Method for inducing 3D cell reprogramming using hyaluronic acid-based hydrogel

Abstract

The present invention relates to a method for inducing 3D cell reprogramming using a hyaluronic acid-based hydrogel, and provides a method for inducing cell reprogramming of adult cells, the method comprising the steps of: preparing a hydrogel solution containing hyaluronic acid and a polymer; suspending adult cells in the hydrogel solution; encapsulating the adult cells by solidifying the hydrogel solution in which the adult cells are suspended; and culturing or injecting into a body the adult cells encapsulated in the hyaluronic acid-based 3D hydrogel.

Description

Method for inducing 3D cell reprogramming using a hyaluronic acid-based hydrogel

[0001] The present invention relates to a method for inducing 3D cell reprogramming using a hyaluronic acid-based hydrogel.

[0002] Pluripotent stem cells (PSCs) refer to cells possessing pluripotency. Because they can differentiate into various tissues, their efficacy in establishing disease models for various conditions, drug screening, and cell therapy is being confirmed. Recently, research in the field of regenerative medicine utilizing PSCs has been active. It has been confirmed that differentiating PSCs into specific cell types and transplanting them into patients increases in vivo survival and proliferation rates compared to transplanting conventional adult cells. However, PSCs, such as conventional embryonic stem cells, present challenges in personalized patient treatment involving immune responses. Furthermore, ethical issues regarding the sacrifice of potential fetuses to obtain these cells necessitate the need for alternative methods.

[0003] Induced pluripotent stem cells (iPSCs) have recently been reported as a means to secure pluripotent stem cells that can replace embryonic stem cells. These iPSCs are reprogrammed to possess pluripotency through an artificial dedifferentiation process of already differentiated cells that lack pluripotency. Since iPSCs can be produced using cells extracted from patients, they offer the advantage of enabling personalized patient treatment and avoiding ethical issues. Furthermore, they present the potential to generate iPSCs through in vivo cell reprogramming, in addition to the existing in vitro methods. However, existing iPSC production methods exhibit a very low induction efficiency of approximately 1%, leading to proposals for improved methods to increase cell volume. While methods utilizing various cytokines and chemicals have been suggested for this purpose, their clinical application is limited due to safety concerns and high costs. Since iPSCs can develop various genetic mutations and abnormal karyotyping depending on culture composition and the introduction of extracellular or intracellular substances, materials with proven safety must be used during the dedifferentiation induction process. Accordingly, the possibility of combinations of external factors that regulate cell characteristics by controlling the external environment without delivering substances into the cell is also being suggested.

[0004] The background description of the invention is provided to facilitate a better understanding of the present invention. The matters described in the background description should not be construed as an acknowledgment that they exist as prior art.

[0005] Meanwhile, conventional methods for producing induced pluripotent stem cells (iPSCs) show a very low induction efficiency of about 1%, and alternative methods for producing iPSCs using various cytokines and chemicals also have the problem of low safety. Therefore, the need for a technology that can improve the induction efficiency of cell reprogramming through external stimulation using a substance with guaranteed safety in both in vitro and in vivo environments was recognized.

[0006] Furthermore, it was noted that components of the extracellular matrix (ECM) can influence the reprogramming process, physical factors directly affect the cell surface to induce morphological changes through cytoskeletal reconstruction, and hyaluronic acid binds to CD44 to regulate various cellular physiological functions such as cell adhesion, migration, proliferation, differentiation, and survival.

[0007] Accordingly, the inventors of the present invention sought to develop a technology for inducing reprogramming from adult cells into induced pluripotent stem cells by constructing a hyaluronic acid microenvironment using a hydrogel with excellent in vivo safety.

[0008] As a result, the inventors of the present invention prepared a hyaluronic acid-based 3D hydrogel containing encapsulated cells, and confirmed that the hyaluronic acid in the 3D hydrogel increases the reprogramming efficiency from adult cells to induced pluripotent stem cells through interaction with the CD44 protein, and that when the 3D hydrogel is exposed to low-power ultrasound, it increases the intracellular expression of CD44 in the hyaluronic acid (HA) microenvironment to promote reprogramming efficiency, thereby completing the present invention.

[0009] Accordingly, the problem that the present invention aims to solve is to provide a hydrogel composition for promoting cell reprogramming of adult cells comprising hyaluronic acid and a polymer.

[0010] In addition, another problem that the present invention aims to solve is to provide a method for manufacturing a hyaluronic acid-based 3D hydrogel comprising encapsulated cells for promoting cell reprogramming.

[0011] In addition, another problem that the present invention aims to solve is to provide a hyaluronic acid-based 3D hydrogel for promoting cell reprogramming, prepared by a method for preparing a hyaluronic acid-based 3D hydrogel containing the encapsulated cells.

[0012] In addition, another problem that the present invention aims to solve is to provide a method for inducing cell reprogramming of adult cells using a hyaluronic acid-based 3D hydrogel containing encapsulated cells.

[0013] In addition, another problem that the present invention aims to solve is to provide induced pluripotent stem cells produced by a method of inducing cell reprogramming of adult cells using a hyaluronic acid-based 3D hydrogel containing the encapsulated cells.

[0014] The problems of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0015] In order to solve the problem described above, a hydrogel composition for promoting cell reprogramming of adult cells comprising hyaluronic acid and a polymer according to one embodiment of the present invention is provided.

[0016] To solve the problem described above, a method for manufacturing a hyaluronic acid-based 3D hydrogel for promoting cell reprogramming is provided, comprising the steps of: preparing a hydrogel solution containing hyaluronic acid and a polymer according to another embodiment of the present invention; suspending adult cells in the hydrogel solution; and solidifying the hydrogel solution in which adult cells are suspended to encapsulate the adult cells.

[0017] According to a feature of the present invention, the hyaluronic acid may be methacrylated hyaluronic acid.

[0018] According to another feature of the present invention, the polymer may be one or more selected from the group consisting of gelatin, alginate, carrageenan, agarose, chitosan, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), and polyurethane.

[0019] According to another feature of the present invention, the gelatin may be methacrylated gelatin.

[0020] According to another feature of the present invention, the hydrogel solution may comprise 0.5 to 1.5 parts by weight of hyaluronic acid, 12 to 16 parts by weight of gelatin, and 8 to 12 parts by weight of polyethylene glycol.

[0021] According to another feature of the present invention, the adult cell may be an adult cell separated from an individual.

[0022] According to another feature of the present invention, the adult cell may be selected from the group consisting of fibroblasts, myofibroblasts, fibro-adipogenic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose-derived stem cells, bone marrow-derived stem cells, neural stem cells, and muscle stem cells.

[0023] According to another feature of the present invention, the adult cells are 2 × 10 5 cells / mL to 2 × 10⁶ 7 It may be included at a cell density of cells / mL.

[0024] According to another feature of the present invention, the 3D hydrogel may promote cell reprogramming into a lineage of cells with different existing epigenetic and differentiation characteristics of adult cells, and may promote the reprogramming of adult cells into at least one cell selected from the group consisting of induced pluripotent stem cells (iPSCs); intermediate reprogrammed cells such as neural stem cells, epithelial stem cells, mesenchymal stem cells, hematopoietic stem cells, pancreatic endoderm, and hepatic endoderm; and progenitor cells.

[0025] According to another feature of the present invention, if the adult cell is a cell transformed to overexpress one or more genes selected from the group consisting of the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene, cell reprogramming into induced pluripotent stem cells may be promoted.

[0026] According to another feature of the present invention, the solidification may be performed by any one method selected from the group consisting of photocrosslinking, chemical crosslinking, thermal gelation, and ionic crosslinking.

[0027] To solve the problem described above, a hyaluronic acid-based 3D hydrogel for promoting cell reprogramming is provided, prepared by a method for preparing a hyaluronic acid-based 3D hydrogel comprising the encapsulated cells according to another embodiment of the present invention.

[0028] To solve the problem described above, a method for inducing cell reprogramming of adult cells is provided, comprising the steps of: preparing a hyaluronic acid-based 3D hydrogel by encapsulating adult cells in a hydrogel comprising hyaluronic acid and a polymer according to another embodiment of the present invention; and culturing or injecting into the body the adult cells encapsulated in the hyaluronic acid-based 3D hydrogel.

[0029] According to a feature of the present invention, the method may promote cell reprogramming into a lineage of cells with different existing epigenetic and differentiation characteristics of adult cells, and may promote the reprogramming of adult cells into at least one cell selected from the group consisting of induced pluripotent stem cells (iPSCs); intermediate reprogrammed cells such as neural stem cells, epithelial stem cells, mesenchymal stem cells, hematopoietic stem cells, pancreatic endoderm, and hepatic endoderm; and progenitor cells.

[0030] According to a feature of the present invention, the adult cell may be an adult cell separated from an individual.

[0031] According to another feature of the present invention, if the adult cell is a cell transformed to overexpress one or more genes selected from the group consisting of the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene, cell reprogramming into induced pluripotent stem cells may be promoted.

[0032] According to another feature of the present invention, the step of preparing a hyaluronic acid-based 3D hydrogel by encapsulating adult cells in a hydrogel comprising hyaluronic acid and a polymer comprises:

[0033] The method may include the steps of preparing a hydrogel solution containing hyaluronic acid and a polymer, suspending adult cells in the hydrogel solution, and solidifying the hydrogel solution in which the adult cells are suspended to encapsulate the adult cells.

[0034] According to another feature of the present invention, the step of culturing or injecting into the body adult cells encapsulated in the hyaluronic acid-based 3D hydrogel comprises:

[0035] It may further include a step of stimulating adult cells encapsulated in the hyaluronic acid-based 3D hydrogel with low-intensity ultrasound (LIUS) during the culture period or after injection into the body.

[0036] According to another feature of the present invention, the step of stimulating adult cells encapsulated in the hyaluronic acid-based 3D hydrogel with low-power ultrasound comprises:

[0037] 20 kHz to 1000 kHz frequency and 30 mW cm -2 Up to 500 mW cm -2 It may involve treating with low-power ultrasound of low intensity for 5 to 25 minutes.

[0038] According to another feature of the present invention, the exposure period of the low-power ultrasound may be 1 to 3 days.

[0039] In order to solve the problem described above, induced pluripotent stem cells are provided by a method for inducing cell reprogramming from adult cells into induced pluripotent stem cells using a hyaluronic acid-based 3D hydrogel containing the encapsulated cells according to another embodiment of the present invention.

[0040] The hyaluronic acid-based 3D hydrogel of the present invention is designed to mimic the physical and chemical properties of the extracellular matrix (ECM) during the cell reprogramming process and has the effect of promoting the reprogramming of adult cells by providing a hyaluronic acid microenvironment to adult cells.

[0041] In addition, the present invention has the effect of significantly improving the reprogramming efficiency of adult cells by providing a step of exposing adult cells encapsulated in a hyaluronic acid-based 3D hydrogel to low-power ultrasound.

[0042] Furthermore, since the present invention utilizes a hydrogel having excellent in vivo safety, it can provide clinically safe induced pluripotent stem cells unlike conventional methods using chemical substances, and the induced pluripotent stem cells produced by the present invention can be utilized in various fields such as regenerative medicine, production of disease models, identification of disease causes, and new drug development.

[0043] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0044] FIG. 1a illustrates a flowchart of a method for preparing a hyaluronic acid-based hydrogel according to one embodiment of the present invention.

[0045] FIGS. 1B and FIGS. 1C illustrate a flowchart of a method for preparing methacrylated hyaluronic acid in a method for preparing a hyaluronic acid-based hydrogel according to one embodiment of the present invention.

[0046] FIG. 1d illustrates a flowchart of a method for preparing methacrylated gelatin in a method for preparing a hyaluronic acid-based hydrogel according to one embodiment of the present invention.

[0047] FIG. 1e illustrates a flowchart of a method for manufacturing a hydrogel in a method for manufacturing a hyaluronic acid-based hydrogel according to one embodiment of the present invention.

[0048] Figure 2 illustrates the results of measuring the DS (Degree of Substitution) of synthesized methacrylated hyaluronic acid according to one embodiment of the present invention.

[0049] FIG. 3 illustrates a flowchart of a method for manufacturing a hyaluronic acid-based 3D hydrogel containing encapsulated cells according to one embodiment of the present invention.

[0050] FIG. 4a illustrates a flowchart of a method for inducing reprogramming from adult cells into induced pluripotent stem cells using a hyaluronic acid-based 3D hydrogel containing encapsulated cells according to one embodiment of the present invention.

[0051] FIGS. 4b and 4c illustrate a schematic diagram of a method for inducing reprogramming from adult cells into induced pluripotent stem cells using a hyaluronic acid-based 3D hydrogel containing encapsulated cells according to one embodiment of the present invention.

[0052] Figures 5a and 5b illustrate the results of measuring cell viability according to LIUS stimulation time for OG-MEF-HA hydrogel and hASC-HA hydrogel according to one embodiment of the present invention.

[0053] FIGS. 6a to 6c illustrate the results of measuring the GFP expression level according to LIUS stimulation time of an OG-MEF-HA hydrogel according to one embodiment of the present invention (fluorescence image, relative fluorescence intensity, number of iPSC colonies).

[0054] FIGS. 7a to 7d illustrate the results of immunofluorescence staining for SOX2, NANOG, SSEA4, and TRA-1-60 proteins and the results of measuring the number of iPSC colonies according to LIUS stimulation time for OG-MEF-HA hydrogel and hASC-HA hydrogel according to one embodiment of the present invention.

[0055] Figure 8 illustrates the results of flow cytometry analysis according to the presence or absence of LIUS stimulation of the OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0056] FIG. 9a shows the results of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis for Oct4, Nanog, and Sox2 according to LIUS stimulation time of OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0057] Figure 9b shows the Western blot results for OCT4, NANOG, and SOX2 proteins according to LIUS stimulation time of the OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0058] FIGS. 10a and 10b illustrate the results of an analysis of changes in MET (Mesenchymal-to-Epithelial Transition) according to LIUS stimulation time of an OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0059] Figure 11 illustrates the results of histone modification analysis according to LIUS stimulation time of an OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0060] FIGS. 12a and 12b illustrate the results of immunofluorescence staining for F-actin according to LIUS stimulation time of an OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0061] FIG. 12c shows the Western blot results for FAK and pFAK according to LIUS stimulation time of the OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0062] Figure 13 illustrates the results of evaluating cell membrane fluidity according to LIUS stimulation time of an OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0063] Figure 14 illustrates the results of an analysis of hydrogel characteristics (shear modulus and swelling ratio) according to LIUS stimulation time of a HA hydrogel according to one embodiment of the present invention.

[0064] FIGS. 15a to 15d illustrate the results of immunofluorescence staining for CD44, OCT4, NANOG, and SOX2 proteins according to the components (gelatin, PEG, hyaluronic acid) of a 3D hydrogel encapsulated with OG-MEF cells according to one embodiment of the present invention and the presence or absence of LIUS stimulation.

[0065] FIG. 16 illustrates a schematic diagram of an experimental process for confirming the efficiency of iPSC reprogramming according to HA hydrogel and LIUS stimulation in an in vivo environment according to one embodiment of the present invention.

[0066] FIGS. 17a to 17c illustrate the results of immunofluorescence staining for OCT4-GFP, NANOG, and SOX2 proteins in vivo with and without LIUS stimulation of the OG-MEF-HA hydrogel according to one embodiment of the present invention.

[0067] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0068] In the following, terms used within this specification are explained for clarity of explanation.

[0069] In this document, expressions such as "have," "can have," "include," or "can include" refer to the existence of the relevant feature (e.g., numerical values, functions, actions, or components, etc.) and do not exclude the existence of additional features.

[0070] In this document, "or" means "and / or" unless otherwise noted. Expressions such as "A or B," "at least one of A or / and B," or "one or more of A or / and B" may include all possible combinations of items listed together. For example, "A or B," "at least one of A and B," or "at least one of A or B" may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0071] The term “hydrogel” as used in this invention refers to a polymeric material containing a large amount of water, which has the characteristic of absorbing water and expanding or contracting within it. Furthermore, it is composed of a polymeric network that is physically or chemically cross-linked, and interacts with water to maintain a solid state while possessing flexible and soft properties. Such hydrogels have high in vivo stability and are used in various biomedical applications, such as drug delivery, tissue engineering, and wound healing.

[0072] The term “hydrogel” as used in the present invention refers to a hydrogel having a three-dimensional network structure. In the present invention, it refers to a hydrogel in which cells are encapsulated within a hydrogel matrix to form a three-dimensional network, providing an environment in which cells can grow or be active. Such a 3D hydrogel is advantageous for promoting cell-cell and cell-matrix interactions.

[0073] The term "adult cell" as used in the present invention encompasses all cells derived from adult tissues and includes all various cell types. Preferably, it may be an adult cell isolated from an individual. In this context, the term "individual" includes not only subjects requiring methods for the prevention, control, or treatment of disease, but also healthy subjects. That is, the adult cell may be the cell of the patient requiring prevention, control, or treatment, or it may be the cell of a healthy donor.

[0074] The term “individual” as used in this invention may be used without limitation and includes humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0075] The term “photoinitiator” as used in the present invention refers to a compound that is activated by absorbing ultraviolet (UV) light or visible light, and promotes the solidification of a hydrogel by inducing a polymerization reaction through the energy generated at that time. It is mainly used to form a hydrogel network structure by causing polymerization or cross-linking reactions of polymers.

[0076] The term “hyaluronic acid microenvironment” as used in the present invention refers to an environment in the extracellular matrix (ECM) where hyaluronic acid acts as a major component, and said hyaluronic acid microenvironment has characteristics such as cell adhesion and migration, cell signaling, cell reprogramming, and regeneration.

[0077] As used in the present invention, the term “vector” refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. A vector may be a plasmid, a phage particle, or simply a potential genomic insert. When transformed into a suitable host cell, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, “plasmid” and “vector” are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferable to use a plasmid vector. A typical plasmid vector that can be used for this purpose has a structure comprising (a) a replication initiation site that enables efficient replication to contain several to hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that enables the host cell transformed into the plasmid vector to be selected, and (c) a restriction enzyme cleavage site into which an alien DNA fragment can be inserted. Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using synthetic oligonucleotide adapters or linkers according to conventional methods. After ligation, the vector must be transformed into a suitable host cell.

[0078] The term “transformation” as used in this invention refers to a change in the genetic properties of a host cell as it accepts DNA provided from the outside.

[0079] In addition, the above transformation may be performed using suitable standard techniques known in the art, such as electroporation, electroinjection, microinjection, calcium phosphate co-precipitation, retroviral infection, cationic liposome method, polyethylene glycol-mediated uptake, gene gun, etc., but is not limited thereto.

[0080] The term "recombination" as used in the present invention refers to any substance (e.g., a vector or an animal cell) that can be formed as a result of genetic manipulation.

[0081] The term “host cell” as used in the present invention refers to a cell capable of utilizing its own transcription system by introducing the vector of the present invention into itself to express the target protein, and may be any known type of cell.

[0082] The term "culture" as used in this invention refers to the growth of a target cell or tissue, etc., under artificially controlled environmental conditions. Representative environmental conditions include nutrients, temperature, osmotic pressure, pH, gas composition, and light; however, the medium exerts the direct influence, and the medium can be broadly classified into liquid and solid media. The culture of the host cell of this invention can be performed using methods widely known in the art.

[0083] The term “expression” as used in the present invention refers to the transcription and / or translation processes occurring within a cell. The transcription level of the target product within the host cell may be determined based on the amount of corresponding mRNA present within the cell. For example, mRNA transcribed from the corresponding sequence may be quantified by PCR, etc. The polypeptide translated by the polynucleotide may be quantified using various methods, e.g., ELISA, analysis of the biological activity of the polypeptide, or analysis independent of said activity, such as radioimmunoassay using an immunoglobulin recognized and bound to the polypeptide, or Western blotting.

[0084] The term “introduction” as used in the present invention may be performed by a process involving the transformation of a protoplast, followed by the production of a protoplast and the regeneration of a cell wall. DNA inserted into the protoplast may be inserted into the chromosomes of a host cell. Commonly known genetic engineering methods may be used to insert genes onto the chromosomes of a host cell, including, for example, electroporation, lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polyvalent cations or lipid:nucleic acid conjugates, naked DNA, artificial virons, and chemically induced DNA infusion. Sonoporation, for example using the Sonitron 2000 system (Rich-Mar), may also be used for nucleic acid delivery, and other representative nucleic acid delivery systems include the methods of Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), and BTX Molecular System (Holliston, MA). Lipofection methods are specified in U.S. Patent No. 5,049,386, U.S. Patent No. 4,946,787, and U.S. Patent No. 4,897,355, and lipofection reagents are commercially available, for example, Transfectam TMand Lipofectin TM Cationic or neutral lipids suitable for the effective receptor-recognition lipofection of polynucleotides include Felgner's lipids (WO91 / 17424 and WO91 / 16024), which can be delivered into cells via ex vivo introduction and into target tissues via in vivo introduction. Methods for preparing lipid:nucleic acid complexes containing targeted liposomes, such as immunolipid complexes, are well known in the industry (Crystal, Science., 270:404-410, 1995; Blaese et al., Cancer Gene Ther., 2:291-297, 1995; Behr et al., Bioconjugate Chem., 5:382389, 1994; Remy et al., Bioconjugate Chem., 5:647-654, 1994; Gao et al., Gene Therapy., 2:710-722, 1995; Ahmad et al., Cancer Res., 52:4817-4820, 1992; U.S. Patent No. 4,186,183; U.S. Patent No. 4,217,344; U.S. Patent U.S. Patent No. 4,235,871; U.S. Patent No. 4,261,975; U.S. Patent No. 4,485,054; U.S. Patent No. 4,501,728; U.S. Patent No. 4,774,085; U.S. Patent No. 4,837,028; U.S. Patent No. 4,946,787). The inserted DNA sequence is maintained stably within the cell. DNA is typically inserted into homologous or non-homologous sites within the chromosome, and is typically inserted in 1 to 20 or more copies.

[0085] In one aspect, the present invention relates to a hydrogel composition for promoting cell reprogramming of adult cells, comprising hyaluronic acid and a polymer.

[0086] In one aspect, the present invention relates to a method for preparing a hyaluronic acid-based 3D hydrogel comprising encapsulated cells for promoting cell reprogramming.

[0087] Hereinafter, with reference to FIG. 3, a method for manufacturing a hyaluronic acid-based 3D hydrogel comprising encapsulated cells according to one embodiment of the present invention will be described. For convenience of explanation, the description will be made with reference to FIG. 1a to 1e.

[0088] Referring to FIG. 3, the present invention comprises the steps of preparing a hydrogel solution containing hyaluronic acid and a polymer (S510), suspending adult cells in the hydrogel solution (S520), and solidifying the hydrogel solution in which adult cells are suspended to encapsulate the adult cells (S530).

[0089] In one embodiment of the present invention, the hyaluronic acid may be methacrylated hyaluronic acid, but is not limited thereto.

[0090] Accordingly, the step (S510) of preparing a hydrogel solution containing hyaluronic acid and a polymer according to the present invention may further include the step (S110) of preparing methacrylated hyaluronic acid.

[0091] Referring to FIG. 1b, the step (S110) of preparing methacrylated hyaluronic acid according to one embodiment of the present invention includes the step of mixing an aqueous solution of hyaluronic acid and a methacrylic anhydride (S210), the step of stirring the mixture under dark conditions to induce a methacrylic group (-CH2=C(CH3)CO-) bonding reaction (S220), the step of dialyzing the reaction-completed mixture with deionized water using a dialysis membrane (S230), and the step of removing residual impurities (S240).

[0092] Meanwhile, the method for producing methacrylated hyaluronic acid is not limited to the chemical synthesis method shown in FIG. 1b and can be produced through methods known in the art, such as enzymatic conversion methods and photochemical methods.

[0093] In one embodiment of the present invention, the polymer may include all of the following: natural polymers such as gelatin, alginate, carrageenan, agarose, and chitosan; synthetic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), and polyurethane; preferably, it may be a combination of natural polymers and synthetic polymers; more preferably, it may be a combination of methacrylated natural polymers and synthetic polymers; and most preferably, it may be methacrylated gelatin and polyethylene glycol, but is not limited thereto.

[0094] Accordingly, the step (S510) of preparing a hydrogel solution containing hyaluronic acid and a polymer according to the present invention may further include the step (S120) of preparing methacrylated gelatin.

[0095] Referring to FIG. 1d, the step (S120) of preparing the methacrylated gelatin comprises the steps of dissolving the gelatin in a phosphate buffer solution (S310), injecting methacrylic anhydride into the solution and stirring under dark conditions to induce a methacrylic group (-CH2=C(CH3)CO-) bonding reaction (S320), filtering the solution after the reaction is completed (S330), and dialyzing the filtered solution with distilled water using a dialysis membrane (S340).

[0096] Meanwhile, the method for producing methacrylated gelatin is not limited to the chemical synthesis method shown in FIG. 1d and can be produced through methods known in the art, such as enzymatic conversion methods and photochemical methods.

[0097] In one embodiment of the present invention, the hydrogel solution may comprise 0.5 to 1.5 parts by weight of hyaluronic acid, 12 to 16 parts by weight of gelatin, and 8 to 12 parts by weight of polyethylene glycol, but is not limited thereto.

[0098] In one embodiment of the present invention, the adult cell may be selected from the group consisting of fibroblasts, myofibroblasts, fibro-adipogenic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose-derived stem cells, bone marrow-derived stem cells, neural stem cells, and muscle stem cells, but is not limited thereto.

[0099] In one embodiment of the present invention, the adult cells are 2 × 10 5 cells / mL to 2 × 10⁶ 7 It may be included at a cell density of cells / mL, preferably 1 × 10 6 cells / mL to 4 × 10⁶ 6 It may be included at a cell density of cells / mL, but is not limited thereto.

[0100] In one embodiment of the present invention, the 3D hydrogel may promote cell reprogramming into a lineage of cells with different existing epigenetic and differentiation characteristics of adult cells, and may promote the reprogramming of adult cells into at least one cell selected from the group consisting of induced pluripotent stem cells (iPSCs); intermediate reprogrammed cells such as neural stem cells, epithelial stem cells, mesenchymal stem cells, hematopoietic stem cells, pancreatic endoderm, and hepatic endoderm; and progenitor cells, but is not limited thereto and may include all various cell types known in the art.

[0101] In one embodiment of the present invention, when the adult cell is a cell transformed to overexpress one or more genes selected from the group consisting of the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene, preferably a cell transformed to overexpress the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene, it may promote cell reprogramming into induced pluripotent stem cells, but is not limited thereto.

[0102] In one embodiment of the present invention, the OCT4 gene may be located on human chromosome 6, 31,164,337-31,170,682 (Genome Reference Consortium: GRCh38.p14, NCBI Reference Sequence: NC_000006.12, Gene ID: 5460).

[0103] In one embodiment of the present invention, the SOX2 gene may be located on human chromosome 3 181,711,925-181,714,436 (Genome Reference Consortium: GRCh38.p14, NCBI Reference Sequence: NC_000003.12, Gene ID: 6657).

[0104] In one embodiment of the present invention, the c-MYC gene may be located on human chromosome 8 127,735,434-127,742,951 (Genome Reference Consortium: GRCh38.p14, NCBI Reference Sequence: NC_000008.11, Gene ID: 4609).

[0105] In one embodiment of the present invention, the KLF4 gene may be located on human chromosome 9 107,484,852-107,489,769 (Genome Reference Consortium: GRCh38.p14, NCBI Reference Sequence: NC_000009.12, Gene ID: 9314).

[0106] In one embodiment of the present invention, if the adult cell is a cell in which the FGF gene and the Noggin gene are induced to express during the redifferentiation process, it may promote the reprogramming of ectoderm-derived cells such as neural stem cells and epithelial stem cells.

[0107] In one embodiment of the present invention, if the adult cell is a cell in which the BMP4 gene and Wnt gene are induced to be expressed during the redifferentiation process, it may promote the reprogramming of mesenchymal-derived cells such as mesenchymal stem cells and hematopoietic stem cells.

[0108] In one embodiment of the present invention, if the adult cell is a cell in which the Activin gene and Nodal gene are induced to be expressed during the redifferentiation process, it may promote endoderm-derived cell reprogramming.

[0109] Referring to FIG. 1e, the step (S530) of encapsulating the adult cells by solidifying the hydrogel solution in which the adult cells are suspended may further include the step (S410) of adding a photoinitiator to the hydrogel solution in which the adult cells are suspended, the step (S420) of injecting the hydrogel solution with the added photoinitiator into a casting mold, and the step (S430) of irradiating with ultraviolet light.

[0110] In one embodiment of the present invention, the photoinitiator may be selected from the group consisting of iriodinone (IrGACURE) 2959, iriodinone (IrGACURE) 651, Lucirin TPO, and benzoin methyl ether (BME), but is not limited thereto, and all various photoinitiators known in the art may be used.

[0111] In one embodiment of the present invention, the casting mold may be a polydimethylsiloxane (PDMS) mold, a polyurethane (PU) mold, a polycarbonate (PC) mold, a silicone mold, or a 3D printing mold, and preferably a polydimethylsiloxane (PDMS) mold, but is not limited thereto and all molds of various materials and shapes known in the art may be used.

[0112] Meanwhile, the method of solidifying the hydrogel solution is not limited to the photocrosslinking method shown in FIG. 1e, and can be produced through methods known in the art, such as chemical crosslinking, thermal gelation, and ionic crosslinking.

[0113]

[0114] In one aspect, the present invention relates to a hyaluronic acid-based 3D hydrogel comprising encapsulated cells for promoting cell reprogramming, prepared by a method for preparing a hyaluronic acid-based 3D hydrogel comprising the encapsulated cells.

[0115] In one embodiment of the present invention, the hyaluronic acid-based 3D hydrogel comprising encapsulated cells for promoting cell reprogramming may provide a hyaluronic acid microenvironment to adult cells to promote the reprogramming of adult cells, but is not limited thereto.

[0116]

[0117] In one aspect, the present invention relates to a method for inducing cell reprogramming of adult cells, comprising the steps of: preparing a hyaluronic acid-based 3D hydrogel by encapsulating adult cells in a hydrogel comprising hyaluronic acid and a polymer; and culturing or injecting into the body the adult cells encapsulated in the hyaluronic acid-based 3D hydrogel.

[0118] In one embodiment of the present invention, the method may promote cell reprogramming into a lineage of cells with different existing epigenetic and differentiation characteristics of adult cells, and may promote the reprogramming of adult cells into at least one cell selected from the group consisting of induced pluripotent stem cells (iPSCs); intermediate reprogrammed cells such as neural stem cells, epithelial stem cells, mesenchymal stem cells, hematopoietic stem cells, pancreatic endoderm, and hepatic endoderm; and progenitor cells, but is not limited thereto.

[0119] In one embodiment of the present invention, the adult cell may be an adult cell separated from an individual, but is not limited thereto.

[0120] In one embodiment of the present invention, cell reprogramming into induced pluripotent stem cells may be promoted when the adult cell is a cell transformed to overexpress one or more genes selected from the group consisting of the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene, preferably a cell transformed to overexpress the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene, but is not limited thereto.

[0121] In one embodiment of the present invention, the step (S610) of preparing a hyaluronic acid-based 3D hydrogel by encapsulating adult cells in a hydrogel comprising hyaluronic acid and a polymer is,

[0122] The method may include, but is not limited to, the step of preparing a hydrogel solution containing hyaluronic acid and a polymer (S510), the step of suspending adult cells in the hydrogel solution (S520), and the step of encapsulating the adult cells by solidifying the hydrogel solution in which the adult cells are suspended (S530).

[0123] In one embodiment of the present invention, the step of culturing or injecting into the body adult cells encapsulated in the hyaluronic acid-based 3D hydrogel comprises:

[0124] The method may further include, but is not limited to, a step of stimulating adult cells encapsulated in the hyaluronic acid-based 3D hydrogel with low-intensity ultrasound (LIUS) during the culture period or after injection into the body.

[0125] In one embodiment of the present invention, the step (S620) of stimulating adult cells encapsulated in the hyaluronic acid-based 3D hydrogel with low-power ultrasound is performed at a frequency of 20 kHz to 1000 kHz and 30 mW cm⁻¹ -2 Up to 500 mW cm -2 Low-power ultrasound of low intensity may be applied for 5 to 25 minutes, but is not limited thereto.

[0126] In one embodiment of the present invention, the exposure period of the low-power ultrasound may be 1 to 3 days, but is not limited thereto.

[0127] In one embodiment of the present invention, the injection into the body may be a subcutaneous injection, an intramuscular injection, an intravenous injection, an intra-articular injection, a skin injection, or an internal injection, and preferably a subcutaneous injection, but is not limited thereto.

[0128] In one embodiment of the present invention, the low-power ultrasound may increase the Mesenchymal-to-Epithelial Transition (MET) and histone modification of adult cells, but is not limited thereto.

[0129] In one embodiment of the present invention, the low-power ultrasound may reduce F-action protein expression to induce a change in the cytoskeletal structure of adult cells, but is not limited thereto.

[0130] In one embodiment of the present invention, the low-power ultrasound may phosphorylate the FAK protein to activate the Focal adhesion signaling pathway, but is not limited thereto.

[0131] In one embodiment of the present invention, the low-power ultrasound may increase the cell membrane fluidity of adult cells, but is not limited thereto.

[0132] In one embodiment of the present invention, the expression amount of CD44, OCT4, SSEA4, NANOG, or TRA-1-60 proteins may be increased, but is not limited thereto.

[0133] In one aspect, the present invention relates to induced pluripotent stem cells produced by a method of inducing cell reprogramming of adult cells using a hyaluronic acid-based 3D hydrogel comprising the encapsulated cells.

[0134] Induced pluripotent stem cells produced by the above method are manufactured using a hydrogel that has excellent in vivo safety. Unlike conventional methods using chemical substances, they are clinically safe and can be utilized in various fields such as regenerative medicine, production of disease models, identification of disease causes, and new drug development.

[0135] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0136] Example 1: Preparation of a hyaluronic acid-based hydrogel

[0137] Example 1-1. Preparation of a photoreactive crosslinked polymer

[0138] Referring to Figures 1a to 1d, photoreactive cross-linked polymers (methacrylated hyaluronic acid (MAHA) and methacrylated gelatin) were prepared.

[0139] Referring to Fig. 1b, methacrylated hyaluronic acid (MAHA) was synthesized by adding 1.5% v / v methacrylic anhydride to an aqueous solution of 1% w / v hyaluronic acid (molecular weight = 500 kDa, Bioland, South Korea) (see Fig. 1c). While stirring the mixture under dark conditions at 4°C for 24 hours, 1.5% v / v 5 N NaOH was added to adjust the pH to 8-9. The mixture was dialyzed with deionized water for 3 days using a limiting dialysis membrane with a molecular weight of 100 kDa, and after removing residual impurities, it was freeze-dried for use. The degree of substitution (DS) of the synthesized MAHA was calculated by the relative integration ratio of the methacrylate proton peaks (peaks at approximately 6.1, 5.6, and 1.85 ppm) and the methyl proton peaks of HA (approx. 1.9 ppm) using proton nuclear magnetic resonance spectroscopy (1H-NMR; 500 MHz FT-NMR Spectrometry, Bruker, USA), and the final degree of substitution (DS) was confirmed to be 10% (Fig. 2).

[0140] Referring to Fig. 1d, methacrylated gelatin was prepared by dissolving 10% w / v type B gelatin (Sigma Aldrich, St. Louis, USA) in phosphate buffer solution (25 mM monopotassium phosphate and 170 mM disodium phosphate) at 50°C. A 10% (v / v) solution of methacrylic anhydride was drip-infused while stirring, and the mixture was reacted for 1 hour under dark conditions at pH 7.5. The reaction solution was filtered through a 100 μm cell filter, dialyzed with distilled water for 3 days using a limiting dialysis membrane with a molecular weight of 10 kDa, and freeze-dried to obtain the final product.

[0141] Example 1-2. Preparation of Hydrogel

[0142] Referring to Figures 1a and 1e, a hydrogel comprising hyaluronic acid (HA), gelatin (Gel), and polyethylene glycol (PEG) was prepared.

[0143] Specifically, to prepare a hydrogel comprising hyaluronic acid (HA), gelatin, and polyethylene glycol (PEG), a polymer solution was prepared by dissolving methacrylated hyaluronic acid (0.5% w / v), methacrylated gelatin (7% w / v), PEG diacrylate (5% w / v, Alfa Aesar, USA), and Irgacure 2959 (final concentration 0.2% w / v, photocrosslinking agent) in Dulbecco's phosphate-buffered saline (DPBS). After placing 40 μL of the polymer solution into a polydimethylsiloxane (PDMS) mold with a depth of 2 mm and a diameter of 5 mm, the sample was subjected to ultraviolet light (365 nm, 60 mW cm⁻¹). -2 A hyaluronic acid-based hydrogel was prepared by exposing it to UV rays (Sei Myung Vactron Co., Ltd., Korea). It is preferable to irradiate methacrylated hyaluronic acid with UV rays for 10 seconds, and methacrylated gelatin and polyethylene glycol with UV rays for 50 seconds or more.

[0144] Example 2: Characterization of Hydrogel

[0145] The swelling characteristics and rheological characteristics of the hydrogel prepared in Example 1 above were analyzed.

[0146] Specifically, the swelling ratio of the hyaluronic acid-based hydrogel was measured after immersing the sample in DPBS under low-intensity ultrasound (LIUS) at 37°C for 3 days. Before weighing the swollen sample, residue on the sample surface was wiped off with paper. The swelling ratio at each time point was defined as the weight ratio of the pure liquid absorption to the dried hydrogel.

[0147] To analyze the mechanical properties of hyaluronic acid-based hydrogels, samples were measured using HAAKE Rheostress1 (Thermo Scientific, USA). The volume of each sample was 500 μL. Vibrational frequency sweeps were applied at frequencies ranging from 0.05 to 20 Hz while maintaining a constant vibrational shear stress of 0.1 Pa. The temperature was maintained at 37°C in all experiments.

[0148] As a result, the swelling properties of the hyaluronic acid-based hydrogel were found to be approximately 130%, and the mechanical properties were found to be approximately 10-100 Pascal.

[0149] Example 3: Preparation of transformed cells into which four genes (OCT4, SOX2, c-MYC, KLF4) were introduced for reprogramming into induced pluripotent stem cells (iPSCs)

[0150] Example 3-1. Cell Culture

[0151] Mouse embryonic fibroblasts (MEFs) and human primary adipose-derived stem cells (ASCs) isolated from the human body were used as adult cells for reprogramming into induced pluripotent stem cells (iPSCs).

[0152] Mouse embryonic fibroblasts (MEFs) used were transformed cells expressing OCT4-GFP (hereinafter, OG-MEF). OG-MEFs were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA) containing 10% v / v fetal bovine serum (FBS, Hyclone) and 1% v / v penicillin / streptomycin (P / S, Hyclone). Cells were subcultured using 0.25% trypsin / EDTA (Hyclone) in an incubator humidified with 5% CO₂ at 37°C.

[0153] Human primary adipose-derived stem cells (ASCs) were isolated from the patient's knee infrapatellar fat pad with the approval of the Dongguk University Institutional Review Board (IRB No. DUIRB-202210-18). Briefly, the infrapatellar fat pad was washed three times with DPBS containing 2% P / S. The washed tissue was [treated] in 0.5 mg mL of low-glucose DMEM -1 The cells were diluted with collagenase (Sigma Aldrich) and 1% P / S and digested at 37°C for 45 minutes. Afterward, the digested tissue was filtered through a 45 μm filter and centrifuged at 1,000 × g for 5 minutes to separate the cell pellet. The ASC cell pellet was resuspended and cultured in a culture medium containing low-glucose DMEM, 10% FBS, and 1% P / S in an incubator humidified with 5% CO2 at 37°C.

[0154] Example 3-2. Transduction of OCT4, SOX2, c-MYC, and KLF4 genes

[0155] One day prior to transformation, GP2-293 cells producing retrovirus particles containing the pMXs-hOCT4, pMXs-hSOX2, pMXs-hKLF4, and pMXs-hc-MYC vectors were prepared. Specifically, GP2-293 packaged cells (Clontech, Germany) were placed in 4 × 10⁶ 6 Cells were inoculated at a certain cell density. Subsequently, GP2-293 packaged cells were transformed with pMXs-hOCT4, pMXs-hSOX2, pMXs-hKLF4, and pMXs-hc-MYC (Addgene, USA) using the retrovirus packaging vector VSV-G (Thermo Scientific) and Lipofectamine 2000 reagent (Thermo Scientific). The transformed GP2-293 packaged cells were cultured for 3 days in an incubator humidified with 5% CO₂ at 37°C to ensure sufficient production of retrovirus particles. The medium containing the culture of the transformed GP2-293 packaged cells was centrifuged at 1,300 rpm for 3 minutes to remove debris, and then filtered through a 0.45 μm syringe filter. The filtered supernatant was loaded onto an Amicon® Ultra-15 10 kDa centrifuge (Merck, USA) and centrifuged at 4,000 × g at 4°C for 20 minutes to filter and concentrate the retrovirus particles. 8 μg mL of the supernatant containing the retrovirus particles was used. -1 It was resuspended in a new culture medium containing polybrene (Sigma Aldrich).

[0156] OG-MEF and hASC, each 1 × 10 6 cells and 2 × 10⁶ 5After inoculating 100 mm culture dishes at a cell density, approximately 10 mL of culture medium containing polybrene in which the retrovirus particles were suspended was added to each culture dish, and the OCT4, SOX2, c-MYC, and KLF4 genes were transfected into OG-MEF cells and hASC cells by culturing for 48 hours. The medium was replaced with fresh culture medium after 24 hours.

[0157] Example 4. Preparation of a cell-encapsulated 3D hydrogel

[0158] Referring to Fig. 3, a 3D hydrogel encapsulated with the transformed cells of Example 3 was prepared.

[0159] Specifically, after confirming the transformation efficiency of OG-MEF (>90%) and ASC (>50%) transduced with the four genes of Example 3 above, the isolated cells were each 2 × 10 6 Cells were suspended in each HA hydrogel solution (polymer solution of Examples 1-2) at a cell density of cells / mL. 40 μL of the polymer solution containing suspended cells was placed into a polydimethylsiloxane (PDMS) mold with a depth of 2 mm and a diameter of 5 mm, and then the sample was subjected to UV light (365 nm, 60 mW cm⁻¹). -2 Encapsulation was performed by solidifying the hydrogel through exposure to (Sei Myung Vactron Co., Ltd., Korea). The 3D hydrogel containing encapsulated adult cells prepared by this method was named HA hydrogel. The 3D hydrogel containing encapsulated OG-MEF cells is OG-MEF-HA hydrogel, and the 3D hydrogel containing encapsulated hASC cells is hASC-HA hydrogel.

[0160] Example 5: Induction of cell reprogramming into iPSCs (induced pluripotent stem cells)

[0161] Referring to Figures 4a to 4c, OG-MEF-HA hydrogels and hASC-HA hydrogels were each placed in 50 mL tubes containing 2 mL of medium. During the culture period, low-power ultrasound (LIUS) was applied every 2 days, and MEF was cultured for 21 days and ASC for 27 days to induce cell reprogramming into iPSCs. The medium was replaced with fresh iPSC medium every day.

[0162] Mouse iPSC medium was used for mouse embryonic fibroblasts (OG-MEF) expressing OCT4-GFP, and the mouse iPSC medium consisted of DMEM containing 15% v / v FBS, 1% v / v P / S, 1% v / v non-essential amino acids (NEAA, Thermo Scientific), 1% v / v N-2-hydroxyethyllipiperazine-N-2-ethane sulfonic acid (HEPES, Thermo Scientific), 1% v / v Glutamax (Thermo Scientific), 1% v / v EmbryoMax Nucleosides (Sigma Aldrich), 0.1% v / v beta-mercaptoethanol (Thermo Scientific), and 1,000 units mL -1 It is a DMEM medium containing leukemia inhibitor factor (LIF; Sigma Aldrich).

[0163] Human iPSC medium was used for human adipose-derived stem cells (hASCs), and said human iPSC medium contained 20% KnockOut™ serum substitute (Thermo Scientific), 1% P / S, 1% non-essential amino acids, 0.1% beta-mercaptoethanol, and 4 ng mL -1 It is a DMEM / F12 (Thermo Scientific) medium containing recombinant human FGF-basic (rhFGF-b; Thermo Scientific).

[0164] Ultrasonic stimulation was performed using a Digital Ultrasonic Set (Daehan, Korea) at a frequency of 40 kHz and 300 mW cm⁻¹ every 2 days during the culture period. -2 Cell-encapsulated 3D hydrogels were exposed to LIUS at intensity for 0, 5, 10, or 20 minutes.

[0165] Example 6: Confirmation of cell viability of iPSC cells reprogrammed from adult cells

[0166] To confirm the cytotoxicity induced by sonication, OG-MEF-HA hydrogel and hASC-HA hydrogel were treated at a frequency of 40 kHz and 300 mW cm⁻¹ -2 Cell viability according to LIUS stimulation time was determined by exposing cells to low-intensity ultrasound (LIUS) for 5, 10, 20, or 30 minutes.

[0167] Live / Dead fluorescence staining was performed to evaluate the cytotoxicity of ultrasonic stimulation. First, the 3D hydrogel containing the transformed cells of Example 5 was washed with DPBS, and then 2 μM Calcein AM (Thermo Scientific) and 4 μM Ethidium Homodimer-1 (Thermo Scientific) were added to the DPBS solution and stained for 30 minutes. Subsequently, fluorescence images of living cells (green) and dead cells (red) were observed using Cytation3 (Biotek, USA).

[0168] As a result, as shown in Figures 5a and 5b, in both OG-MEF and hASC cells, cell viability after 7 days of LIUS stimulation (2-day cycle) was high in the experimental group stimulated by LIUS for 0 to 20 minutes, and no apoptotic cells were observed. However, in cells stimulated by LIUS for 30 minutes, apoptosis occurred, and cell viability decreased to approximately 71% (OG-MEF) and 68% (hASC). After 14 days, cell viability remained high in the experimental group stimulated by LIUS for 0 to 20 minutes, and no apoptotic cells were observed.

[0169] These results indicate that cell viability can be maintained for 14 days when the LIUS stimulation time does not exceed 20 minutes, and accordingly, the LIUS stimulation time was limited to 20 minutes for subsequent experiments.

[0170] Example 7: Effect of LIUS Stimulation on iPSC Reprogramming in 3D Hydrogels

[0171] To observe the effect of LIUS stimulation on cell reprogramming, fluorescence microscopy, qRT-PCR analysis, Western blot, immunofluorescence staining, and flow cytometry were performed on the OG-MEF-HA hydrogel and hASC-HA hydrogel in which cell reprogramming of Example 5 was induced during the culture period.

[0172] First, GFP expression was visually observed using a fluorescence microscope during the culture period of the 3D hydrogel encapsulated with the cell reprogramming induced in Example 5, and on day 21 of culture, the GFP expression level and OCT4-GFP positive colonies (iPSC colonies) were quantified using ImageJ.

[0173] As a result, as shown in Figures 6a to 6c, it was found that the longer the LIUS stimulation time, the more OCT4-GFP positive cells were induced. While the unstimulated group began OCT4-GFP expression 14 days after transduction, the group treated with LIUS for 20 minutes showed accelerated GFP expression after 7 days after transduction (Figure 6a). In addition, the LIUS-stimulated group produced more iPSC colonies in terms of colony number and GFP intensity. In particular, the group treated with LIUS for 20 minutes showed an increase in the number of iPSC colonies by approximately 4.0 times and GFP intensity by approximately 6.25 times compared to the unstimulated group.

[0174] Subsequently, immunofluorescence staining was performed as follows. The 3D hydrogel was washed three times with DPBS and fixed in 4% paraformaldehyde (Biosesang, Korea) at room temperature for 1 hour. The fixed 3D hydrogel was permeated with a solution of 0.3% Triton X-100 (PBS-T) added to DPBS at room temperature for 30 minutes. Afterward, it was blocked with a blocking solution of 1% BSA added to PBS-T at room temperature for 1 hour. The 3D hydrogel was incubated overnight at 4°C in a primary antibody solution diluted 1:200.

[0175] After washing the samples with DPBS, they were incubated in a solution of fluorescently labeled secondary antibody (Thermo Scientific) diluted with 1% BSA in PBS-T (1:200) at room temperature under dark conditions for 2 hours. Texas red-X phalloidin (Thermo Scientific) was used for F-actin staining. Unbound antibodies were washed with DPBS and counterstained with 4′Thermo Scientific to observe cell nuclei. Fluorescence images were observed using Cytation3. Details regarding the primary and secondary antibodies used for immunofluorescence staining are shown in Table 1 below.

[0176] [Table 1]

[0177]

[0178] The expression levels of each protein and OCT4-positive colonies (iPSC colonies) were quantified using ImageJ.

[0179] As a result, as shown in Figures 7a to 7d, it was observed that the fluorescence intensity of the pluripotency markers NANOG and SOX2 increased as the LIUS stimulation time increased in the 3D hydrogel encapsulated with OG-MEF cells (Figure 7a), and similarly, in the 3D hydrogel encapsulated with hASC cells, the fluorescence intensity of the pluripotency markers SSEA4, TRA-1-60, and NANOG increased as the LIUS stimulation increased (Figures 7b and 7c). In addition, the number of colonies formed also significantly increased in the group treated with LIUS for 20 minutes (Figure 7d).

[0180] These results confirmed that treatment with LIUS increases the efficiency of reprogramming adult cells into iPSC cells.

[0181] Subsequently, cell surface antigens were identified via flow cytometry, which was performed as follows. OG-MEF cells encapsulated in a 3D hydrogel were washed twice with DPBS, dissociated using Type Is hyaluronidase derived from bovine testes (Sigma Aldrich), and collected after centrifugation at 1,300 rpm for 3 minutes. The cell pellet was washed twice with DPBS and blocked with a DPBS solution containing 2% FBS (FACS buffer). Specific antibodies (antibodies diluted 1:100 in FACS buffer) were incubated at 4°C for 30 minutes, followed by three washes with FACS buffer. Finally, fluorescence was detected using BD Accuri C6 (BD Bioscience, Japan). The expression ratio of cell surface antigens was calculated based on 10,000 gated cell events. The antibody used in the experiment is anti-SSEA1 (125608, Biolegend).

[0182] As a result, as shown in Figure 8, the ratio of OCT4+ / SSEA1+ cells was found to be 15.5%, which is an improvement compared to 11.1% in the group not treated with LIUS.

[0183] These results confirmed that treatment with LIUS increases the efficiency of reprogramming adult cells into iPSC cells.

[0184] Subsequently, for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, the 3D hydrogels in which reprogramming from OG-MEF cells to iPSC cells was induced were frozen in liquid nitrogen and then disrupted using a homogenizer with 200 μL of TRIzol™ Scientific. After complete homogenization, an additional 800 μL of TRIzol and 200 μL of chloroform were added. After mixing, the samples were centrifuged at 13,000 rpm at 4°C for 20 minutes. The supernatant was mixed with an equal amount of isopropanol and centrifuged at 13,000 rpm at 4°C for 20 minutes. The supernatant was removed from the pellet, washed with 75% ethanol, and then centrifuged for an additional 10 minutes at 13,000 rpm at 4°C. After completely drying the pellet, it was resuspended in RNase-free water (Thermo Scientific). RNA quantification was performed using Cytation3.

[0185] For cDNA synthesis, complementary DNA was synthesized from 1 μg of total RNA using the PrimeScript™RT reagent kit (Takara, Japan). Subsequently, qRT-PCR was performed using Power SYBR®Green PCR Master Mix (Applied Biosystems, UK), with the specific PCR conditions as follows: initial denaturation was performed at 95°C for 20 seconds, followed by 40 cycles of denaturation at 95°C for 3 seconds and annealing at 60°C for 30 seconds, and a melting curve stage at 95°C for 15 seconds and 60°C for 60 seconds. The primer sequences used for PCR are shown in Table 2 below.

[0186] [Table 2]

[0187]

[0188] As a result, as shown in Fig. 9a, mRNA expression of Oct4, Nanog, and Sox2 pluripotency markers increased with longer LIUS stimulation times, and compared to cells not stimulated by LIUS, 20 minutes of LIUS stimulation increased the expression of Oct4, Nanog, and Sox2 markers by approximately 2.7 times, 6.4 times, and 2.0 times, respectively (Fig. 9a).

[0189] Next, Western blotting was performed as follows. Prior to Western blotting, all 3D hydrogel samples were washed three times with DPBS and frozen in liquid nitrogen. Subsequently, the samples were homogenized using 50 μL of 5X RIPA buffer (Sigma Aldrich) supplemented with a proteolytic inhibitor (Merck) and a kinase inhibitor (Sigma Aldrich). The extracts were centrifuged at 13,000 rpm at 4 °C for 20 minutes, and the supernatant was collected. Total protein concentration was quantified using the bicinchonic acid protein assay with the Pierce BCA Protein Assay Kit (Thermo Scientific). Approximately 20 μg of each protein sample was denatured and separated by 10% polyacrylamide gel electrophoresis.

[0190] The isolated proteins were transferred to a nitrocellulose membrane and blocked for 1 hour with a solution of 5% skim milk in Tris-buffered saline and 0.05% Tween-20 (TBS-T). The membrane was incubated overnight at 4 °C with a primary antibody containing 5% bovine serum albumin (BSA) in TBS-T. Subsequently, the membrane was washed three times with TBS-T and then incubated at room temperature for 2 hours with an appropriate HRP-conjugated secondary antibody diluted 1:5000 in 5% skim milk TBS-T.

[0191] After washing three times with TBS-T, protein bands were detected using a Chemi-doc detection system (Bio-Rad, USA), and images were visualized using Image Lab (Bio-Rad) software. Details regarding the primary and secondary antibodies are shown in Table 3 below.

[0192] [Table 3]

[0193]

[0194] As a result, as shown in Figure 9b, similar to the qRT-RCR results, the expression of OCT4, NANOG, and SOX2 pluripotency markers increased with longer LIUS stimulation times, and when LIUS was stimulated for 20 minutes, the intensities of the OCT4, NANOG, and SOX2 bands were found to be up to 2.5 times, 5.1 times, and 4.0 times higher, respectively, compared to cells not treated with LIUS stimulation.

[0195] Example 8: Confirmation of cell changes in 3D hydrogel following LIUS stimulation

[0196] Example 8-1. Change in MET (Mesenchymal-to-Epithelial Transition)

[0197] The gene and protein expression levels of the epithelial marker E-cadherin and the mesenchymal marker N-cadherin were measured through quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis and Western blot. The primer sequences used for PCR are shown in Table 4 below, and details regarding the primary and secondary antibodies used for Western blot are shown in Table 5.

[0198] As a result, as shown in Figures 10a and 10b, as the LIUS stimulation time increased, the mRNA and protein expression levels of the epithelial marker E-cadherin were significantly increased, while the expression of the mesenchymal marker N-cadherin was slightly decreased, and the ratio of E-cadherin to N-cadherin expression increased as the LIUS stimulation time increased.

[0199] These results further clearly demonstrated that LIUS stimulation increases Mesenchymal-to-Epithelial Transition (MET) during iPSC reprogramming.

[0200] [Table 4]

[0201]

[0202] [Table 5]

[0203]

[0204] Example 8-2. Epigenetic modification

[0205] To observe the expression of active H3 acetylation, H3K4 dimethylation, and H3K4 trimethylation during the initial reprogramming process, immunofluorescence staining was performed on ACH3, H3K4ME2, and H3K4ME3, and the fluorescence intensity of each protein was quantified using ImageJ. Details regarding the primary and secondary antibodies used for immunofluorescence staining are shown in Table 6 below.

[0206] [Table 6]

[0207]

[0208] As a result, as shown in Figure 11, LIUS stimulation was found to increase the expression of all histone markers regardless of stimulation time, and in particular, when LIUS was stimulated for 20 minutes, the fluorescence intensity of ACH3, H3K4ME2, and H3K4ME3 increased by 6.26 times (ACH3), 4.61 times (H3K4ME2), and 2.75 times (H3K4ME3), respectively, compared to cells not treated with LIUS stimulation.

[0209] Epigenetic modifications are another aspect of iPSC reprogramming that plays a role in activating or repressing genes. In particular, histone modifications are major transformations that occur early in the reprogramming process and are important checkpoint molecules in iPSC reprogramming.

[0210] Therefore, these results indicate that when LIUS is treated during the culture of the 3D hydrogel in which the adult cells of Example 4 are encapsulated, it increases the MET and histone modification of the cells, thereby promoting reprogramming into iPSCs.

[0211] Example 9: Changes in the cytoskeletal structure of adult cells in a 3D hydrogel following LIUS stimulation

[0212] First, immunofluorescence staining for F-actin was performed to observe changes in the expression of actin stress fibers following LIUS stimulation.

[0213] As a result, as shown in Figure 12a, F-actin filament expression decreased as the LIUS stimulation time increased, and the fluorescence intensity of F-actin decreased by approximately 37%, 43%, and 75% when LIUS was stimulated for 5, 10, and 20 minutes, respectively, compared to the group without LIUS stimulation.

[0214] These results indicate that LIUS stimulation increases the efficiency of reprogramming into iPSCs by regulating the cytoskeletal structure of adult cells encapsulated within a hydrogel.

[0215] Next, further analysis was performed to determine whether the mechanical changes in F-actin stress fibers permanently reduced F-actin expression due to LIUS stimulation.

[0216] As a result, as shown in Fig. 12b, F-actin expression decreased after applying LIUS for 20 minutes, and fluorescence intensity remained decreased until 40 minutes after LIUS stimulation. However, F-actin expression gradually re-expressed after 60 minutes, and the cytoskeletal structure was rapidly rearranged. These results suggest that the mechanical force induced by LIUS induces transient changes in the rearrangement of the cytoskeleton of reprogrammed cells.

[0217] Next, the phosphorylation level of FAK (Focal adhesion kinase) according to LIUS stimulation time was measured through Western blot for FAK and pFAK. Detailed information on the primary and secondary antibodies is shown in Table 7 below.

[0218] [Table 7]

[0219]

[0220] As a result, as shown in Figure 12c, it was found that as the LIUS stimulation time increased, the phosphorylation of FAK (Focal adhesion kinase) also increased, and compared to cells not stimulated by LIUS, the pFAK level was found to increase by up to 2.7 times.

[0221] FAK plays an important role in cells attaching to the extracellular matrix or other cells.

[0222] These results suggest that changes in the cytoskeletal structure of adult cells induced by LIUS activate the Focal adhesion signaling pathway associated with mechanical changes, thereby contributing to the cell reprogramming process by helping cells undergo the morphological changes necessary for reprogramming.

[0223] Example 10: Changes in membrane fluidity of adult cells in a 3D hydrogel following LIUS stimulation

[0224] The cell membrane fluidity of adult cells in a 3D hydrogel was evaluated using the diffusion rate of a lipid-like probe in a fluidized cell membrane.

[0225] Specifically, cell membrane fluidity was analyzed using a membrane fluidity analyzer (Abcam, UK) according to the manufacturer's instructions. LIUS-stimulated cells were labeled with pyrenedecanoic acid (PDA) for 1 hour under dark conditions at 25°C. Unbound PDA was washed, and the ratio of monomer (excimeter: 400 nm) to excimer (excimeter: 470 nm) fluorescence was normalized to the values ​​of cells not stimulated by LIUS.

[0226] As a result, as shown in Figure 13, the relative fluorescence unit (RFU) increased as the LIUS stimulation time increased, which means that more lipid probes diffused, indicating that the cell membrane was highly fluidized.

[0227] Through these results, it was confirmed that LIUS stimulation sequentially rearranges cytoskeletal fibers and regulates membrane fluidity.

[0228] Example 11: Analysis of 3D Hydrogel Characteristics According to LIUS Stimulation

[0229] The physical properties (elastic modulus, swelling ratio) of the 3D hydrogel according to LIUS stimulation time were analyzed.

[0230] As a result, as shown in Figure 14, no significant change was observed between cells that were not stimulated by LIUS and cells that were stimulated by LIUS, and this result means that LIUS stimulation does not affect the physical properties of the hydrogel.

[0231] Example 12: Confirmation of the reprogramming efficiency of adult cells into iPSCs according to a hydrogel

[0232] To determine the reprogramming efficiency of adult cells into iPSCs according to hydrogel components, control and experimental groups were prepared as shown in Table 8, and immunofluorescence staining was performed for CD44, OCT4-GFP, NANOG, and SOX2 proteins, and the fluorescence intensity of each protein was quantified using ImageJ.

[0233] [Table 8]

[0234]

[0235] The above PEG hydrogel and Gel hydrogel are prepared in the same manner as the 3D hydrogel preparation method of Example 4, wherein the PEG hydrogel is prepared by using a hydrogel solution containing PEG diacrylate, and the Gel hydrogel is prepared by using a hydrogel solution containing methacrylated gelatin.

[0236] Details regarding the primary and secondary antibodies used for immunofluorescence staining are shown in Table 9 below.

[0237] [Table 9]

[0238]

[0239] As a result, immunofluorescence-stained CD44 showed high expression only in the HA hydrogel, and CD44 expression increased twofold upon LIUS stimulation. In contrast, no significant increase in CD44 expression was observed in the PEG hydrogel and Gel hydrogel before and after LIUS stimulation, and the amount of CD44 expression was only about 16% of that in the HA hydrogel group. Although significant changes in CD44 were observed in the PEG hydrogel after LIUS stimulation, the increase in CD44 expression was only about 20% of the increase in CD44 expression in the HA hydrogel (Fig. 15a). These differences in protein expression levels according to the hydrogel components showed the same trend in OCT4-GFP fluorescence intensity (Fig. 15b).

[0240] Hyaluronic acid (HA) is a high-molecular-weight polysaccharide that plays an important role in the extracellular matrix (ECM), and CD44 is a receptor protein expressed in various cells.

[0241] Through these results, it was confirmed that hyaluronic acid increases the efficiency of reprogramming adult cells into induced pluripotent stem cells through interaction with the CD44 protein.

[0242] In addition, the expression levels of OCT4-GFP protein following LIUS stimulation showed a distinct difference only in the HA hydrogel, while there was no change in the PEG hydrogel and Gel hydrogel (Fig. 15b). Similarly, immunofluorescence-stained SOX2 and NANOG also showed an approximately 3.6-fold increase in the HA hydrogel upon LIUS stimulation, while no change in expression levels was observed in the other hydrogels (Fig. 15c, d).

[0243] These results indicate that LIUS plays a role in promoting reprogramming efficiency by increasing the intracellular expression of CD44 in the hyaluronic acid (HA) microenvironment.

[0244] Example 13: Confirmation of iPSC reprogramming efficiency following HA hydrogel and LIUS stimulation in vivo

[0245] Referring to Fig. 16, the efficiency of reprogramming adult cells into iPSCs following LIUS stimulation in an in vivo environment was analyzed according to the ARRIVE guidelines (https: / / arriveguidelines.org / arrive-guidelines) with the approval of the Dongguk University Integrated Animal Care and Treatment Committee (IACUC-2021-018-3). Hydrogels encapsulated with transgenic OG-MEFs (OG-MEF-HA hydrogels) were implanted into a single subcutaneous space of randomly selected 6-8 week old male BALB / c immunodeficient mice (Orient Bio, Inc., Korea). Each hydrogel was stimulated with ultrasound therapy (ST-10A, StraTek Co., Ltd., Korea), and the ultrasound was applied at a frequency of 1 MHz and 300 mW cm⁻¹ on the skin where the hydrogel was implanted. -2 Treatment was performed for 20 minutes at a 2-day intensity. On the day of analysis, each hydrogel was detached from the skin and analyzed. Subsequently, to measure the expression levels of pluripotency markers, immunofluorescence staining for OCT4-GFP, NANOG, and SOX2 proteins was performed and quantified using ImageJ. Details regarding the primary and secondary antibodies used for immunofluorescence staining are shown in Table 10 below.

[0246] [Table 10]

[0247]

[0248] As a result, as shown in Figures 17a to 17c, the expression levels of the pluripotency markers OCT4-GFP, NANOG, and SOX2 proteins were found to increase significantly when LIUS was treated, and in the analysis of relative fluorescence intensity, it was confirmed that the expression of OCT4, NANOG, and SOX2 increased by 3.87 times, 1.77 times, and 2.33 times, respectively, when LIUS was treated compared to when LIUS was not treated.

[0249] Through these results, it was confirmed that encapsulating adult cells in a hyaluronic acid-based hydrogel and stimulating the cells encapsulated in the hydrogel with LIUS can significantly improve the reprogramming efficiency of adult cells into iPSCs under in vivo conditions.

[0250]

[0251] [National R&D projects that supported this invention]

[0252] [Project ID] 2710011307

[0253] [Assignment No.] 2022R1A2C3004850

[0254] [Ministry Name] Ministry of Science and ICT

[0255] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0256] [Research Project Name] Individual Basic Research (Mid-career Researcher)

[0257] [Project Title] Development of Technology to Enhance Therapeutic Efficacy for Inflammatory Skeletal Diseases through Mass Production and Modification of Patient-Specific Stem Cell-Derived Multifunctional Exosomes Using a 3D Fusion System

[0258] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0259] [Research Period] 2022.03.01 ~ 2027.02.28

[0260]

[0261] [Project ID] 2710018148

[0262] [Project No.] RS-2024-00405381

[0263] [Ministry Name] Ministry of Science and ICT

[0264] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0265] [Research Project Name] Basic Research Laboratory Pioneer Type

[0266] [Project Title] Basic Research Laboratory for the Development of Multimodal Induced Regeneration Biomaterials

[0267] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0268] [Research Period] 2024.08.01 ~ 2027.04.30

[0269]

[0270] [Project ID] 2710001426

[0271] [Project No.] RS-2023-00214410

[0272] [Ministry Name] Ministry of Science and ICT

[0273] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0274] [Research Project Name] Individual Basic Research (Sejong Science Fellowship)

[0275] [Research Project Title] For the Promotion of Rejuvenation of Aging Cells and Treatment of Degenerative Diseases

[0276] Mass production of functional exosomes derived from induced pluripotent stem cells

[0277] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0278] [Research Period] 2023.03.01 ~ 2028.02.29

Claims

1. A hydrogel composition for promoting cell reprogramming of adult cells, comprising hyaluronic acid and a polymer.

2. A step of preparing a hydrogel solution containing hyaluronic acid and a polymer, Step of suspending adult cells in the hydrogel solution and A method comprising the step of solidifying a hydrogel solution in which adult cells are suspended to encapsulate the adult cells. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

3. In Paragraph 2, The above hyaluronic acid is characterized as being methacrylated hyaluronic acid, Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

4. In Paragraph 2, The above polymer is one or more selected from the group consisting of gelatin, alginate, carrageenan, agarose, chitosan, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), and polyurethane. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

5. In Paragraph 4, The above gelatin is characterized as being methacrylated gelatin. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

6. In Paragraph 4, The above hydrogel solution is characterized by comprising 0.5 to 1.5 parts by weight of hyaluronic acid, 12 to 16 parts by weight of gelatin, and 8 to 12 parts by weight of polyethylene glycol. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

7. In Paragraph 2, The above adult cells are selected from the group consisting of fibroblasts, myofibroblasts, fibro-adipogenic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose-derived stem cells, bone marrow-derived stem cells, neural stem cells, and muscle stem cells. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

8. In Paragraph 2, The above adult cells are 2 × 10 5 cells / mL to 2 × 10⁶ 7 Included in a cell density of cells / mL, Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

9. In Paragraph 2, The above 3D hydrogel promotes cell reprogramming from adult cells into at least one cell selected from the group consisting of induced pluripotent stem cells (iPSCs), intermediate reprogrammed cells, and progenitor cells. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

10. In Paragraph 2, In the case where the above adult cell is a cell transformed to overexpress one or more genes selected from the group consisting of the OCT4 gene, SOX2 gene, c-MYC gene, and KLF4 gene, cell reprogramming into induced pluripotent stem cells is promoted. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

11. In Paragraph 2, The above solidification is performed by any one method selected from the group consisting of photocrosslinking, chemical crosslinking, thermal gelation, and ionic crosslinking. Method for preparing a hyaluronic acid-based 3D hydrogel containing encapsulated cells for promoting cell reprogramming.

12. A hyaluronic acid-based 3D hydrogel comprising encapsulated cells for promoting cell reprogramming, prepared by the method of any one of claims 2 to 11.

13. A step of preparing a hyaluronic acid-based 3D hydrogel by encapsulating adult cells in a hydrogel containing hyaluronic acid and a polymer, and A method for inducing cell reprogramming of adult cells, comprising the step of culturing or injecting into the body adult cells encapsulated in the above hyaluronic acid-based 3D hydrogel.

14. In Paragraph 16, The above method is a method for inducing cell reprogramming of an adult cell, wherein the method promotes cell reprogramming from an adult cell into at least one cell selected from the group consisting of induced pluripotent stem cells (iPSCs), intermediate reprogrammed cells, and progenitor cells.

15. In Paragraph 13, A method for inducing cell reprogramming of adult cells, wherein cell reprogramming into induced pluripotent stem cells is promoted when the adult cells are cells transformed to overexpress one or more genes selected from the group consisting of the OCT4 gene, the SOX2 gene, the c-MYC gene, and the KLF4 gene.

16. In Paragraph 13, The step of preparing a hyaluronic acid-based 3D hydrogel by encapsulating adult cells in a hydrogel containing the above hyaluronic acid and polymer is: A step of preparing a hydrogel solution containing hyaluronic acid and a polymer; Step of suspending adult cells in the hydrogel solution and A method for inducing cell reprogramming of adult cells, comprising the step of solidifying a hydrogel solution in which adult cells are suspended to encapsulate the adult cells.

17. In Paragraph 13, The step of culturing or injecting into the body adult cells encapsulated in the above hyaluronic acid-based 3D hydrogel is: A method for inducing cell reprogramming of adult cells, further comprising the step of stimulating adult cells encapsulated in the hyaluronic acid-based 3D hydrogel with low-intensity ultrasound (LIUS) during the culture period or after injection into the body.

18. In Paragraph 16, The step of stimulating adult cells encapsulated in the hyaluronic acid-based 3D hydrogel with low-power ultrasound is as follows: 20 kHz to 1000 kHz frequency and 30 mW cm -2 Up to 500 mW cm -2 A method for inducing cell reprogramming of adult cells, wherein the cell is treated with low-power ultrasound of low intensity for 5 to 25 minutes.

19. In Paragraph 16, A method for inducing cell reprogramming of adult cells, wherein the exposure cycle of the low-power ultrasound is 1 to 3 days.

20. Induced pluripotent stem cells produced by the method of any one of paragraphs 13 to 19.

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