Compounds for maintaining stem cell stemness and organoid culture and uses thereof

Abstract

This invention proposes compounds for maintaining stem cell stemness and organoid culture, and their uses. The compounds proposed in this invention are photostable, exhibiting superior performance in relevant applications without requiring deliberate light avoidance, and can maintain stem cell stemness and significantly promote organoid culture from multiple tissue sources. Specifically, this invention proposes uses of the compounds in at least one of the following: maintaining stem cell stemness; organoid culture; preparation of drugs; drugs for treating and / or preventing stem cell failure or related diseases; preparation of culture media for culturing organoids; the compounds being compounds of formula (I) or their stereoisomers, tautomers, solvates, or pharmaceutically acceptable salts.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to compounds for maintaining stem cell stemness and organoid culture and their uses, and more specifically, to a compound and its use in maintaining stem cell stemness, organoid culture, drug preparation, culture medium preparation, drugs, culture media, and methods for in vitro culture of organoids. Background Technology

[0002] Blebbistatin is a classic non-muscle myosin II inhibitor. Its unique molecular mechanism, which specifically binds to the intermediate complex formed by myosin II and ADP-phosphate, blocking its phosphate release step, locks myosin II in a low-energy state dissociated from actin filaments. This allows it to precisely interfere with the dynamics of non-muscle myosin II in the cytoskeleton, thereby broadly affecting cell morphology maintenance, adhesion and migration, division and differentiation, and related signal transduction networks. Under ideal conditions, Blebbistatin shows great promise for applications: in stem cell biology, it can maintain stem cell stemness and regulate the stem cell microenvironment.

[0003] However, the practical application potential of Blebbistatin is severely hampered by a fatal flaw: it exhibits extreme photochemical instability to light, especially in the short wavelength range (365 nm–490 nm) of ultraviolet and visible light. Under normal laboratory or medical lighting conditions, this compound rapidly photodegrades, generating a series of photodegradation products with significant cytotoxicity. These products can lead to cell death through non-specific pathways such as inducing DNA damage, mitochondrial dysfunction, and oxidative stress. This characteristic necessitates that its practical application must be carried out in completely dark environments or in dark rooms equipped with specific wavelength filters, greatly increasing technical complexity and cost. More importantly, the phototoxic products severely interfere with the interpretation of experimental results, fundamentally hindering the translation of Blebbistatin from in vitro cell research to clinical treatment.

[0004] Therefore, it is still necessary to further develop a novel small-molecule Blebbistatin derivative with photostability that can exhibit superior performance in relevant applications without the need for deliberate light avoidance; it should also be non-phototoxic and able to maintain the stem cell nature and organoid culture function, in order to further advance research in related regenerative medicine and related disease fields. Summary of the Invention

[0005] This application is based on the inventor's discoveries and understanding of the following facts and problems: Blebbistatin is highly sensitive to blue and ultraviolet light, rapidly photodegrading and inactivating under irradiation, while also exhibiting phototoxicity. During their exploration of photostable Blebbistatin derivatives, the inventors synthesized a novel small molecule, MT-330, through extensive creative work. Subsequent culturing of organoids from multiple tissues revealed that MT-330 effectively promotes the culture of organoids from various tissues and also demonstrates its ability to maintain the stemness of corresponding stem cells and serve as a supplement to organoid culture media.

[0006] Therefore, in a first aspect, the present invention provides a compound. The compound is a compound of formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of a compound of formula (I): Equation (Ⅰ).

[0007] According to embodiments of the present invention, the compound is photostable and can exhibit superior performance in relevant application scenarios without the need for deliberate light avoidance. It can also maintain stem cell stemness and has a significant promoting effect in the culture of organoids from multiple tissue sources, promoting the growth and proliferation of organoids in a dose-dependent manner.

[0008] In a second aspect of the invention, the invention proposes the use of the foregoing compounds in at least one of the following: (1) maintaining stem cell stemness; (2) organoid culture; (3) preparing a medicament for treating and / or preventing stem cell failure or related diseases; and (4) preparing a culture medium for culturing organoids.

[0009] According to embodiments of the present invention, the stem cells are derived from at least one of the small intestine, bile duct, stomach, or trachea; and / or the organoids include at least one of the small intestinal organoids, bile duct organoids, gastric organoids, or tracheal organoids; and / or the stem cell failure-related diseases include at least one of the following: malabsorption syndrome, intestinal barrier dysfunction, inflammatory bowel disease repair disorder, liver regeneration disorder, bile duct atrophy syndrome, refractory gastric ulcer, atrophic gastritis, chronic obstructive pulmonary disease, bronchiolitis obliterans, and airway ciliary movement disorder; and / or maintaining stem cell stemness includes upregulating the expression level of stemness genes of the stem cells, said genes including at least one of the following: LGR5 , HES1 , SOX9 , KRT5 , AXIN2 or Ki67 ; and / or in the drug, the dosage of the compound is 40-1200 μg / kg body weight / day.

[0010] In a third aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises the compounds described above. The medicament proposed by the present invention can maintain stem cell stemness, maintain stem cell self-renewal, multi-lineage differentiation potential, and genomic stability, thereby effectively treating or preventing diseases caused by impaired stem cell stemness.

[0011] According to an embodiment of the present invention, the drug is used to upregulate the expression level of stem genes, wherein the stem genes include at least one of the following: LGR5 , HES1 , SOX9 , KRT5 , AXIN2 or Ki67 .

[0012] In a fourth aspect, the present invention provides a culture medium. According to embodiments of the invention, the culture medium comprises the compounds described above. The culture medium is capable of promoting the growth and proliferation of organoids from multiple tissue sources, providing a nutritional basis and growth factors for the growth and proliferation of organoids.

[0013] According to embodiments of the present invention, the final concentration of the compound is 1 μM to 20 μM; and / or the culture medium further includes at least one of the following: basal culture medium, B27, N2, Noggin, EGF, L-glutamine, HEPES, or antibiotics.

[0014] According to an embodiment of the present invention, the basal culture medium is selected from at least one of the following: Advanced DMEM / F12 medium, DMEM medium, or MEM medium.

[0015] According to embodiments of the present invention, the final concentration of B27 is 100×~50×; and / or the final concentration of N2 is 200×~100×; and / or the final concentration of Noggin is 50 ng / mL~150 ng / mL; and / or the final concentration of EGF is 10 ng / mL~80 ng / mL; and / or the final concentration of L-glutamine is 200×~50×; and / or the final concentration of HEPES is 200×~50×; and / or the final concentration of the antibiotic is 200×~50×.

[0016] In a fifth aspect, the present invention provides a method for in vitro culture of organoids. According to embodiments of the present invention, the method includes: contacting the aforementioned compound or culture medium with stem cells and / or the organoid to be cultured to obtain cultured organoids. The organoids obtained by the method proposed according to the present invention exhibit good growth, and the method can effectively promote the growth and proliferation of organoids.

[0017] According to embodiments of the present invention, during the culture process, the concentration of the compound is 1 μM to 20 μM; and / or the stem cells include at least one of the following: embryonic stem cells, adult stem cells, or induced pluripotent stem cells; and / or the organoids include at least one of small intestinal organoids, bile duct organoids, gastric organoids, or tracheal organoids.

[0018] The beneficial effects of this invention are at least as follows: The compounds proposed in this invention are photostable and can exhibit superior performance in relevant application scenarios without the need for deliberate light avoidance. They can also maintain stem cell stemness and have a significant promoting effect in the culture of organoids from multiple tissue sources. Attached Figure Description

[0019] Figure 1 This is the chemical synthesis route diagram for compound MT-330.

[0020] Figure 2 These are bright-field images of organoids from multiple tissue sources treated with different concentrations of MT-330, with a scale bar of 200 µm. Among them, A is a bright-field image of mouse small intestinal organoids; B is a bright-field image of mouse bile duct organoids; C is a bright-field image of mouse gastric organoids; and D is a bright-field image of mouse tracheal organoids.

[0021] Figure 3 This is a statistical chart showing the relative expression levels of stem genes in various mouse tissues after injection of different concentrations of MT-330. A represents the expression of stem genes corresponding to mouse small intestinal stem cells; B represents the expression of stem genes corresponding to mouse bile duct stem cells; C represents the expression of stem genes corresponding to mouse gastric stem cells; and D represents the expression of stem genes corresponding to mouse tracheal stem cells.

[0022] Figure 4 Bright-field images of mouse small intestine, bile duct, stomach, and trachea organoids in a culture system using MT-330 instead of R-Sondin1, with a scale bar of 200 µm. Among them, A is the bright-field image of mouse small intestine organoids; B is the bright-field image of mouse bile duct organoids; C is the bright-field image of mouse stomach organoids; and D is the bright-field image of mouse trachea organoids.

[0023] Figure 5 The graphs show the relative expression levels of stem genes in mouse small intestinal stem cells after injection of Bebbistatin and MT-330 in different pretreatment groups (Control group is the control group, 1 is the light-protected group; 2 is the light-exposed group). Among them, A is the relative expression level of stem genes in mouse small intestinal stem cells treated with Bebbistatin; B is the relative expression level of stem genes in mouse small intestinal stem cells treated with MT-330. Detailed Implementation

[0024] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0027] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. In the description of this invention, the terms used herein have been explained and described; these explanations and descriptions are merely for the purpose of facilitating understanding and should not be construed as limiting the scope of protection of this invention.

[0028] In this article, the term "stem cell stemness" or "stemness" refers to the ability of stem cells to maintain their "undifferentiated state" and differentiate into specific functional cells under appropriate conditions, which refers to the self-renewal and multi-directional differentiation potential of stem cells.

[0029] In this document, the terms “MT-330” or “(3aR)-6,7-dimethyl-1-(6-quinolinyl-2-methyl)-1,2,3,4-tetrahydro-3aH-pyrrolo[2,3-b]quinoline-3a,4-diol” are synonymous, both having the chemical structure shown in formula (I).

[0030] In this article, the term "stemness gene" refers to the gene that encodes proteins that maintain the core characteristics (stemness) of stem cells, which is the molecular basis for maintaining the undifferentiated state and functionality.

[0031] The technical solution of this application will be described in detail below.

[0032] Uses of compounds In some embodiments of the present invention, a compound is provided. According to embodiments of the present invention, the compound is a compound of formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound of formula (I): Formula (I). The compound is photostable and can exhibit superior performance in relevant application scenarios without the need for deliberate light avoidance. It can also maintain stem cell stemness and has a significant promoting effect in the culture of organoids from multiple tissue sources, promoting the growth and proliferation of organoids in a dose-dependent manner.

[0033] In some embodiments of the invention, the use of the aforementioned compounds in maintaining stem cell stemness is proposed. According to embodiments of the invention, the stem cells are derived from at least one of the small intestine, bile duct, stomach, or trachea.

[0034] According to embodiments of the present invention, maintaining stem cell stemness includes upregulating the expression levels of stemness genes in the stem cells, said stemness genes including at least one of the following: LGR5 , HES1 , SOX9 , KRT5 , AXIN2 or Ki67 .

[0035] According to an embodiment of the present invention, the LGR5 The gene is a hallmark stem cell gene for small intestinal stem cells; HES1 The gene is a hallmark stem gene for bile duct stem cells; SOX9 The gene is a hallmark stem gene for gastric stem cells; KRT5 The gene is a tracheal marker stem gene; AXIN2 Gene expression levels reflect the activation state of stem cells; Ki67 Genes represent the proliferative activity of cells.

[0036] According to embodiments of the present invention, the stem cells include at least one of the following: embryonic stem cells, adult stem cells, or induced pluripotent stem cells.

[0037] According to an embodiment of the present invention, the compound activates the expression of downstream stem cell proliferation-related genes by enhancing the Wnt signal intensity.

[0038] In some embodiments of the present invention, the use of the aforementioned compounds in organoid culture is proposed.

[0039] According to embodiments of the present invention, the organoids include at least one of small intestinal organoids, bile duct organoids, gastric organoids, or tracheal organoids. The compounds are capable of significantly inducing the expression of stem genes in the corresponding organoid culture systems.

[0040] In some embodiments of the present invention, the use of the foregoing compounds in the preparation of medicaments for the treatment and / or prevention of stem cell failure or related diseases is proposed.

[0041] According to embodiments of the present invention, the stem cell failure-related diseases include at least one of the following: malabsorption syndrome, intestinal barrier dysfunction, inflammatory bowel disease repair disorder, liver regeneration disorder, bile duct atrophy syndrome, refractory gastric ulcer, atrophic gastritis, chronic obstructive pulmonary disease, bronchiolitis obliterans, and airway ciliary movement disorder.

[0042] According to embodiments of the present invention, the dosage of the compound in the drug is 40-1200 μg / kg body weight / day; exemplaryly, the dosage of the compound is 41, 50, 60, 80, 85, 90, 100, 200, 400, 600, 800, 820, 850, 900, 1000, 1100, or 1200 μg / kg body weight / day; or a range between any two of the above values. According to some preferred embodiments of the present invention, the dosage of the compound is 80-850 μg / kg body weight / day. The present invention, through the conversion of mouse and human dosages, demonstrates that the drug, after dosage conversion, can effectively treat and / or prevent stem cell failure or related diseases. Those skilled in the art will understand that the dosage of MT-330 is not particularly limited. For example, the dosage can be set according to the characteristics of different target populations, such as the weight of the target population (e.g., 40 kg, 60 kg, 70 kg, 80 kg, 90 kg, or 100 kg), or it can be set according to the characteristics of the target population and the MT-330 dosage. For example, the conversion factor between mouse and human dosage is 12.3. Based on the mouse dosage of MT-330 (500-15000 μg / kg body weight / day), the human dosage = mouse dosage / 12.3, resulting in a human dosage of MT-330 of 40-1200 μg / kg body weight / day.

[0043] In some embodiments of the invention, the compound is used in the preparation of a culture medium for culturing organoids.

[0044] According to embodiments of the present invention, the compound can drive stem cell proliferation while improving cell viability and maintaining its undifferentiated state, and possesses at least the same function as the traditional R-Sondin1 growth factor during organoid culture. Furthermore, MT-330 is a synthetically produced small molecule, exhibiting more stable batch-to-batch effects compared to R-Sondin1 (secreted by the R-Spo1-HEK293 cell line); and the synthesis of MT-330 is chemically synthesized, offering a cost advantage over R-Sondin1; MT-330 also has a smaller molecular weight, resulting in better tissue penetration compared to R-Sondin1.

[0045] drug In some embodiments of the present invention, a medicament is proposed. According to embodiments of the present invention, the medicament comprises the compounds described above. The medicament proposed by the present invention can maintain stem cell stemness, maintain stem cell self-renewal, multi-lineage differentiation potential, and genomic stability, thereby effectively treating or preventing diseases caused by stem cell stemness failure or damage.

[0046] According to embodiments of the present invention, the stem cell failure-related diseases include at least one of the following: malabsorption syndrome, intestinal barrier dysfunction, inflammatory bowel disease repair disorder, liver regeneration disorder, bile duct atrophy syndrome, refractory gastric ulcer, atrophic gastritis, chronic obstructive pulmonary disease, bronchiolitis obliterans, and airway ciliary movement disorder.

[0047] According to an embodiment of the present invention, the drug is used to upregulate the expression level of stem genes, wherein the stem genes include at least one of the following: LGR5 , HES1 , SOX9 , KRT5 , AXIN2 or Ki67 .

[0048] According to a specific embodiment of the present invention, the drug is used to upregulate the expression level of stem genes by 3 to 11 times, wherein the stem genes include at least one of the following: LGR5 , HES1 , SOX9 , KRT5 , AXIN2 or Ki67 .

[0049] According to embodiments of the present invention, the dosage of the compound in the drug is 40-1200 μg / kg body weight / day; exemplaryly, the dosage of the compound is 41, 50, 60, 80, 85, 90, 100, 200, 400, 600, 800, 820, 850, 900, 1000, 1100, or 1200 μg / kg body weight / day; or a range between any two of the above values. According to some preferred embodiments of the present invention, the dosage of the compound is 80-850 μg / kg body weight / day. The present invention, through the conversion of mouse and human dosages, demonstrates that the drug, after dosage conversion, can effectively treat and / or prevent stem cell failure or related diseases. Those skilled in the art will understand that the dosage of MT-330 is not particularly limited. For example, the dosage can be set according to the characteristics of different target populations, such as the weight of the target population (e.g., 40 kg, 60 kg, 70 kg, 80 kg, 90 kg, or 100 kg), or it can be set according to the characteristics of the target population and the MT-330 dosage. For example, using the body surface area method, the dose conversion factor between mice and humans is calculated as human Km factor / animal Km factor. According to FDA guidelines, the human Km factor is 37, and the mouse Km factor is 3, resulting in a conversion factor of 12.3 for mouse and human dosages. Based on the mouse dosage of MT-330 (500–15000 μg / kg body weight / day), the human dosage is calculated as mouse dosage / 12.3, resulting in a human dosage of MT-330 of 40–1200 μg / kg body weight / day.

[0050] According to embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier, including any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, the pharmaceutical composition includes isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Of course, a pharmaceutically acceptable carrier may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to prolong the shelf life or potency of the antibody.

[0051] According to embodiments of the present invention, the dosage form of the drug is at least one of an injectable formulation, an oral formulation, or an inhaled formulation.

[0052] According to an embodiment of the present invention, the injectable formulation is an injection solution or a lyophilized powder for injection.

[0053] According to embodiments of the present invention, the oral preparation is a tablet, capsule, granule, oral liquid / solution, or droplet.

[0054] According to embodiments of the present invention, the inhaled formulation is a solution, powder, or suspension thereof.

[0055] culture medium In some embodiments of the present invention, a culture medium is provided. According to embodiments of the present invention, the culture medium comprises the compounds described above. The culture medium is capable of promoting the growth and proliferation of organoids from multiple tissue sources, providing a nutritional basis and growth factors for the growth and proliferation of organoids.

[0056] According to embodiments of the present invention, the final concentration of the compound is 1 μM to 20 μM. Exemplarily, the final concentration of the compound is 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 15 μM, 16 μM, 18 μM, or 20 μM, or a range between any two of the above values. According to some preferred embodiments of the present invention, the final concentration of the compound is 2 μM to 12 μM.

[0057] According to embodiments of the present invention, the culture medium further includes at least one of the following: basal culture medium, B27, N2, Noggin, EGF, L-glutamine, HEPES, or antibiotics. B27 provides basic nutritional support and microenvironment regulation for organoid growth; N2 maintains the stem cell characteristics and undifferentiated state of the organoids, and the combination of B27 and N2 promotes stem cell proliferation and self-renewal more effectively than either alone. Noggin inhibits BMP signaling, maintaining stem cell self-renewal; EGF, by activating EGFR (epidermal growth factor receptor), triggers downstream signaling pathways and is an essential component for maintaining organoid growth; when R-spondin1 and EGF are used alone, intestinal organoids exhibit poor growth and low survival rates, but with the addition of Noggin, organoids show significantly enhanced growth and survival capabilities; this synergistic effect is a necessary condition for maintaining the self-renewal of Lgr5+ intestinal stem cells. L-glutamine supports stem cell proliferation and tissue differentiation, and is often replaced with GlutaMAX to improve stability; HEPES maintains pH stability, which is particularly important for 3D structured organoids; and the antibiotics prevent microbial contamination.

[0058] According to embodiments of the present invention, the organoid culture medium proposed in this invention, compared with traditional organoid culture media, has the compound MT-330 replacing R-spondin1 in activating the Wnt / β-catenin signaling pathway in the organoid culture system. It works synergistically with other culture medium additives to induce the expression of stem genes, thereby promoting the growth and proliferation of the corresponding organoids.

[0059] According to an embodiment of the present invention, the basal culture medium is selected from at least one of the following: Advanced DMEM / F12 medium, DMEM medium, or MEM medium.

[0060] According to an embodiment of the present invention, the final concentration of B27 is 100×~50×. Exemplarily, the final concentration of B27 is 100×, 90×, 80×, 70×, 60× or 50×, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of B27 is 60×~50×.

[0061] According to an embodiment of the present invention, the final concentration of N2 is 200×~100×. Exemplarily, the final concentration of N2 is 200×, 180×, 160×, 140×, 120× or 100×, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of N2 is 120×~100×.

[0062] According to an embodiment of the present invention, the final concentration of Noggin is 50 ng / mL to 150 ng / mL. Exemplarily, the final concentration of Noggin is 50 ng / mL, 80 ng / mL, 100 ng / mL, 120 ng / mL, 140 ng / mL or 150 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of Noggin is 80 ng / mL to 120 ng / mL.

[0063] According to an embodiment of the present invention, the final concentration of EGF is 10 ng / mL to 80 ng / mL. Exemplarily, the final concentration of EGF is 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, or 80 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of EGF is 40 ng / mL to 60 ng / mL.

[0064] According to an embodiment of the present invention, the final concentration of L-glutamine is 200×~50×. Exemplarily, the final concentration of L-glutamine is 200×, 150×, 100× or 50×, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of L-glutamine is 150×~100×.

[0065] According to an embodiment of the present invention, the final concentration of HEPES is 200×~50×. Exemplarily, the final concentration of HEPES is 200×, 150×, 100× or 50×, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of HEPES is 150×~100×.

[0066] According to an embodiment of the present invention, the final concentration of the antibiotic is 200×~50×. Exemplarily, the final concentration of the antibiotic is 200×, 150×, 100× or 50×, or a range between any two of the above values. According to a preferred embodiment of the present invention, the final concentration of the antibiotic is 150×~100×.

[0067] According to an embodiment of the present invention, the culture medium comprises: 1× basal medium, 100× L-glutamine, 100× HEPES, 100× Penicillin / Streptomycin, 1 mM N-acetylcysteine, 50 ng / mL EGF, 500 ng / mL MT330 and 100 ng / mL Noggin.

[0068] According to an embodiment of the present invention, the culture medium comprises: 1× basal medium, 100× L-glutamine, 100× HEPES, 100× Penicillin / Streptomycin, 1mM N-acetylcysteine, 10mM Nicotinamide, 500ng / mL MT330, 50ng / mL EGF, 100ng / mL FGF10, 50ng / mL HGF, and 10nM Gastrin-1.

[0069] According to an embodiment of the present invention, the culture medium comprises: 1× basal medium, 100× L-glutamine, 100× HEPES, 100× Penicillin / Streptomycin, 100 ng / mL Noggin, 500 ng / mL MT330, 50 ng / mL LEGF, 100 ng / mL Wnt3a, 100 ng / mL FGF10, and 50× B27.

[0070] According to an embodiment of the present invention, the culture medium comprises: 1× basal medium, 100× L-glutamine, 100× HEPES, 100× Penicillin / Streptomycin, 1mM N-acetylcysteine, 50× B27Supplement, 0.5µM SB202190, 0.5µM A8301, 5µM Y-27632, 10mM Nicotinamide, 500ng / mL L-spondin1, 00ng / mL Noggin, 100ng / mL Wnt3a, 25ng / mL FGF7, 100ng / mL FGF10 and 50ng / mL LEGF.

[0071] In vitro organoid culture methods In some embodiments of the present invention, a method for in vitro culture of organoids is proposed. According to embodiments of the present invention, the method includes: contacting the aforementioned compound or culture medium with stem cells and / or the organoid to be cultured to obtain cultured organoids. The organoids obtained by the method proposed according to the present invention exhibit good growth, and the method can effectively promote the growth and proliferation of organoids.

[0072] According to embodiments of the present invention, during the culture process, the concentration of the compound is 1 μM to 20 μM. Exemplarily, the concentration of the compound is 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 15 μM, 16 μM, 18 μM, or 20 μM, or a range between any two of the above values. According to some preferred embodiments of the present invention, the concentration of the compound is 2 μM to 10 μM.

[0073] According to embodiments of the present invention, the stem cells include at least one of the following: embryonic stem cells, adult stem cells, or induced pluripotent stem cells.

[0074] According to embodiments of the present invention, the organoids include at least one of small intestinal organoids, bile duct organoids, gastric organoids, or tracheal organoids.

[0075] Embodiments of the present invention will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0076] Example 1: MT-330 significantly promotes the proliferation of organoids from multiple tissue sources in vitro. The specific chemical synthesis route for the preparation of MT-330 is attached. Figure 1As shown, the structure of the compound is as shown in formula (Ⅰ): Equation (Ⅰ).

[0077] Mouse small intestinal organoid culture medium formulation: 1× Advanced DMEM / F12 (ThermoFisher, catalog number 11320033), 100× GlutaMAX-100 (ThermoFisher, catalog number 35050061), 100× HEPES (ThermoFisher, catalog number 15630130), 100× Penicillin / Streptomycin (ThermoFisher, catalog number 15140122), 1 mM N-acetylcysteine ​​(Sigma-Aldrich, catalog number A0737), 50 ng / mL EGF (Biotech, catalog number 568-EGF), 500 ng / mL R-spondin1 (Biotech, catalog number 861-RS1), 100 ng / mL Noggin (purchased from Bozhen Biotechnology, product number 807-NOG).

[0078] Mouse bile duct organoid culture medium formulation: 1× Advanced DMEM / F12, 100× GlutaMAX-100, 100× HEPES, 100× Penicillin / Streptomycin, 1mM N-acetylcysteine, 10mM Nicotinamide (purchased from MedChemExpresss, catalog number HY-B0150), 500ng / mL R-spondin1, 50ng / mL EGF, 100ng / mL LFGF10 (purchased from Bozhen Biotechnology, catalog number 816-FGF), 50ng / mL HGF (purchased from Bozhen Biotechnology, catalog number 871-HGF), 10nM GSastrin-1 (purchased from Bozhen Biotechnology, catalog number 031-GAS).

[0079] Mouse gastric organoid culture medium formulation: 1× Advanced DMEM / F12, 100× GlutaMAX-100, 100× HEPES, 100× Penicillin / Streptomycin, 100 ng / mL Noggin, 500 ng / mL R-spondin1, 50 ng / mL EGF, 100 ng / mL Wnt3a (purchased from ThermoFisher, catalog number PHG0401), 100 ng / mL FGF10, 50× B27Supplement (purchased from Bozhen Biotechnology, catalog number S223151-10).

[0080] Mouse tracheal organoid culture medium formulation: 1× Advanced DMEM / F12, 100× GlutaMAX-100, 100× HEPES, 100× Penicillin / Streptomycin, 1mM N-acetylcysteine, 50× B27Supplement, 0.5µM SB202190 (purchased from Bozhen Biotechnology, catalog number C152121), 0.5µM A8301 (purchased from Bozhen Biotechnology, catalog number C909910), 5µM Y-27632 (purchased from Bozhen Biotechnology, catalog number C629401), 10mM Nicotinamide, 500ng / mL R-spondin1, 100ng / mL Noggin, 100ng / mL Wnt3a, 25ng / mL FGF7 (purchased from Bozhen Biotechnology, catalog number 923-FG7), 100ng / mL FGF10, 50 ng / mL EGF.

[0081] Primary construction of mouse small intestinal organoids: Small intestinal tissue was obtained from 6-week-old male mice weighing 20 g (purchased from Jicui Pharmaceutical, strain C57BL / 6JGpt). The intestines were longitudinally dissected and unfolded, rinsed with pre-cooled DPBS solution (purchased from Bozhen Biotechnology, catalog number E238005), and the residue and villi in the intestines were scraped off with coverslips and other tools. The cleaned small intestines were cut into 3-5 mm segments and added to a digestive solution containing 5 mmol / L EDTA (purchased from Invitrogen, catalog number AM9912). The digestion was carried out at 4°C in a shaker for 20-30 minutes. After digestion, discard EDTA and add pre-cooled DPBS containing 0.1% BSA (Sigma-Aldrich, catalog number 9048-46-8). Vortex or gently pipette to detach the intestinal crypts from the tissue. Filter the suspension containing the crypts through a 70 µm cell filter, collect the filtrate, and centrifuge at 1000-1200 rpm for 5 minutes. Discard the supernatant (wash and centrifuge repeatedly as needed to remove excess single cells or villus fragments). Mix with an appropriate volume of matrix gel (Bio-Zhen Biotech, catalog number M315077) at 4°C, and spot 20 μL of the mixture (cell clusters and matrix gel mixture) in the center of a 24-well plate. Incubate at 37°C for 30 minutes until the matrix gel completely solidifies. Then, add the prepared mouse small intestinal organoid culture medium for further culture.

[0082] Primary construction of mouse bile duct organoids: Liver tissue was collected from 6-week-old male mice weighing 20 g. The tissue was washed twice with organoid basal culture medium (purchased from Bozhen Biotechnology, catalog number B213151), and the supernatant was discarded. The tissue was then minced into small pieces (approximately 2-3 mm³) in 2 mL EP tubes using scissors. The tissue fragments were resuspended in 1 mL of tissue digestion solution (purchased from Bozhen Biotechnology, catalog number K601008). The tissue fragments were then placed in an intelligent tissue dissociation apparatus (purchased from Bozhen Biotechnology) and dissociated using the preset program. 100 μL of fetal bovine serum was added to terminate the digestion. The tissue suspension was then passed through a 70 µm filter. The cells were filtered through a cell filter, and the filtrate was collected and centrifuged at 300g for 3 minutes. The supernatant was discarded, and 400μL of erythrocyte lysis buffer (purchased from Bozhen Biotechnology, catalog number E238010) was added. The cells were incubated on ice for 3 minutes, and 1mL of organoid basal culture medium was added. The cells were centrifuged at 300g for 2 minutes, and the supernatant was discarded. An appropriate volume of matrix gel was added and mixed at 4℃. 20μL of the mixture (cell cluster and matrix gel mixture) was placed in the center of a 24-well culture plate and incubated at 37℃ for 30 minutes. After the matrix gel had completely solidified, the prepared mouse bile duct organoid culture medium was added for further culture.

[0083] Primary construction of mouse gastric organoids: Liver and stomach tissues from 6-week-old male mice weighing 20 g were collected and placed in pre-cooled DPBS solution. The stomach was cut open and the contents were rinsed clean. The outer layer of fat and blood vessels were carefully dissected. The tissues were washed twice with organoid basal culture medium, and the supernatant was discarded. The tissue fragments were minced into a homogenate in 2 mL EP tubes, and 1 mL of tissue digestion solution was added to resuspend the tissue fragments. The tissue fragments were placed in an intelligent tissue dissociator and dissociated using the preset program. After dissociation, 100 μL of fetal bovine serum was added to terminate the digestion. The tissue suspension was then passed through a 70 µm filter. The cells were filtered through a cell filter, and the filtrate was collected. After centrifugation at 300g for 3 minutes, the supernatant was removed, 400μL of erythrocyte lysis buffer was added, and the mixture was incubated on ice for 3 minutes. 1mL of organoid basal culture medium was added, and the mixture was centrifuged at 300g for 2 minutes, and the supernatant was removed. An appropriate volume of matrix gel was added and mixed at 4℃. 20μL of the mixture (cell cluster and matrix gel mixture) was placed in the center of a 24-well culture plate and incubated at 37℃ for 30 minutes. After the matrix gel had completely solidified, the prepared mouse gastric organoid culture medium was added for further culture.

[0084] Primary construction of mouse tracheal organoids: Intact tracheal tissue was collected from 6-week-old male mice weighing 20 g. The tissue was placed in pre-chilled DPBS solution and washed twice with organoid basal culture medium, discarding the supernatant. The tissue fragments were minced to approximately 1-2 mm³ in 2 mL EP tubes, and 1 mL of tissue digestion solution was added to resuspend the fragments. The tissue fragments were then placed in an intelligent tissue dissociator and dissociated using the preset program. 100 μL of fetal bovine serum was added to terminate the digestion. The tissue suspension was then passed through a 70 µm filter. The cells were filtered through a cell filter, and the filtrate was collected. After centrifugation at 300g for 3 minutes, the supernatant was removed, 400μL of erythrocyte lysis buffer was added, and the mixture was incubated on ice for 3 minutes. 1mL of organoid basal culture medium was added, and the mixture was centrifuged at 300g for 2 minutes, and the supernatant was removed. An appropriate volume of matrix gel was added and mixed at 4℃. 20μL of the mixture (a mixture of cell clusters and matrix gel) was placed in the center of a 24-well culture plate and incubated at 37℃ for 30 minutes. After the matrix gel had completely solidified, the prepared mouse tracheal organoid culture medium was added for further culture.

[0085] The successfully constructed mouse small intestine, mouse bile duct, mouse stomach, and mouse trachea P0 generation organoids were collected using 300 μL of DPBS solution.

[0086] Mouse small intestinal P0 generation organoids were gently dispersed with a pipette tip, centrifuged at 300×g for 3 min at room temperature, and then mixed with 60 μL of Matrigel (Bio-Zhen Biotechnology Co., Ltd., catalog number: M315077) and kept on ice for later use. Mouse bile duct, stomach, and trachea P0 generation organoids were directly centrifuged at 300×g for 3 min at room temperature, and then 200 μL of organoid digestion solution (Bio-Zhen Biotechnology Co., Ltd., catalog number: K601010) was added. Digestion was carried out at 37°C for 3 min, gently pipetted, and then an equal volume of DPBS solution was added to stop digestion. Afterward, they were centrifuged again at 300×g for 3 min at room temperature, mixed with 60 µL of Matrigel, and kept on ice for later use.

[0087] 20 μL of cell clusters and matrix gel mixture was added to each well of a 24-well plate, and 500 μL of the corresponding organoid culture medium was added to each well. After culturing for 5 days in a 37°C incubator, bright-field images of organoids were collected from each well using a star map microscope (Bio-Zhen Biotechnology Co., Ltd., Star-Map F1 organoid localization imaging system).

[0088] The corresponding organoid culture media were: MT-330 at different concentrations (2µM, 10µM) was added to the culture media of mouse small intestine, mouse bile duct, mouse stomach and mouse trachea, and DMSO (purchased from ThermoFisher, catalog number 20688) was added to the control group.

[0089] The specific results are attached. Figure 2As shown, compared with the control group, MT-330 significantly promoted the proliferation of multi-tissue-derived organoids at working concentrations of 2 μM and 10 μM, indicating that MT-330 has a significant promoting effect on multi-tissue-derived organoid culture at these two concentrations.

[0090] Example 2: MT-330 promotes stem gene expression in stem cells from multiple tissue sources. Six-week-old male mice of the C57BL / 6JGpt strain, weighing 20 g (purchased from Jicui Pharmaceutical), were selected and divided into groups of three. Each group received intraperitoneal injections of MT-330 at concentrations of 1 mg / kg and 5 mg / kg, while the control group received the same dose of solvent. Forty-eight hours later, appropriate amounts of small intestine, bile duct, stomach, and trachea tissues were collected, and total RNA was extracted using the RNAprep Pure Microsample Total RNA Extraction Kit (TIANGEN). Then, GoScript was used to extract the RNA. TM The reverse transcription system reagent reverses the transcription of DNA into cDNA, which is then used for timed and quantitative PCR to detect the expression of the corresponding gene.

[0091] The solvent used in this embodiment is hydroxypropyl-β-cyclodextrin (HPβCD, CAS No.: 128446-35-5) solution, which is prepared by dissolving 9 g of hydroxypropyl-β-cyclodextrin powder in 21 mL of 100 mM acetate buffer (pH 5.4).

[0092] The corresponding gene for the small intestine is LGR5 , AXIN2 , Ki67 The corresponding gene for bile ducts is HES1 , AXIN2 , Ki67 The corresponding gene for the stomach is SOX9 , AXIN2 , Ki67 The gene corresponding to the trachea is KRT5 , AXIN2 , Ki67 .

[0093] Specific experimental results are attached. Figure 3 As shown, compared with the control group, intraperitoneal injection of MT-330 at concentrations of 1 mg / kg and 5 mg / kg significantly increased the expression of stem genes corresponding to the small intestine, bile duct, stomach and trachea by 4 to 12 times, indicating that MT-330 has the function of enhancing the proliferation of stem cells from multiple tissue sources.

[0094] Example 3: Application of MT-330 as an additive in organoid culture media The construction and collection of primary (P0 generation) organoids (mouse small intestine, bile duct, stomach and trachea) from multiple tissue sources are shown in Example 2.

[0095] Mouse small intestinal P0 generation organoids were gently dispersed with a pipette tip, centrifuged at 300×g for 3 min at room temperature, and then mixed with 60 μL of Matrigel (Bio-Zhen Biotechnology Co., Ltd., catalog number: M315077) and kept on ice for later use. Mouse bile duct, stomach, and trachea P0 generation organoids were directly centrifuged at 300×g for 3 min at room temperature, and then 200 μL of organoid digestion solution (Bio-Zhen Biotechnology Co., Ltd., catalog number: K601010) was added. Digestion was carried out at 37°C for 3 min, gently pipetted, and then an equal volume of DPBS solution was added to stop digestion. Afterward, they were centrifuged again at 300×g for 3 min at room temperature, mixed with 60 µL of Matrigel, and kept on ice for later use.

[0096] 20 μL of a mixture of cell clusters and matrix gel was added to each well of a 24-well plate, and 500 μL of the corresponding organoid culture medium was added to each well. After culturing for 5 days in a 37°C incubator, bright-field images of the organoids were collected from each well using a star map microscope (Bio-Zhen Biotechnology Co., Ltd., Star-Map F1 organoid localization imaging system).

[0097] The corresponding organoid culture media in this embodiment are: the mouse small intestine, mouse bile duct, mouse stomach and mouse trachea culture media (the corresponding components and concentrations are as shown in Example 2) with R-Spondin1 removed from the corresponding culture system (-RS group); 2 μM MT-330 added to the -RS group (-RS+MT-330 group); and the corresponding culture media without any adjustment (+RS-MT-330 group).

[0098] Specific experimental results are attached. Figure 4 As shown, the growth of mouse small intestine, bile duct, stomach and tracheal organoids in the -RS group was severely restricted, while the addition of MT-330 to the culture system (-RS+MT-330 group) can ensure the normal growth of mouse small intestine, bile duct, stomach and tracheal organoids, and their growth status is comparable to that of the traditional culture medium (+RS-MT-330 group).

[0099] Comparative Example 1: Comparison of the effects of MT-330 and Blebbistatin Six-week-old male mice of the C57BL / 6JGpt strain, weighing 20 g (purchased from Jicui Yaokang), were selected and divided into groups (n=3 per group). Each group received an intraperitoneal injection of 5 mg / kg of Blebbistatin and MT-330 in the light-protected group (1) and the light-exposed group (2), while the control group received the same dose of solvent. Forty-eight hours later, appropriate amounts of small intestinal tissue were collected, and total RNA was extracted using the RNAprep Pure Microsample Total RNA Extraction Kit (TIANGEN). Then, GoScript was used to extract the RNA. TM The Reverse Transcription System reverses the transcription of DNA into cDNA, which is then used for timed and quantitative PCR to detect the expression of the corresponding gene.

[0100] The solvent used in this embodiment is hydroxypropyl-β-cyclodextrin solution, which is prepared by dissolving 9 g of hydroxypropyl-β-cyclodextrin powder in 21 mL of 100 mM acetate buffer (pH 5.4).

[0101] To make the results of this comparative experiment more representative, we selected a standard bright-field light source (380nm~750nm) from a star chart microscope for illumination processing. The specific procedures are as follows: Pretreatment for the light-shielded group: EP tubes containing the corresponding concentrations of Blebbistatin and MT-330 solutions were wrapped in aluminum foil and irradiated for 5 min under a standard bright-field light source of a star chart microscope. Pretreatment for the light-illuminated group: EP tubes containing the corresponding concentrations of Blebbistatin and MT-330 solutions were directly irradiated for 5 min under a standard bright-field light source of a star chart microscope. The stem gene corresponding to the small intestine is... LGR5 , AXIN2 , Ki67 .

[0102] Specific experimental results are attached. Figure 5 As shown, unlike the light-shielded group where Blebbistatin significantly promoted the expression of small intestinal stem genes (3-6 times), the promotion effect of Blebbistatin on small intestinal stem gene expression was significantly reduced in the light-exposed group (1-2 times); however, there was no significant difference in the promotion effect of MT-330 on small intestinal stem gene expression between the light-shielded and light-exposed groups (4-7 times). This indicates that, unlike Blebbistatin, which is easily degraded under normal experimental conditions, MT-330 possesses excellent photostability.

[0103] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0104] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A compound, characterized in that, The compound is a compound of formula (I) or a pharmaceutically acceptable salt thereof: Formula (I).

2. Use of the compound of claim 1 in at least one of the following: (1) Organoid culture; (2) Prepare a drug for treating and / or preventing stem cell failure or related diseases; (3) Prepare a culture medium for culturing organoids.

3. The use according to claim 2, characterized in that, The organoids include at least one of small intestinal organoids, bile duct organoids, gastric organoids, or tracheal organoids; and / or The stem cell failure-related diseases include at least one of the following: malabsorption syndrome, intestinal barrier dysfunction, inflammatory bowel disease repair disorder, liver regeneration disorder, bile duct atrophy syndrome, refractory gastric ulcer, atrophic gastritis, chronic obstructive pulmonary disease, bronchiolitis obliterans, and airway ciliary movement disorder.

4. A drug, characterized in that, The drug includes the compound as described in claim 1.

5. A culture medium, characterized in that, The culture medium comprises the compound as described in claim 1.

6. The culture medium according to claim 5, characterized in that, The final concentration of the compound is 1 μM to 20 μM.

7. The culture medium according to claim 6, characterized in that, The culture medium also includes at least one of the following: basal culture medium, B27, N2, Noggin, EGF, L-glutamine, or an antibiotic.

8. The culture medium according to claim 7, characterized in that, The basal culture medium is selected from at least one of the following: DMEM medium or MEM medium.

9. The culture medium according to claim 7, characterized in that, The culture medium comprises at least one of the following: The final concentration of B27 is 100×~50×; The final concentration of N2 is 200×~100×; The final concentration of Noggin is 50 ng / mL to 150 ng / mL; The final concentration of EGF is 10 ng / mL to 80 ng / mL; The final concentration of L-glutamine is 200×~50×; The final concentration of the antibiotic is 200×~50×.

10. A method for in vitro culture of organoids, characterized in that, include: The compound as defined in claim 1 or the culture medium as described in any one of claims 5 to 9 is contact cultured with stem cells and / or organoids to be cultured to obtain cultured organoids.

11. The method according to claim 10, characterized in that, During the culture process, the concentration of the compound is 1 μM to 20 μM; and / or The stem cells include at least one of the following: adult stem cells or induced pluripotent stem cells; and / or The organoids include at least one of small intestinal organoids, bile duct organoids, gastric organoids, or tracheal organoids.

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