Method for inducing reprogramming of alveolar type 1 cell into alveolar type 2 cell using ML141

Abstract

The present invention relates to a composition for inducing the reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising ML141 as an active ingredient. According to the present invention, by reprogramming AT1 cells into AT2 cells using ML141, 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 ML141 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 ML141, a Cdc42 / Rac1 GTPase inhibitor.

[0009] Accordingly, the objective of the present invention is to provide a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising ML141 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 ML141 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 ML141 may be included at a concentration of 1 to 100 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, 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 ML141 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 simply reprogramming type 1 alveolar cells into type 2 alveolar cells with only the treatment of ML141, without the process of overexpression of signaling proteins and transcription factors via viruses, pDNA, etc., which are generally required for reprogramming, it is possible to efficiently obtain type 2 alveolar cells, which were difficult to obtain in sufficient numbers. Furthermore, it is expected that by reprogramming type 1 alveolar cells into type 2 alveolar 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 obtained. Brief explanation of the drawing

[0027] Figure 1 is a schematic diagram showing a method for producing patient-derived airway organoids. Figure 2 shows the results of confirming the culture status after treating airway organoids with 5 μM, 10 μM, and 50 μM of ML141 ((A) after ML141 treatment, (B) 5 days after ML141 treatment). Figure 3 is a diagram showing the identification of AT2 (Alveolar Type 2) cells using LysoTracker after treating airway organoids with ML141 5 μM, 10 μM, and 50 μM for 5 days. Figure 4 is a diagram showing the identification of AT2 (Alveolar Type 2) cells using LysoTracker after treating airway organoids with ML141 5 μM, 10 μM, and 50 μM for 5 days. Figure 5 is a diagram showing the results of identifying AT1 cells after treating airway organoids with 10 μM of ML141. 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 ML141, a Cdc42 / Rac1 GTPase inhibitor.

[0029] Specifically, according to one embodiment of the present invention, when airway organoids are produced (cultured) in a medium mixed (added, treated) with ML141 compared to airway organoids cultured in a medium conventionally used for producing (culture) airway organoids, it was confirmed that type 1 alveolar cells (AT1 cells) decrease and type 2 alveolar cells (AT2 cells) increase, and from this, it was confirmed that ML141 can reprogram type 1 alveolar cells into type 2 alveolar cells.

[0031] The present invention will be described in detail below.

[0033] The present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells comprising ML141 as an active ingredient, wherein the reprogramming of type 1 alveolar cells (AT1 cells) into type 2 alveolar cells (AT2 cells) may be induced by adding (treating, mixing) ML141 to airway organoids being cultured in the medium.

[0034] In this specification, the term “ML141” belongs to the class of intracellular signaling inhibitors and selectively inhibits Cdc42 and Rac1. Cdc42 and Rac1 are known to be involved in the formation of actin within cells. Additionally, ML141 increases p38 within cells and induces p38-mediated apoptosis and senescence.

[0035] In the present invention, the ML141 is 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 1 to 5 μM, 5 to 50 μM, 5 to 40 μM, 5 to 30 μM, 5 to 20 μM, 5 to 10 μM, 8 to 10 μM, 10 to 13 μM, 10 to 15 μM, 9 to 11 μM, 9 to 10 μM, 10 to 11 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM relative to the total composition. It may be included at a concentration of μM, and according to one embodiment of the present invention, the ML141 may be included at a concentration of 10 μM relative to the total composition, but is not limited thereto, and may be included at a concentration within an appropriate range relative to the total composition that can reprogram AT1 cells into AT2 cells without inducing apoptosis.

[0036] According to one embodiment of the present invention, the ML141 is characterized by reprogramming type 1 alveolar cells into type 2 alveolar cells.

[0037] In this specification, the term "reprogramming" refers to a method of converting a specific cell into a target cell by controlling 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, and for the purposes of the present invention, said reprogramming may be performed by treating type 1 alveolar cells with ML141. For example, reprogramming may include, but is not limited to, cell dedifferentiation, direct reprogramming (or direct conversion), or direct transdifferentiation.

[0038] 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).

[0039] That is, transformative differentiation 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, and in the present invention, whether differentiation into type 2 alveolar cells can be confirmed through the expression of LysoTracker, which is specifically expressed in type 2 alveolar cells.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] According to one embodiment of the present invention, the type 1 alveolar cells are characterized by expressing the Aquaporin 5 (AQP5) gene. Accordingly, by confirming the expression of the AQP5 gene within the airway organoid, the concentration of ML141 applied to the airway organoid can be adjusted to produce an organoid having a desired ratio of type 1 alveolar cells 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.

[0044] 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.

[0046] 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.

[0047] 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.

[0048] The form of the kit according to the present invention may be a single container containing ML141, a nutrient mixture, a growth factor, or a basic medium and other reagents in appropriate amounts and / or forms, or may be provided by separate containers.

[0049] 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.

[0051] 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.

[0052] 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.

[0053] 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, 4 to 7 days, 5 to 7 days, 4 to 6 days, 5 to 6 days, 4 to 5 days, 1 day, 2 days, 3 days, 4 days, or 5 days. According to one embodiment of the present invention, airway organoids or type 1 alveolar cells may be cultured for 5 days in a medium composition containing ML141 at a concentration of 10 μM, but are not limited thereto. The above airway organoid is an airway organoid containing type 1 alveolar cells.

[0054] 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 secure 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.

[0056] 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.

[0058] [Experimental Method]

[0059] 1. Production of patient-derived airway organoids

[0060] 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.

[0061] The specific composition of the AO medium used for the production of airway organoids is as follows:

[0062] 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. Treatment of organoids with ML141

[0065] After constructing airway organoids, the Cdc42 / Rac1 GTPase inhibitor ML 141 (Tocris, 4266) was added to the organoid medium at predetermined concentrations. In this case, DMSO (Dimethylsulfoxide) was used as the solvent for ML 141, while the vehicle group among the controls was treated only with DMSO. The organoids treated with either ML 141 or DMSO were cultured for 5 days in an incubator under 5% CO2 and 37°C conditions.

[0067] 3. Immunofluorescence staining

[0068] 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). The nuclei were stained with Hoechst 33342 (ThermoFisher Scientific, 62249) and then washed with PBS (Phosphate Buffered Saline). After the staining process was completed, the medium was replaced with organoid medium, and images were taken using a fluorescence microscope.

[0069] Additionally, 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 with 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 sample was treated with the primary antibody Aquaporin 5 (1:200, abcam, ab92320) overnight at 4°C. After treatment with the primary antibody, the sample was washed with PBS at room temperature for 15 minutes, and then treated with the secondary antibody goat anti-rabbit IgG 488 (1:1000, Invitrogen, A-11008) 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.

[0071] [Example]

[0072] Example 1. ML141 reprogramming into AT2 (Alveolar Type 2) cells in airway organoids

[0073] Since high concentrations of compounds can induce apoptosis, we determined the concentration of ML141 that can reprogram into AT2 cells without inducing apoptosis. Specifically, we observed cultures of airway organoids treated with ML141 at concentrations of 5 μM, 10 μM, and 50 μM and cultured for 5 days.

[0074] As a result, as shown in Figure 2, the airway organoids proliferated normally and maintained their morphology even after reacting for 5 days at each ML141 treatment concentration. Therefore, we decided to check for the generation of AT2 cells using LysoTracker on airway organoids treated with ML141 at concentrations of 5 μM, 10 μM, and 50 μM. As a result, as shown in Figure 3, it was confirmed that AT2 cells increased when treated with ML141 at concentrations of 5 μM and 10 μM. On the other hand, when treated at the highest concentration of 50 μM, the cells constituting the organoid died entirely due to cytotoxicity. Thus, it was confirmed that ML141 generates AT2 cells under appropriate concentration conditions.

[0075] In addition, the presence of AT2 cells was more specifically confirmed using LysoTracker in airway organoids treated with ML141 at concentrations of 5 μM, 10 μM, and 50 μM. As shown in Figure 4, at the highest concentration of 50 μM, a significant number of organoids died due to stress caused by treatment with the high concentration compound, but a high proportion of LysoTracker+ cells, i.e., AT2 cells, were observed in the remaining individuals. Furthermore, it was confirmed that organoids treated with ML141 at 10 μM showed no abnormalities in culture and that the production of LysoTracker+ cells, specifically AT2 cells, also increased.

[0077] Example 2. Confirmation of reprogramming from AT1 (Alveolar Type 1) cells to AT2 cells

[0078] To confirm that AT2 cells were reprogrammed from AT1 cells, 10 μM of ML141, which efficiently induced AT2 cells in Example 1, was treated to airway organoids, and the remaining AT1 cells were identified. As a result, as shown in Figure 5, it was confirmed that the number of cells expressing Aquaporin 5 (AQP5) in organoids treated with 10 μM of ML141 was significantly lower compared to the control group not treated with ML141.

[0079] AQP5 is an AT1 marker, and from the above results, it was confirmed that AT1 cells inside the airway organoid were converted into AT2 cells by ML141 treatment, and the number of remaining AT1 cells decreased.

[0081] 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 An airway organoid medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising ML141 as an active ingredient, wherein the ML141 is included at a concentration of 5 to 10 μM relative to the total composition. Claim 2 delete Claim 3 A culture medium composition according to claim 1, characterized in that the type 1 alveolar cells express the Aquaporin 5 gene. Claim 4 An airway organoid culture kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a culture medium composition according to either claim 1 or 3. Claim 5 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. 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. Claim 8 delete

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