Method for inducing reprogramming of alveolar type 1 cell into alveolar type 2 cell using Rhosin

Abstract

The present invention relates to a composition for inducing the reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising rhosin as an active ingredient. According to the present invention, by reprogramming AT1 cells into AT2 cells using rhosin, it is possible to efficiently secure AT2 cells, which were difficult to obtain in sufficient numbers. Furthermore, it is expected that by reprogramming AT1 cells into AT2 cells within the organoid while culturing the organoid itself rather than culturing a single cell, cells with physiological activity similar to actual tissue can be secured.

Description

Technology Field

[0001] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising Rhosin as an active ingredient. Background Technology

[0003] Among the various cells that make up the lungs, the cells that perform major functions include adult cells, specifically AT1 (alveolar type 1) cells, and stem cells, specifically AT2 (alveolar type 2) cells. AT1 cells are cells that constitute the alveoli and perform the primary function of mediating gas exchange, while AT2 cells protect and maintain the alveoli by secreting surfactant, and as progenitor cells, they repair damage to epithelial cells by generating AT1 cells.

[0004] AT1 and AT2 cells differ in their internal structures, most notably in F-actin. In AT1 cells, actin forms structures extending from the cytoplasm to the outside of the nucleus, whereas in AT2 cells, actin is not observed within the cytoplasm and does not come into contact with the outside of the nucleus. Additionally, regarding contact with actin, the nucleus of AT1 cells, where actin is in contact with the outside of the nucleus, has an oval shape, whereas the nucleus of AT2 cells, where actin is not in contact, has a shape that is relatively closer to a circle.

[0005] Meanwhile, AT2 cells are generally widely used in research where they are established as iPSCs (induced pluripotent stem cells) and then differentiated into various cells that make up the respiratory tract, and they are typically obtained by isolating them from surgical tissues. However, the number of AT2 cells obtained by isolating them from surgical tissues is very limited, and there is a problem that AT2 cells are difficult to culture because they take on a phenotype similar to that of AT1 cells when cultured in 2D. Consequently, it is difficult to obtain a sufficient number of AT2 cells compared to their utility. Prior art literature

[0007] Republic of Korea Registered Patent No. 10-153913 The problem to be solved

[0008] The inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with Rhosin, a Rho GTPase inhibitor.

[0009] Accordingly, the objective of the present invention is to provide a medium composition for inducing the reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising rhosin as an active ingredient.

[0010] Another objective of the present invention is to provide a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells comprising a culture medium composition according to the present invention.

[0011] Another objective of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in a culture medium composition according to the present invention to induce them into type 2 alveolar cells.

[0012] Another objective of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a culture medium composition according to the present invention to induce type 1 alveolar cells of the airway organoid into type 2 alveolar cells.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0016] To achieve the above objective, the present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising rhosin as an active ingredient.

[0017] In addition, the present invention provides a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells comprising a culture medium composition according to the present invention.

[0018] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in a culture medium composition according to the present invention to induce them into type 2 alveolar cells.

[0019] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing an airway organoid in a culture medium composition according to the present invention to induce type 1 alveolar cells of the airway organoid into type 2 alveolar cells.

[0020] In one embodiment of the present invention, the rosine may be included at a concentration of 1 to 500 μM relative to the total composition, but is not limited thereto.

[0021] In another embodiment of the present invention, the type 1 alveolar cells may express the Aquaporin 5 gene, and the type 2 alveolar cells may express the SFTPC gene, but are not limited thereto.

[0022] In another embodiment of the present invention, the culture may be performed for 1 to 10 days, but is not limited thereto.

[0023] In addition, the present invention provides a use of Rhosin for inducing the reprogramming of type 1 alveolar cells into type 2 alveolar cells. Effects of the invention

[0025] According to the present invention, by reprogramming type 1 alveolar cells into type 2 alveolar cells using Rhosin, it is possible to efficiently obtain type 2 alveolar cells, which were difficult to obtain in sufficient numbers. Furthermore, by culturing the organoid itself rather than culturing a single cell and reprogramming type 1 alveolar cells into type 2 alveolar cells within the organoid, it is expected that cells with physiological activity similar to actual tissue can be obtained. Brief explanation of the drawing

[0027] Figure 1 is a schematic diagram showing a method for producing patient-derived airway organoids. Figure 2a shows the expression of AQP5 (Aquaporin 5) to confirm AT1 cells in airway organoids cultured for 20 days, and the bottom image is an enlarged view of the area marked with a white box in the top image (D20: Day 20). Scale bar = 50 µm (top), 100 µm (bottom). Figure 2b shows the identification of AT2 cells in airway organoids cultured for 20 days using LysoTracker (arrow: airway organoid in which AT2 cells were confirmed to be present via LysoTracker). Scale bar = 200 µm. Figure 3a shows the identification of AT2 cells using LysoTracker after treating airway organoids with Rhosin 100 μM, 500 μM, and 1 mM for 7 days. Figure 3b shows the results of confirming the proportion of AT2 cells by FACS (Fluorescence-Activated Cell Sorting) analysis after treating airway organoids with 100 μM Rhosin for 7 days. Figure 4a shows the results of observing airway organoids after treating them with Rhosin 10 μM, 50 μM, and 100 μM for 2 days (195N: Airway organoid classification number, AO: Airway organoid, P.6: passage number 6, D2: Day 2). Figure 4b shows the results of identifying AT2 cells using LysoTracker after treating airway organoids with Rhosin 10 μM and 50 μM for 2 days. Figure 4c shows the results of identifying AT2 cells using LysoTracker after treating airway organoids with Rhosin 5 μM, 10 μM, and 50 μM for 7 days. Figure 5 is a diagram showing the results of confirming the expression of SFTPC, a biomarker of AT2 cells, after treating airway organoids with Rhosin 5 μM, 10 μM, and 50 μM for 7 days. Specific details for implementing the invention

[0028] The inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with Rhosin, a Rho GTPase inhibitor.

[0029] Specifically, according to one embodiment of the present invention, when airway organoids were prepared (cultured) in a medium mixed (added, treated) with Rhosin compared to airway organoids cultured in a medium conventionally used for preparing (culture) airway organoids, it was confirmed that type 2 alveolar cells (AT2 cells) increased (see Examples 2 and 4).

[0030] In addition, according to another embodiment of the present invention, since Rhosin can induce apoptosis, in order to find the optimal conditions for reprogramming AT1 cells into AT2 cells using this, airway organoids were cultured by adjusting the concentration and culture period of Rhosin, and it was confirmed that reprogramming into AT2 cells proceeded with only low concentrations of 5 μM or 10 μM for 2 days (see Example 3).

[0032] The present invention will be described in detail below.

[0034] The present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising rhosin as an active ingredient. The reprogramming of type 1 alveolar cells (AT1 cells) into type 2 alveolar cells (AT2 cells) may be induced by adding (treating, mixing) rhosin to airway organoids being cultured in the medium, but is not limited thereto, and may also be induced by adding (treating, mixing) rhosin to type 1 alveolar cells being cultured in the medium.

[0035] In this specification, the term “Rhosin” refers to a RhoA inhibitor that has the function of inhibiting cancer proliferation and promoting nerve cell regeneration, and is known to specifically target Rho GTPases to prevent the growth of cancer-related cells. Rhosin has been confirmed to inhibit the growth of cancer cells in breast cancer cells in a dose-dependent manner, and has also been proven to be effective against asthma and diabetes.

[0036] In the present invention, the rosine may be included at a concentration of 1 to 500 μM, 5 to 500 μM, 10 to 500 μM, 50 to 500 μM, 100 to 500 μM, 1 to 100 μM, 5 to 100 μM, 10 to 100 μM, 50 to 100 μM, 1 to 50 μM, 5 to 50 μM, 10 to 50 μM, 1 to 10 μM, 5 to 10 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM relative to the total composition, and according to one embodiment of the present invention, the rosine may be included at a concentration of 5 μM or 10 μM relative to the total composition, but is not limited thereto. It can be included in an appropriate range of concentrations capable of reprogramming AT1 cells into AT2 cells without inducing apoptosis relative to the total composition.

[0037] According to one embodiment of the present invention, the rosine is characterized by reprogramming type 1 alveolar cells into type 2 alveolar cells.

[0038] In this specification, the term "reprogramming" refers to a method of converting a specific cell into a target cell by regulating its global gene expression pattern. Specifically, in the present invention, reprogramming refers to a method of converting a specific cell into a cell having completely different characteristics by artificially manipulating it. For the purposes of the present invention, said reprogramming may be performed by treating type 1 alveolar cells or airway organoids containing them with rosine. For example, reprogramming may include, but is not limited to, cell dedifferentiation, direct reprogramming (or direct conversion), or direct transdifferentiation.

[0039] In this specification, the term “trans-differentiation,” also known as lineage reprogramming, refers to differentiation in which a mature somatic cell transforms into another mature somatic cell without passing through an intermediate pluripotent or progenitor cell stage (Graf, T.; Enver, T. (2009). "Forcing cells to change lineages". Nature 462 (7273): 587-594).

[0040] That is, transdifferentiation (direct transdifferentiation) is the conversion of one type of mature cell into another type of mature cell, and whether the conversion has occurred can be confirmed by detecting the morphology of each cell or the expression of specific genes. In the present invention, whether differentiation into type 2 alveolar cells can be confirmed through the expression of SFTPC (Surfactant Protein C), a marker specifically expressed in type 2 alveolar cells.

[0041] In this specification, the term “direct-reprogramming” is a technology that is differentiated from the technology of producing induced pluripotent stem cells (iPSCs) with pluripotency through a reprogramming process, and is a technology that induces direct conversion into a desired target cell through reprogramming culture. Conventional somatic cell nuclear transfer has the disadvantage of requiring the use of oocytes, which limits its applicability compared to other cell reprogramming technologies. Furthermore, when using iPSC reprogramming technology, there is a disadvantage in that the presence of undifferentiated cells and the assurance of safety must be verified because it passes through pluripotent stem cells by nature. However, the present invention is expected to provide an alternative that can overcome the problems of the aforementioned technologies, such as production time, cost, efficiency, and safety, by directly producing the target cell, Type II alveolar cells, from Type I alveolar cells through direct reprogramming. For the purposes of the present invention, direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct transdifferentiation, transdifferentiation, etc. In the present invention, direct reprogramming may mean direct dedifferentiation or transdifferentiation into type 2 alveolar cells.

[0042] As used herein, the term "medium" refers to a mixture of nutrient substances that enables the growth, survival, expansion, or differentiation of cells or organoids in vitro, and includes all appropriate conventional media used in the art. Depending on the type of cell or organoid, the type of medium and culture conditions may be selected at the technical level of the art.

[0043] In the present invention, the medium composition may additionally include a basal media for cell or organoid culture comprising a carbon source, a nitrogen source, and trace elements, and the basal media may include, for example, DMEM (Dulbecco's Modified Eagle Medium), Ham's F12, DMEM / F12, and Advanced DMEM / F12. According to one embodiment of the present invention, the basic medium may be Advanced DMEM / F12, and preferably, it may be a culture medium for airway organoids comprising, but is not limited to, a Wnt agonist (R-Spondin-1), growth factors (FGF-7 and FGF-10), TGF-β inhibitors (Noggin and A83-01), ROCK inhibitor (Y-27632), p38 MAPK inhibitor (SB 202190), serum substitute (B-27), NAC (N-Acetyl-L-cysteine), nicotinamide, glutamine substitute (L-alanyl-L-glutamine dipeptide; product name GlutaMax), zwitterionic buffer (HEPES), and antibiotics (Penicillin-Streptomycin and Primocin) in Advanced DMEM / F12 medium. Any medium commonly used for cell or AT2 cell culture can be used without restriction.

[0044] According to one embodiment of the present invention, the type 1 alveolar cells express the Aquaporin 5 (AQP5) gene, and the type 2 alveolar cells express the SFTPC gene. Accordingly, by confirming the expression of the AQP5 and SFTPC genes within the airway organoid, the concentration of Rhosin applied to the airway organoid can be adjusted to produce an organoid having a desired ratio of type 1 and type 2 alveolar cells. Therefore, the culture medium composition of the present invention can be utilized in various ways to produce organoids suitable for desired purposes, such as drug screening and cell therapy.

[0045] In this specification, “active ingredient” means an ingredient that exhibits the intended activity alone or can exhibit the intended activity together with a carrier, etc., which is inactive itself.

[0047] In addition, the present invention provides, in another embodiment, a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells comprising a culture medium composition according to the present invention. The type of the kit of the present invention is not particularly limited, and any type of kit commonly used in the art may be used.

[0048] The above kit is not limited to the culture medium composition according to the present invention and may include other reagents or equipment. For example, it may include a culture plate for culturing target cells or organoids, or a reagent capable of evaluating the reprogramming of type 1 alveolar cells into type 2 alveolar cells, and may also include cells or organoids to be cultured.

[0049] The form of the kit according to the present invention may be a single container containing all of the rosine, nutrient mixture, growth factor, or basic medium and other reagents in appropriate amounts and / or forms, or may be provided by separate containers.

[0050] The kit according to the present invention may include an instruction manual describing the sequence, etc., for carrying out the method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells according to the present invention described below.

[0052] In addition, the present invention provides, in another aspect, a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in a culture medium composition according to the present invention to induce them into type 2 alveolar cells.

[0053] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing an airway organoid (containing type 1 alveolar cells) in a culture medium composition according to the present invention to induce type 1 alveolar cells of the airway organoid into type 2 alveolar cells.

[0054] In the present invention, the method involves culturing type 1 alveolar cells or airway organoids in the medium composition to induce reprogramming of type 1 alveolar cells into type 2 alveolar cells, and the culture may be performed for 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 6 to 10 days, 7 to 10 days, 1 to 7 days, 2 to 7 days, 3 to 7 days, 1 to 3 days, 2 to 3 days, 1 to 2 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. According to one embodiment of the present invention, airway organoids or type 1 alveolar cells may be cultured for 2 days or 7 days in a medium composition containing Rhosin at a concentration of 5 or 10 μM, but are not limited thereto. The above airway organoid is an airway organoid containing type 1 alveolar cells.

[0055] The present invention allows for reprogramming type 1 alveolar cells into type 2 alveolar cells by culturing type 1 alveolar cells in a culture medium composition according to the present invention. Specifically, by culturing an airway organoid containing type 1 alveolar cells in a culture medium composition according to the present invention, the type 1 alveolar cells within the airway organoid are reprogrammed into type 2 alveolar cells, thereby making it easier to obtain type 2 alveolar cells, which are difficult to obtain in sufficient numbers. Furthermore, by reprogramming while culturing the organoid itself rather than culturing a single cell, type 2 alveolar cells with physiological activity similar to actual tissue can be obtained.

[0057] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0059] [Experimental Method]

[0060] 1. Production of patient-derived airway organoids

[0061] Patient-derived airway organoids were prepared as shown in the schematic diagram in Figure 1. Specifically, airway tissue obtained from the patient's lungs was washed with a PBS (Phosphate Buffered Saline) solution containing 100 U / mL Penicillin-Streptomycin to remove red blood cells (RBCs). After finely separating the airway tissue, 2 mg / mL collagenase (Roche, 11097113001) was added and incubated at 37°C for 1 hour. After filtering the reacted cell solution, it was treated with RBC Lysis Buffer (Invitrogen, 00-4333-57) and incubated at room temperature for 2 minutes, after which single cells derived from the airway tissue were obtained through centrifugation and washing. 5,000–10,000 single cells derived from airway tissue were prepared as a cell suspension with 20 μL of Matrigel (50%) solution and incubated at 37°C for 15 minutes to form a Matrigel dome. Then, the single cells were embedded into the Matrigel dome, and AO medium, an airway organoid medium, was added. The cells were cultured in an incubator under 5% CO2 and 37°C conditions. The formation of organoids was monitored by replacing half of the medium with fresh medium every 2–3 days.

[0062] The specific composition of the AO medium used for the preparation of airway organoids is as follows: Advanced DMEM / F12 (Invitrogen, 12634-034) supplemented with 500 ng / mL R-Spondin-1 (PeproTech, 120-38), 25 ng / mL FGF-7 (PeproTech, 100-19), 100 ng / mL FGF-10 (PeproTech, 100-26), 100 ng / mL Noggin (PeproTech, 120-10C), 500 nM A83-01 (Tocris, 2939), 5 μM Y-27632 (Abmole, M1817), 500 nM SB 202190 (Sigma, S7067), 2% (v / v) B-27 (Gibco, 17504044), 1.25mM N-Acetyl-L-cysteine ​​(Sigma-Aldrich, A9165), 5mM Nicotinamide (Sigma, N0636), 1% GlutaMax (Invitrogen, 35050061), 10mM HEPES (Gibco, 15630056), 100 U / mL Penicillin-Streptomycin (Gibco, 15140122), 50 μg / mL Primocin (Invivogen, Ant-pm-1).

[0063]

[0064] 2. Rhosin treatment of organoids

[0065] After preparing airway organoids, the organoids were treated with Rhosin (Sigma-Aldrich (Merck), 555460-M), a Rho inhibitor, by mixing it into the organoid medium at predetermined concentrations. At this time, DMSO (Dimethylsulfoxide) was used as the solvent for Rhosin, while the vehicle group among the controls was treated only with DMSO. The organoids treated with either Rhosin or DMSO were cultured for a predetermined period in an incubator under a 5% CO2, 37°C environment.

[0067] 3. Immunofluorescence staining

[0068] After removing the external organoid medium, the Matrigel dome was washed with PBS (Phosphate Buffered Saline) solution. The organoids inside the Matrigel dome were fixed in 2% PFA (paraformaldehyde) containing 0.05% glutaraldehyde (Sigma-Aldrich, G6257) at room temperature for 20 minutes. After fixation, the Matrigel dome and organoids were washed three times with PBS, and a frozen OCT block was prepared using cryomold (Sakura Finetek, 4565) and Optimal Cutting Temperature (OCT) (Sakura Finetek, 4583) compounds. The frozen OCT block was prepared into a sectioning slide using a Cryocut Microtome (Leica, CM1850) and then washed with PBS at room temperature for 15 minutes. After quenching with 0.3M glycine at room temperature for 20 minutes, the sample was washed with PBS at room temperature for 15 minutes. After blocking with 5% normal goat serum (Vector Laboratories, Inc., S-1000) at room temperature for 1 hour, the primary antibodies Aquaporin 5 (1:200, abcam, ab92320) and SFTPC (1:100, ThermoFisher Scientific, PA5-102493) were applied overnight at 4°C. After treatment with the primary antibodies, the sample was washed with PBS at room temperature for 15 minutes, and the secondary antibody goat anti-rabbit IgG 488 (1:1000, Invitrogen, A-11008) was applied at room temperature for 1 hour. After washing with PBS at room temperature for 15 minutes, the sample was stained with DAPI (Invitrogen, D1306) at room temperature for 2 minutes.After mounting the organoid samples onto a cover slide, images were taken using a Carl Zeiss LSM800 or LSM900 confocal microscope.

[0069] To stain AT2 cells with LysoTracker, the medium was removed from the cultured organoids, and 50 nM LysoTracker Green DND-26 (Invitrogen, L7526) was added using organoid medium prewarmed at 37°C. After treatment for 1 hour in an incubator under 5% CO2 and 37°C conditions, the medium containing LysoTracker was removed, and the cells were washed three times with PBS (Phosphate Buffered Saline). Organoid medium was then added, and images were taken using a fluorescence microscope.

[0071] [Example]

[0072] Example 1. Identification of AT1 (Alveolar Type 1) cells and AT2 (Alveolar Type 2) cells in airway organoids

[0073] Since AT2 cells are present in airway organoids by default even without reprogramming with Rhosin, AT1 and AT2 cells within the airway organoid were identified before reprogramming AT1 (Alveolar Type 1) cells into AT2 (Alveolar Type 2) cells. Specifically, as shown in Figure 2a, the presence of Aquaporin 5 (Aqp5) in airway organoids cultured for 20 days was confirmed, and it was found that AT1 cells, which are the initiating cells for reprogramming into AT2 cells, were present within the airway organoid.

[0074] In addition, as shown in Figure 2b, the presence of AT2 cells in the airway organoid was confirmed using LysoTracker, which specifically stains only AT2 cells among the cells constituting the respiratory tract. As a result, it was confirmed that a certain level of AT2 cells is basically present when the airway organoid is cultured for a sufficient amount of time, even before reprogramming into AT2 cells.

[0076] Example 2. Rhosin for reprogramming AT1 (Alveolar Type 1) cells into AT2 (Alveolar Type 2) cells in airway organoids

[0077] To reprogram AT1 cells into AT2 cells within airway organoids, the prepared airway organoids were treated with Rhosin, a Rho inhibitor, and the presence of AT2 was confirmed using LysoTracker. As a result, as shown in Figure 3a, when treated with Rhosin at 100 μM, 500 μM, and 1 mM for 7 days, a significant increase in AT2 cells was observed at 100 μM Rhosin. However, when treated with 500 μM and 1 mM Rhosin, it was observed that the cells died due to the high concentration of Rhosin.

[0078] Furthermore, FACS (Fluorescence-Activated Cell Sorting) analysis was performed on AT2 cells generated after Rhosin treatment in airway organoids. Specifically, LysoTrackers of the control group and the Rhosin 100 μM treatment group + As a result of comparing the proportion of cells (AT2 cells) by FACS, as shown in Figure 3b, a higher proportion of LysoTrackers were produced when Rhosin was treated compared to the control group. + We were able to observe the cells.

[0080] Example 3. Confirmation of Rhosin conditions capable of reprogramming AT1 (Alveolar Type 1) cells into AT2 (Alveolar Type 2) cells in airway organoids

[0081] As seen in Example 2 above, high concentrations of Rhosin actually induced cell death, so a concentration of Rhosin capable of reprogramming AT1 cells into AT2 cells without inducing cell death was determined.

[0082] To determine the specific conditions for AT2 cell reprogramming using Rhosin, airway organoids were treated with Rhosin at low concentrations of 10 μM, 50 μM, and 100 μM compared to Example 3, and cultured for 2 days, and the cultures were observed. As a result, as shown in Figure 4a, the airway organoids proliferated normally and maintained their morphology even after reacting for 2 days with each Rhosin treatment concentration; therefore, it was decided to check whether AT2 cells were generated in airway organoids treated with Rhosin at a concentration of 100 μM or less.

[0083] Reprogramming conditions were confirmed by treating with Rhosin at a concentration of 100 μM or less for a short period of time, compared to Example 2, in which Rhosin was treated for 7 days. Specifically, after treating with Rhosin 10 μM and 50 μM for 2 days, the presence of AT2 cells was compared and confirmed using LysoTracker, and as shown in Figure 4b, it was confirmed that AT2 cells were generated in the airway organoid.

[0084] Furthermore, to confirm the effect of Rhosin at low concentrations, Rhosin was treated at 5 μM, 10 μM, and 50 μM for 7 days and then stained with LysoTracker. As shown in Figure 4c, it was confirmed that AT2 cells increased in airway organoids treated with 5 μM Rhosin.

[0086] Example 4. Confirmation of AT2 (Alveolar Type 2) cell reprogramming using Rhosin in airway organoids

[0087] LysoTracker is known to specifically stain only AT2 cells among the cells constituting the respiratory tract, but AT2 cell reprogramming using Rhosin, SFTPC, a biomarker of AT2 cells + Further verification was performed using cells. Specifically, after treating airway organoids with Rhosin at 5 μM, 10 μM, and 50 μM for 7 days, SFTPC, a biomarker of AT2 cells, + The cells were identified.

[0088] As a result, as shown in Figure 5, when Rhosin is treated, SFTPC, similar to the results of confirming AT2 cells using LysoTracker, + It was possible to confirm that the number of cells had increased.

[0089] When Rhosin is administered to airway organoids, LysoTracker + SFTPC + It was confirmed that the cells were generated by Rhosin, and from this, it was determined that AT2 cells were generated by Rhosin in the airway organoids.

[0091] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising rhosin as an active ingredient. Claim 2 A culture medium composition according to claim 1, characterized in that the rosine is included at a concentration of 1 to 500 μM relative to the total composition. Claim 3 A culture medium composition according to claim 1, characterized in that the type 1 alveolar cells express the Aquaporin 5 gene and the type 2 alveolar cells express the SFTPC gene. Claim 4 A kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a culture medium composition according to any one of claims 1 to 3. Claim 5 A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in the culture medium composition of claim 1 to induce them into type 2 alveolar cells. Claim 6 A method according to claim 5, characterized in that the culture is performed for 1 to 10 days. Claim 7 A method according to claim 5, characterized in that the type 1 alveolar cells express the Aquaporin 5 gene and the type 2 alveolar cells express the SFTPC gene. Claim 8 A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing an airway organoid in the culture medium composition of claim 1 to induce type 1 alveolar cells of the airway organoid into type 2 alveolar cells.

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