

Example 1. MSC regeneration promoting ability improvement miRNA screening

In mesenchymal stromal cells (MSC), when the EMT (epithelial-mesenchymal transition) gradient increases, the regeneration promoting ability is improved. Possible miRNA groups were screened. The target genes of the screened miRNAs were tracked through miRNA databases, and miRNAs regulating the expression of genes related to stemness in cells were screened through a similar miR database (Fig. 1). ).

As a result, it was confirmed that miR 203a-3p, miR124-3p, miR181-5p and 128-3p could enhance the EMT gradient in human bone marrow-derived MSCs, miR145-5p, miR-128-3p and miR 34a-5P. It was confirmed that they commonly target pluripotency genes (oct-4, nanog, Klf-4 and Sox-2) ( FIG. 2 ).

Example 2. miRNA candidates Confirmation of inducibility of EMT-related genes and pluripotency-related genes

In order to determine whether the antigomir combination of miRNA candidates selected in Example 1 can increase the expression of the EMT gradient and pluripotency-related genes required for cell regeneration, gene expression patterns for each combination were compared. Specifically, the antagomir (Table 1) of each miRNA selected above was ordered from Shanghai Genetics, Inc., and after transfection of various combinations of miR-antagomirs into MSCs derived from human bone marrow, Snai1, an EMT gradient related gene , Slug, Twist1, Zeb1 and Zeb2 and the expression levels of pluripotency-related genes Oct4, Nanog, Sox2 and KLF4 were confirmed by qRT-PCR.

As a result, four combinations of miR-antagomir (miR145 antagomir+miR124 antagomir, miR145 antagomir+miR203a antagomir, miR145 antagomir+miR124 antagomir+miR34a antagomir) and miR145 antagomir+miR124 antagomir+miR106b antagomir of the selected candidate groups were used as controls (miR145 antagomir+miR124 antagomir+miR106b antagomir). : NC), it was confirmed that the expression of EMT gradient-related genes and pluripotency-related genes can be induced (FIG. 3).

Example 3. Confirmation of EMT-hyper MSC treatment effect on myocardial infarction in permanent ligation model

3-1. Effect of EMT-MSC on myocardial stem cell proliferation stimulation

In order to observe the effect of EMT-MSC on the proliferation of myocardial stem cells involved in cardiac muscle regeneration, MSC derived from human bone marrow was attached to a plastic dish in DMEM (Dulbecco's Modified Eagles Media) supplemented with 10% FBS. After subculture (Ikebe and Suzuki, 2014), the antagomir for the miRNA combination group was transfected into BM-MSCs to produce EMT-enhanced MSCs (EMT-MSCs), which were derived from human fetal myocardium myocardial stem cells (c). -kit+) (Cardiac Progenitor Cell, CPC) was co-cultured, and then myocardial stem cells were stained with CFSE to determine the extent to which the intensity of CFSE stained on the cell membrane was weakened according to the proliferation of CPC (Cardiac Progenitor Cell).

As a result, compared to myocardial stem cells co-cultured with control naive MSC (+NC), myocardium co-cultured with EMT-enhanced MSC (+1, +2, +3 and +4) by each combination of miRNA antagomir Stem cells showed a higher percentage and number of P4 cell fractions, and it was confirmed that they had a significantly higher proliferative capacity (FIG. 4).

3-2. Cell therapy effect of EMT-MSC

In order to confirm the cell therapy effect of EMT-MSC on myocardial infarction, the effect of EMT-enhanced MSC on myocardial infarction animal model was analyzed after direct intramyocardial injection of EMT-MSC on recovery of cardiac function. Specifically, after thoracic incision of Fischer 344 (F344) rat, left anterior descending artery (LAD) ligation in the heart coronary artery 1 hour later, by removing the ligation, myocardial ischemia-reperfusion (ischemic reperfusion) model An animal model of myocardial infarction was prepared by inducing myocardial infarction. In addition, EMT-enhanced MSCs were prepared by transfecting together antagomir for miR124-3p, one of the EMT-related miRNAs, and antagomir for miR-145-5p, one of the pluripotency-related miRNAs, and the myocardial infarction animal model production process Upon induction of reperfusion by removal of the middle LAD ligation, each cell group was injected into the myocardium around the necrosis-induced myocardial tissue (infarct area) with a 31G insulin syringe. One week after ischemia-reperfusion, each cell group was injected a second time into the myocardium after open thoracic surgery in the same way. After 8 weeks, changes in cardiac function were confirmed using echocardiography. The contractility of the heart was evaluated through left ventricular ejection fraction (LVEF) and fractional shortening (FS), left ventricular internal systolic dimension (LVISd) and diastolic internal diameter (Left ventricular internal dimension). The change in heart size due to adverse remodeling was quantitatively evaluated through diastolic dimension LVIDd).

As a result, in myocardial infarction animal model, EMT-MSC significantly increased the left ventricular ejection fraction (% LVEF: left ventricular ejection fraction) and fractional shortening (% FS: fractional shortening) compared to the control MSC, and it was found that the diastolic internal diameter ( LVIDd) and systolic internal diameter (LVISd) were significantly decreased compared to the untreated group and the control group injected with MSC ( FIG. 5 ). It has also been shown to increase left ventricular wall thickness and increase relative wall thickness (RWT) associated with left ventricular dysfunction.

3-3. Myocardial protective effect of EMT-MSC

In order to analyze the mechanism by which EMT-MSC (EMT-enhanced MSC prepared by transfection with antagomir for miR124-3p and antagomir for miR-145-5p) in a permanent ligation model exhibits a therapeutic effect on myocardial infarction, myocardial infarction Fibrosis and survival rate of myocardial tissue after infarction were confirmed. For this purpose, immunofluorescence staining with hybridization-CHP (collagen hybridization peptide) and TNNT2 (troponin, cardiac muscle troponin T, cTnT) was performed, and quantitative analysis and comparison with Image J were used to confirm remodeling of myocardial tissue and recovery of cardiac function histologically. .

As a result, fibrosis was significantly reduced in the group injected with EMT-enhanced MSC (EMT-MSC) compared to the untreated group (CON) or control MSC (BM-MSC), and the viable myocardium (TNNT2+) was also increased. appeared (Fig. 6).

Through the above results, it can be seen that EMT-MSC has a cardiac function recovery and myocardial protective effect in an animal model of myocardial infarction (permanent ligation model).

Example 4. Confirmation of EMT-stimulated MSC myocardial infarction treatment effect in ischemic reperfusion (IR) model

4-1. Cell therapy effect of EMT-MSC

In order to confirm the effect of EMT-MSC treatment of myocardial infarction in a model similar to a clinical environment, EMT-MSC cell treatment in an ischemic reperfusion (IR) model in which the coronary artery of the myocardium is ligated for 1 hour and then recirculated. The effect was analyzed. Specifically, as shown in FIG. 7 , each EMT-MSC cell group (antagomir for miR124-3p and antagomir for miR-145-5p was transfected together in the periphery of myocardial infarction immediately after IR induction and 1 week after induction. EMT-enhanced MSC) was directly injected, and as a control, cardiac function 4 hours after IR occurred was set as the baseline, and the treatment effect was confirmed by echocardiography.

As a result, in the echocardiographic-based functional evaluation analyzed with LVEF%, FS%, LVIDd, and LVISd, the control MSC did not show a significant difference compared to the untreated group (CON), but the EMT-MSC treated group showed no significant difference between these two types. Compared to the control group, it showed a significant improvement in cardiac function (FIG. 8).

4-2. Hemodynamic analysis of cell therapy effect of EMT-MSC

After EMT-MSC was treated in the IR model, changes in the hemodynamic dimension were observed by analyzing the improvement of cardiac function through a cardiac pressure-volume curve using an intracardiac catheter.

As a result, it was confirmed that the contractile force of the EMT-MSC-treated group was significantly increased compared to the control and MSC-treated groups in the systolic and diastolic pressure/volume ratio, and stroke volume and cardiac output were also significantly increased. (Fig. 9).

Through this, it was confirmed that EMT-MSC significantly restored cardiac function compared to the untreated group or control group MSC even in myocardial infarction of the IR model, which is a model optimized for the clinical environment.

4-3. Myocardial protective effect of EMT-MSC

To analyze the mechanism of EMT-MSC's therapeutic effect on myocardial infarction in the IR model, myocardial necrosis, the amount of viable myocardium, and myocardial fibrosis after treatment for myocardial infarction were investigated. For this, EMT-MSC was injected into the IR model and myocardial necrosis (measured by Evan's blue & TTC test) was prevented within 24 hours, and myocardial cells ( cardiomyocyte), the survival rate (TNNT2+ cell number) was confirmed, and fibrosis was confirmed at the end of the experiment.

As a result, it was shown that EMT-MSCs significantly protected cardiomyocytes compared to control MSCs ( FIG. 10 ).

4-4. Confirmation of necrotic myocardial regeneration effect of EMT-MSC

To determine whether EMT-MSC promotes regeneration of necrotic myocardial tissue, EMT-stimulator produced by co-transfection of EMT-MSC injected into an IR model (antagomir for miR124-3p and antagomir for miR-145-5p) MSCs) can be directly differentiated into vascular cells or cardiomyocytes. For this purpose, after the injected MSCs are labeled with Dil, these cells are administered to the tissue where myocardial infarction has occurred, and the amount of differentiation into vascular cells (Isolectin B4: IB4+) or cardiomyocytes (TNNT2+/Dil+ or CX43+/Dil+) is fluoresced. It was confirmed by immunostaining.

As a result, compared to the group treated with the control MSC, the number of cells differentiating into vascular cells (IB4+/Dil+) and cardiomyocytes (TNN2+/Dil+ or CX43+/Dil+) in the myocardial tissue of the EMT-MSC-treated group was significantly higher appeared to increase (Fig. 11). Through this, it was confirmed that EMT-MSC promoted myocardial regeneration by differentiating into vascular cells and cardiomyocytes at a higher rate in the microenvironment where myocardial infarction occurred.

Through the above results, MSCs with enhanced EMT by the combination of antagomir for miR124-3p and antagomir for miR-145-5p significantly reduced cardiac function lost through myocardial protection and myocardial regeneration in the permanent ligation model and the IR model. It was confirmed that there was a significantly more effective treatment of myocardial infarction compared to conventional MSC.

Claims (9)

EMT (epithelial-mesenchymal transition) gradient (gradient) related gene and pluripotency (pluripotency) production method of a stem cell induced expression of a gene comprising the step of treating the stem cells with a mir145 inhibitor and a miR124 inhibitor. The method of claim 1 , wherein mir145 is mir-145-5p and miR124 is miR-124-3P. The method of claim 1 , wherein the mir145 inhibitor or miR124 inhibitor is at least one selected from the group consisting of an inhibitory RNA molecule, an antagonist microRNA, an enzymatic RNA molecule, and an anti-miRNA antibody. The method according to claim 1, wherein the mir145 inhibitor or miR124 inhibitor is an antisense oligonucleotide, antagomir, ribozyme, aptamer, siRNA, miRNA that specifically binds to mir145 or miR124. , shRNA (short hairpin RNA), PNA (peptide nucleic acid) and LNA (locked nucleic acid) at least one selected from the group consisting of, the method. The method of claim 1, wherein the stem cells are mesenchymal stem cells. A pharmaceutical composition for preventing or treating myocardial infarction comprising stem cells treated with mir145 inhibitor and miR124 inhibitor. A stem cell prepared by the method of claim 1 . A method of inducing the expression of an epithelial-mesenchymal transition (EMT) gradient-related gene and pluripotency-related gene in stem cells, comprising the step of treating the stem cells with a mir145 inhibitor and a miR124 inhibitor. The method of claim 8, wherein the EMT gradient-related gene is Snai1, Slug, Twist1, Zeb1 or Zeb2, and the pluripotency-related gene is Oct4, Nanog, Sox2 or KLF4.

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