A method for chemically inducing fibroblast reprogramming into lung stem cells

Abstract

This invention relates to a method for chemically inducing fibroblast reprogramming into lung stem cells. The method includes: inducing fibroblast reprogramming into lung stem cells using a small chemical molecule composition to prepare lung stem cells; and preparing a reprogramming culture medium or reagent / formulation. During the induction and reprogramming process, no exogenous genes / transcription factors / microRNA (miRNA) genes, their RNA, proteins, or polypeptides, or exogenous cytokines or growth factors are introduced or used. The small chemical molecule composition comprises only GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds; or, the small chemical molecule composition consists only of GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds. This invention also provides applications of the method for preparing lung stem cells, providing a source of lung stem cells for clinical and research purposes.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary fields of cell biology, stem cell biology (cell reprogramming), medicine, and pharmacy; more specifically, this invention relates to a method for chemically inducing fibroblasts to reprogram into lung stem cells using only a chemical small molecule (hereinafter referred to as small molecule) composition without introducing or using any exogenous genes / transcription factors / microRNA (miRNA) genes, or their RNA, protein, or polypeptide inducing factors; nor using any exogenous cytokines or growth factors. Background Technology

[0002] For lung injury and chronic lung diseases, most current treatments, except for lung transplantation, can only alleviate symptoms but cannot achieve a cure. Due to limitations in donor availability and surgical techniques, research focus in lung disease treatment has shifted to adult stem cell and embryonic stem cell transplantation for lung diseases, as well as related research in lung regenerative medicine. Adult stem cells have received more attention because they avoid the ethical, sourcing, and immune rejection issues associated with embryonic stem cells. In particular, the global pandemic of COVID-19 has led to a large number of deaths due to acute respiratory distress syndrome (ARDS). Besides ARDS, pulmonary fibrosis from various causes is a leading cause of death worldwide. Effective treatments are lacking for both ARDS and pulmonary fibrosis. Stem cell therapy, applicable to both acute and chronic lung diseases, represents a promising treatment approach.

[0003] Pluripotent stem cells (PSCs) are a type of pluripotent cell with self-renewal capacity and multipotent differentiation potential. Under certain conditions, they can differentiate into various functional cells. Stem cells are classified according to their differentiation potential: stem cells with strong differentiation potential include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); stem cells with weak or limited differentiation potential are adult stem cells or tissue stem cells, including pluripotent stem cells with weak differentiation potential such as mesenchymal stem cells (MSCs), and unipotent stem cells that can only differentiate into a certain type of tissue functional cell, such as lung stem cells.

[0004] Adult stem cells or tissue stem cells have gained more attention in lung regenerative medicine and its clinical applications because they can avoid the ethical, sourcing, in vivo transplantation carcinogenic and immune rejection risks associated with embryonic stem cells and induced pluripotent stem cells.

[0005] Lung stem cells are adult or tissue stem cells capable of continuous self-renewal and differentiating into functional lung tissue cells under specific conditions. Due to the complexity of the lung structure, it is currently believed that lung injury relies on the differentiation, renewal, and repair of lung tissue stem cells from different anatomical locations within the lung. Studies have shown that there is no single type of lung tissue stem cell; instead, different types of lung stem cells exist in different anatomical locations within the lung tissue. These mainly include: basal cells (BCs) expressing specific markers Trp63 (p63) and keratin 5 (Krt5); distal airway stem cells (DASCs; hereinafter referred to as airway stem cells in this article) also expressing Trp63 (p63) and keratin 5 (Krt5); bronchoalveolar stem cells (BASCs; hereinafter referred to as alveolar stem cells in this article) expressing specific markers SPC and CC10; and alveolar type 2 cells (AT2Cs) expressing the specific marker SPC. These types of lung tissue stem cells are crucial for maintaining lung homeostasis, function, and promoting lung injury repair. Lung stem cells are currently considered unipotent stem cells, capable of directly differentiating into functional cells of lung tissue. Lung basal cells, in particular, can be extracted from the human trachea and bronchi, expanded and cultured in vitro, and then transplanted to treat ARDS and pulmonary fibrosis (fibroblasts do not possess this function). Although this method has proven to be an effective treatment, its sourcing is relatively complex, and the number of lung stem cells obtained is limited, usually requiring allogeneic sources, which cannot meet the needs of acute respiratory injuries. For chronic pulmonary fibrosis, whether a sufficient number of lung basal cells can be obtained, and the potential for immune rejection due to allogeneic stem cells, remain problematic. Therefore, developing new sources of lung stem cells remains a crucial issue that urgently needs to be addressed.

[0006] Directed differentiation of stem cells is one way to obtain lung stem cells. Stepwise differentiation of human pluripotent stem cells (hPSCs), including directed induced pluripotent stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), into lung stem cells is another source of lung stem cells. hPSCs can differentiate into proximal airway cells and distal lung epithelial cells. However, the application of directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) into lung stem cells is currently limited by ethical concerns and the potential tumorigenic risks of residual hESCs and hiPSCs.

[0007] Mesenchymal stem cells (MSCs), with relatively low differentiation potential, belong to adult stem cells. Currently, there are no reports of their directed differentiation into functional lung cells, but transplanted MSCs can potentially exert therapeutic effects by secreting several immunomodulatory factors. There are numerous reports on the use of MSCs for treatment, both for improving acute respiratory distress syndrome (ARDS) caused by acute respiratory injury and for improving pulmonary fibrosis. Reports also indicate that MSCs can transform under different internal environmental conditions. It has been reported that most COVID-19 patients worldwide died from cytokine storm syndrome (CSS). CSS is caused by the rapid production of multiple inflammatory factors following viral or bacterial infection, leading to ARDS and multiple organ failure. However, whether MSCs are a safe and effective source of transplanted cells for improving acute respiratory injury or pulmonary fibrosis requires further research. Therefore, obtaining a sufficient number of transplantable lung stem cells through other pathways or methods, such as cell reprogramming, is a significant need for improving the health of patients with lung diseases.

[0008] Cell reprogramming is the transformation of cells from one type to another. It is the process by which exogenous transcription factors (genes) or small chemical molecules and other inducing factors are introduced to target and induce changes in specific cell signaling pathways or epigenetics, thereby transforming one type of cell into another. Epigenetics refers to heritable changes in gene expression without changes in DNA sequence or structure. Induced cell reprogramming includes: (1) reprogramming of induced pluripotent stem cells to reverse the differentiation of cells back to a pluripotent or totipotent state; (2) direct reprogramming of cells that directly transform from one type of differentiated cell to another without going through the pluripotent stem cell stage (also known as transdifferentiation or lineage reprogramming).

[0009] Chemically induced cell reprogramming is a process of transforming one type of cell into another without introducing or using any exogenous genes / transcription factors / microRNA (miRNA) genes, their RNA, proteins and peptides, or exogenous cytokines or growth factors; using only chemical small molecules to target and induce changes in cell signaling pathways and epigenetic modifications, without altering the cell's structural genes, but only changing the cell's gene expression profile. Chemically induced cell reprogramming includes: (1) chemically induced pluripotent stem cell reprogramming (Hongkui Deng et al., Science. 341, 651-4, 2013); (2) chemically induced direct cell reprogramming (transdifferentiation, lineage reprogramming) (Li X et al., Cell Stem Cell; 17(2): 195-203, 2015; Hu W et al., Cell Stem Cell. 17(2): 204-212, 2015).

[0010] This invention uses only a small chemical molecule composition to chemically induce fibroblasts to reprogram into lung stem cells, which belongs to stem cell reprogramming. In order to distinguish this reprogramming from induced pluripotent stem cell (iPSC) reprogramming, this reprogramming is referred to as "lung stem cell reprogramming" or "reprogramming" in this article.

[0011] Since Shinya Yamanaka introduced four transcription factors (genes)—Oct4 (also known as Oct, Oct3, or OCT-3 polypeptide expression factor, etc.), Sox2, Klf4, and c-Myc—into differentiated somatic cells in 2006 to induce pluripotent stem cell reprogramming and obtain iPS cells, reprogramming methods have been continuously improved due to drawbacks such as disrupting the stability of the original gene sequence, leading to mutations and cancer risks, low cell reprogramming conversion rates, poor stability, and complex operations. These improvements have gradually shifted to inducing reprogramming with only 1-2 exogenous transcription factors, combined with microRNA (miRNA) genes and their RNA, protein, and polypeptide components, as well as chemical small molecules, cytokines, and growth factors to create multi-factor induction compositions. Currently, it is possible to induce reprogramming using only chemical small molecule combinations. Differentiated cells Reprogramming into iPS cells (CN201010296987.8. Deng Hongkui et al.). However, there are no reports to date of chemically inducing fibroblasts to reprogram into lung tissue stem cells using only small chemical molecules.

[0012] Because small molecules (or simply small molecules) are targeted molecular compounds, they can target and induce the regulation of specific signaling pathways and epigenetics, transforming one type of cell into another. Furthermore, they are easy to manufacture, low in cost, stable, and simple to operate, making them the best candidates to replace inducible transcription factors.

[0013] Based on this, the inventors explored a method for inducing fibroblasts to reprogram into lung stem cells (including BASCs, AT2Cs, BCs, and DASCs) using only small chemical molecule compositions. Summary of the Invention

[0014] The purpose of this invention is to provide a method for chemically inducing fibroblast reprogramming into lung stem cells. This method does not introduce or use any exogenous genes / transcription factors / microRNA (miRNA) genes, their RNA, proteins or peptides, or exogenous cytokines or growth factors; it only uses a composition of small chemical molecules: GSK3β inhibitors, G9a histone methyltransferase (G9aHMT) inhibitors, and retinoic acid compounds to chemically induce fibroblast reprogramming into lung stem cells (including BASCs, AT2Cs, BCs, DASCs, etc.); or, based on this, the small molecule composition may further include a small chemical molecule TGFβ inhibitor.

[0015] Through extensive experimental research, the inventors discovered that although the three small chemical molecules constituting this small molecule composition—GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compound, as well as the optional TGFβ inhibitor—do not have the function of inducing fibroblast reprogramming into lung stem cells when they exist alone; and the combination of GSK3β inhibitor and G9aHMT inhibitor, and the combination of GSK3β inhibitor and TGFβ inhibitor, also do not have the function of inducing fibroblast reprogramming into lung stem cells; however, the small chemical molecule composition consisting of GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compound has the unique function of inducing fibroblast reprogramming into lung stem cells.

[0016] In the method of the present invention, no exogenous genes / transcription factors / microRNA (miRNA) genes, or their RNA, protein, or polypeptide inducing factors, are introduced or used; nor are exogenous cytokines or growth factors used; only a method for chemically inducing fibroblasts to reprogram into lung stem cells using small chemical molecules is used. The method includes: applying any of the above-described small chemical molecule compositions to chemically induce fibroblasts to reprogram into lung stem cells to prepare lung stem cells; and a method for preparing reprogramming culture medium or reagents / formulations.

[0017] The method of chemically inducing fibroblast reprogramming into lung stem cells using the small molecule composition includes: a method of chemically inducing fibroblast reprogramming into lung stem cells in vitro using the small molecule composition; or, a method of promoting the repair of lung damage and improving and alleviating pulmonary fibrosis by using the small molecule composition to chemically induce in situ reprogramming of fibroblasts into lung stem cells in vivo.

[0018] The advanced and innovative aspects of this invention: This invention is the first to provide a method for chemically inducing fibroblast reprogramming into lung stem cells (including BASCs, AT2Cs, BCs, and DASCs) without introducing or using any exogenous genes / transcription factors / microRNA (miRNA) genes, their RNA, proteins, or polypeptides, or exogenous cytokines or growth factors; using only a composition composed of small chemical molecules such as GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds. Its advanced and innovative advantages are as follows:

[0019] (1) The lung stem cells prepared by the method of this invention are unipotent stem cells, avoiding the carcinogenic risk caused by reprogramming into induced pluripotent stem cells (iPSCs); (2) The fibroblasts induced by this reprogramming method can be obtained from the patient himself, so the lung stem cells obtained by reprogramming have personalized characteristics and have two major advantages: first, they are easier to enter clinical applications; second, they minimize or avoid the risk of immune rejection caused by transplantation of lung stem cells transformed from allogeneic fibroblasts; (3) In the process of reprogramming lung stem cells, no exogenous genes / transcription factors / microRNA genes are introduced or used; this avoids the new carcinogenic risk caused by the introduction of exogenous genes, making it safer and more reliable; moreover, no exogenous cytokines or growth factors are used, resulting in lower costs and simpler operation; therefore, it can provide a sufficient quantity and safe quality of lung stem cell sources for clinical and scientific research. (4) The lung stem cells obtained by chemically induced fibroblast reprogramming transformation can be further developed and prepared as cell preparations or drugs for clinical transplantation treatment or repair of lung injury and improvement and relief of pulmonary fibrosis; (5) Although the chemical small molecules GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, and TGFβ inhibitor that make up the small molecule composition do not have the function of inducing fibroblast reprogramming into lung stem cells when they exist alone; and the combination of GSK3β inhibitor and G9aHMT inhibitor, and the combination of GSK3β inhibitor and TGFβ inhibitor do not have the function of inducing fibroblast reprogramming into lung stem cells; but when they are combined into the chemical small molecule composition of the present invention, they have the overall function of inducing fibroblast reprogramming into lung stem cells. (6) Chemical small molecules are targeted small molecule compounds with stable properties. The timing, dosage and combination of their effects are easy to control, and the effects are stable and reliable with good drug properties. (7) Chemical small molecules are classified and named according to their common function of inducing and regulating a specific cell signaling pathway and its epigenetic changes. Differences or differences in the compound types, structures and physicochemical properties between chemical small molecules within the same category do not affect their unique functions. Therefore, it is convenient to screen and optimize small molecule combination components.

[0020] In a first aspect of the invention, a method for chemically inducing fibroblasts to reprogram into lung stem cells using a small chemical molecule composition is provided, comprising: treating fibroblasts with the small chemical molecule composition to induce fibroblast differentiation into lung stem cells; wherein, during the induction and reprogramming process, no exogenous genes / transcription factors / microRNA genes, their RNA, proteins, polypeptides, or exogenous cytokines or growth factors are introduced or used; wherein the small chemical molecule composition comprises: a GSK3β inhibitor, a G9aHMT inhibitor, and a retinoic acid compound; or, the small chemical molecule composition consists only of a GSK3β inhibitor, a G9aHMT inhibitor, and a retinoic acid compound.

[0021] In a preferred embodiment, the chemical small molecule composition may further include a TGFβ inhibitor; or, the chemical small molecule composition may consist only of a GSK3β inhibitor, a G9aHMT inhibitor, a retinoic acid compound, and a TGFβ inhibitor.

[0022] In another preferred embodiment, the method described above is characterized in that the chemical small molecule composition contains:

[0023] The term "GSK3β inhibitor" refers to a general term for inhibitors capable of targeting and inhibiting the GSK3β signaling pathway, including but not limited to: CHIR-99021, BIO, LiCl, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, LY2090314, SB216763, AZD1080, other small molecule GSK3β inhibitors that target and induce inhibition of the GSK3β signaling pathway, or pharmaceutical preparations, analogues, isomers, salts, hydrates or precursors equivalent to them, or combinations thereof; preferably, the GSK3β inhibitors CHIR-99021, LiCl, BIO, and LY2090314.

[0024] In another preferred embodiment, the G9aHMT inhibitor refers to the general term for inhibitors capable of targeting and inhibiting G9aHMT, including but not limited to: BIX01294, UNC0638, A-366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321, BRD4770, HKMTI-1-247, HKMTI-1-248, CPUY074020, DCG066, other small molecule inhibitors of G9aHMT that target and inhibit G9aHMT, pharmaceutical preparations, analogues, isomers, salts, hydrates or precursors equivalent to them, or combinations thereof; preferably, it is the G9aHMT inhibitor BIX01294, UNC0638 or UNC0642.

[0025] In another preferred embodiment, the retinoic acid compound refers to a small chemical molecule capable of specifically binding to retinoic acid response elements (RAREs) to induce and regulate the RA signaling pathway; including but not limited to: retinoic acid (RA), 13-cis-retinoic acid, 9-cis-retinoic acid, UAB7, UAB8, TTNPB, 3-methyl-TTN PB, AM80, AM580, CD437, Targretin, LGD1069, isotretinoin, isotretinoin, isotretinoin, isotretinoin, isotretinoin, isotretinoin, isotretinoin ester, tazarotene, adapalene, and other retinoic acid compounds that induce and regulate the RA signaling pathway; or pharmaceutical preparations, analogs, salts, hydrates, precursors, or combinations thereof equivalent to them; preferably retinoic acid (RA), 13-cis-retinoic acid, or 9-cis-retinoic acid.

[0026] In another preferred embodiment, the optional or addable TGFβ inhibitor refers to the general term for inhibitors capable of targeting and inhibiting the TGFβ signaling pathway, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388, SB505124, EW-7197, Galunisertib, and other small molecule TGFβ inhibitors that induce inhibition of the TGFβ signaling pathway, or pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors equivalent to them, or combinations thereof; preferably, the TGFβ inhibitors SB431542, A83-01 or RepSox.

[0027] In a preferred embodiment, the small molecule chemical composition in the method comprises:

[0028] GSK3β inhibitor: 5-80 parts by weight, preferably 10-70 parts by weight; or a final concentration of 0.1-20 μM / mM in solution, preferably 0.5-15 μM / mM (preferably, the molar concentration is calculated based on the molecular weight of the GSK3β inhibitor; μM is used for large molecular weights and mM is used for small molecular weights).

[0029] G9aHMT inhibitor: 0.1-50 parts by weight, preferably 0.5-40 parts by weight; or a final concentration of 0.01-20 μM in solution, preferably 0.05-10 μM;

[0030] Retinoic acid compounds: 0.05-20 parts by weight; preferably 0.15-15 parts by weight; or a final concentration of 0.1-20 μM in solution; preferably 0.5-10 μM.

[0031] In a preferred embodiment, the chemical small molecule composition may contain an optional chemical small molecule TGFβ inhibitor at a concentration of 0.1-50 parts by weight, preferably 0.5-40 parts by weight; or at a final concentration of 0.01-20 μM, preferably 0.05-10 μM in solution.

[0032] In a preferred embodiment, the small molecule composition contains GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compound in a weight ratio of (5-80):(0.1-50):(0.05-20); or in solution, a molar concentration ratio of (0.1-20):(0.01-20):(0.1-20).

[0033] In a preferred embodiment, the small molecule composition of the method comprises GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, and TGFβ inhibitor in a weight ratio of (5-80):(0.1-50):(0.05-20):(0.1-50); or in solution, a molar concentration ratio of (0.1-20):(0.01-20):(0.1-20):(0.01-20).

[0034] In a preferred embodiment, the components of the chemical small molecule composition of the method are, in parts by weight, the GSK3β inhibitor (such as CHIR99021, LiCl, BIO, or Ly2090314), the G9aHMT inhibitor (such as BIX01294, UNC0638, or UNC0642), the retinoic acid compound (such as retinoic acid, 13-cis-retinoic acid, or 9-cis-retinoic acid), and the TGFβ inhibitor (such as SB431542, A83-01, or Rep). The molar concentration ratio of Sox is (5-80):(0.1-50):(0.05-20):(0.1-50); preferably (10-70):(0.5-40):(0.15-15):(0.5-40); or the molar concentration ratio in solution is (0.1-20):(0.01-20):(0.1-20):(0.01-20); preferably (0.5-15):(0.05-10):(0.5-10):(0.05-10).

[0035] In another preferred embodiment, the small molecule GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, and optional TGFβ inhibitor, together account for 0.01 to 99.9% of the total weight of the composition; more preferably 50 to 99.9%; and 0.01 to 50% in solution, such as 0.01%, 1%, 5%, 10%, 20%, 30%, etc.

[0036] The weight units for the above weight ratios can be any weight unit such as kilogram (kg), milligram (mg), or microgram (μg); the molar units for the molar concentration ratios can be any molar concentration unit such as mole (M), millimole (mM), or micromole (μM).

[0037] When the small molecule composition is applied to large animals, the effective dosage for large animals (including dosage conversion for solid or solution states) is calculated using appropriate professional conversion formulas based on the dosage used in small animals. This dosage conversion based on animal dosage is a well-known technique in the art. For example, it can be calculated using the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W² / 3) / 10,000. Where A is the body surface area, calculated in m²; W is the body weight, calculated in g; and K is a constant that varies depending on the animal species. Generally, it is 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that the dosage conversion can vary depending on the composition and the animal being treated, based on the assessment of an experienced technician.

[0038] In another preferred embodiment, the method of chemically inducing fibroblasts to reprogram into lung stem cells using the small molecule composition described above includes: a method of chemically inducing fibroblasts to reprogram into lung stem cells in vitro using the small molecule composition; or, a method of promoting the repair of lung damage and improving and alleviating pulmonary fibrosis by using the small molecule composition to chemically induce in situ reprogramming of fibroblasts into lung stem cells in vivo.

[0039] In another preferred embodiment, the method for chemically inducing fibroblast reprogramming into lung stem cells using the small molecule composition described above includes: a method for preparing lung stem cells by chemically inducing fibroblast reprogramming using a small molecule composition; or a method for preparing reprogramming culture medium or reagent by adding a small molecule composition to a cell-based culture medium, or an organic solvent or physiological saline, respectively.

[0040] In another preferred embodiment, the method described above is characterized in that the method for reprogramming lung stem cells and the method for preparing reprogramming culture medium or reagents include:

[0041] (1) Preparation of concentrated reagent: According to any of the small molecule compositions described above, each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrated reagent (in the range of 1:50 to 1:10,000); preferably, the organic solvent includes dimethyl sulfoxide; preferably, the aqueous solvent includes water, physiological saline, and phosphate buffer.

[0042] (2) Obtaining the reprogramming medium for reprogramming fibroblasts into lung stem cells: Dilute the concentrated reagent in step (1) into a cell basal medium containing 5-20% fetal bovine serum (so that the concentration of each component meets the final concentration defined in any of the small molecule compositions described above) to obtain the reprogramming medium; wherein, the percentage content of each component in the medium may fluctuate by 50%; preferably by 30%; more preferably by 20%, such as 10% or 5%;

[0043] (3) Prepare a 100mg / ml concentrate by mixing the weight ratio of each component of the small molecule combination according to the weight ratio of each mouse: dilute it into 0.9% NaCl physiological saline (50μl / mouse) and mix it to prepare a chemically induced in situ reprogramming of fibroblasts into lung stem cells for in vivo reprogramming.

[0044] (4) Inducing fibroblasts to reprogram into lung stem cells: Fibroblasts were suspended in a cell basal medium containing 5-20% fetal bovine serum, plated, and after the cells adhered, the medium was replaced with the reprogramming medium from step (2), cultured at 37°C, and the medium was changed every 2-4 days; the cells were passaged every 3-15 days.

[0045] (5) Passaging of fibroblasts to lung stem cells: Discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, incubate at 37°C for 1-5 minutes, terminate cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and passage at a ratio of 1:1-1:3. Use the transdifferentiation medium from step (2) and culture according to the above method, changing the medium every 2-4 days. The digestion solution used includes trypsin, EDTA, acutase, TrypleE, etc. Passage every 3-15 days.

[0046] (6) Harvesting of transformed lung stem cells: After reprogramming lung stem cells through the above experimental steps (4) and (5) and passage culture for 2-4 weeks, fibroblasts can be reprogrammed into lung stem cells, and transformed lung stem cells can be obtained (the obtained lung stem cells). Transformation Lung stem cells This includes ciBASCs, ciAT2Cs, ciBCs, and ciDASCs, which have essentially the same or similar shapes.

[0047] The above method does not introduce or use exogenous genes / transcription factors / microRNA genes, or their RNA, protein, or polypeptide inducing factors; nor does it add or use any exogenous cytokines or growth factors; it only uses chemical small molecule compositions to induce fibroblasts to reprogram into lung stem cells.

[0048] In another aspect of the present invention, the use of a method for chemically inducing fibroblast reprogramming into lung stem cells using a small molecule composition is provided, comprising: reprogramming fibroblasts into lung stem cells to prepare transformed lung stem cells to provide a source of lung stem cells for clinical and scientific research; or, preparing a reagent or culture medium for inducing fibroblast reprogramming into lung stem cells.

[0049] In this invention, the fibroblasts include: human fibroblasts or mammalian fibroblasts; preferably, including but not limited to: skin fibroblasts, liver fibroblasts (hepatic stellate cells, HSCs), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts of other tissues or organs of humans or mammals; preferably skin fibroblasts.

[0050] In this invention, the lung stem cells obtained by chemically induced fibroblast reprogramming include: alveolar type 2 cells (AT2Cs), bronchoalveolar stem cells (BASCs), basal cells (BCs), distal airway stem cells (DASCs), or mixtures thereof (mixed cells).

[0051] In another preferred embodiment, the method is one that does not have the direct purpose of treating the disease.

[0052] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0053] Figure 1 Morphological comparison of lung stem cells transformed from small molecule chemically induced fibroblast (mouse) reprogramming. Morphological comparison of lung stem cells (ciAT2Cs) transformed from chemically induced fibroblast (MF) reprogramming with primary alveolar type II cells (pAT2Cs) and MF cells; Figure 1 The results showed that lung stem cells (ciAT2Cs) transformed by chemically induced fibroblast (MF) reprogramming of small molecule compositions had the same or similar morphology as primary alveolar type II cells (pAT2Cs); however, their morphology was completely different from that of their inducing initiator cells MF.

[0054] Figure 2 The induced alveolar type II cells (ciAT2Cs) have the lamellar structure unique to AT2Cs. Figure 2The results showed that the reprogrammed lung stem cells had the lamellar structure characteristic of alveolar type II cells (AT2Cs) (electron microscopy observation results), confirming that the lung stem cells chemically induced and reprogrammed by this small molecule composition were alveolar type II cells (AT2Cs).

[0055] Figure 3 Immunostaining and nuclear staining (DAPI) were performed on lung stem cell-like cells transformed by chemically induced fibroblast (mouse) reprogramming to obtain AT2Cs-specific marker SPC. Figure 3 The results showed that the reprogrammed lung stem cell-like cells were positive for the alveolar type II cell-specific marker SPC via immunofluorescence staining. The experimental results also indicated that the small molecule composition induced positive SPC-specific marker immunofluorescence staining in reprogrammed fibroblast-transformed lung stem cells. This is further supported by the fact that its morphology is similar to or identical to pAT2Cs (Example 2). Figure 1 ), possessing the layered structure unique to AT2Cs (Example 3) Figure 2 Indicators such as these further indicate that the transformed lung stem cells belong to alveolar type II cells (AT2Cs), that is, chemically induced alveolar type II cells (ciAT2Cs).

[0056] Figure 4 Comparison of gene expression of specific markers (SPC, CC10) of alveolar stem cells (BASCs) in reprogrammed lung stem cell-like cells. Figure 4 The results showed that the expression of BASCs-specific marker genes SPC and CC10 in the lung stem cell-like cells obtained from the treatment group was upregulated; the expression of the epithelial cell-specific marker gene E-cad was also upregulated. Meanwhile, the expression of fibroblast-related genes such as Vimentin, aSMA, and Timp was downregulated. This indicates that the small molecule composition induced fibroblasts to undergo MET transformation, reprogramming them into lung stem cells, and that the obtained lung stem cell-like cells belong to alveolar stem cells, i.e., chemically induced alveolar stem cells (ciBASCs).

[0057] Figure 5 Immunostaining and nuclear staining (DAPI) of alveolar stem cell (BASC) specific markers (SPC, CC10) were performed on reprogrammed lung stem cell-like cells. Figure 5 The results showed that the lung stem cells obtained through induced transformation were positive for SPC and CC10 immunostaining, specific markers for BASCs. This further indicates that the lung stem cells obtained through fibroblast reprogramming transformation induced by this small molecule composition are alveolar stem cells, specifically chemically induced alveolar stem cells (ciBASCs).

[0058] Figure 6Reprogrammed lung stem cell-like cells were subjected to immunostaining and nuclear staining (DAPI) of p63 and Krt5, markers specific to lung basal cells or lung airway stem cells. Figure 6 The results showed that the lung stem cell-like cells obtained by induced transformation were positive for p63 and Krt5, specific markers for lung basal cells (BCs) or lung airway stem cells (DASCs), respectively, by routine immunostaining. This indicates that the lung stem cell-like cells obtained by fibroblast reprogramming transformation induced by this small molecule composition belong to lung basal cells or lung airway stem cells; that is, chemically induced transformed lung basal cells (ciBCs) or lung airway stem cells (ciDASCs).

[0059] Figure 7 Immunostaining and nuclear staining (DAPI) of specific markers (SPC, CC10) of alveolar stem cells (BASCs) were performed on reprogrammed lung stem cell-like cells. Figure 7 This shows that chemical induction Human fibroblasts The lung stem cells obtained through reprogramming transformation were positive for BASCs-specific markers SPC and CC10 immunostaining. This indicates that the lung stem cells obtained through reprogramming transformation of human fibroblasts induced by this small molecule composition belong to alveolar stem cells (BASCs), specifically chemically induced alveolar stem cells (ciBASCs).

[0060] Figure 8 Reprogrammed lung stem cell-like cells were subjected to immunostaining and nuclear staining (DAPI) of p63 and Krt5, markers specific to lung basal cells or lung airway stem cells. Figure 8 This shows that chemical induction Human fibroblasts The lung stem cell-like cells obtained by reprogramming transformation were positive for p63 and Krt5, markers specific to lung basal cells (BCs) or lung airway stem cells (DASCs), respectively, using conventional immunostaining. This fully demonstrates that the lung stem cell-like cells obtained by reprogramming human fibroblasts induced by this small molecule composition belong to lung basal cells or lung airway stem cells; that is, chemically induced transformed lung basal cells (ciBCs) or lung airway stem cells (ciDASCs).

[0061] Figure 9 The nebulized inhalation reagent 1 has the effect of chemically inducing the reprogramming of in situ fibroblasts into lung stem cells, promoting the repair of lung damage and improving and alleviating pulmonary fibrosis. Figure 9Immunohistochemical staining results of the treatment group (Treat) and the control group (Control) showed that the lung tissue of the control group mice was consolidated and lacked normal alveolar structure; the marker of activated pulmonary fibroblasts, αSMA (brown staining), was highly expressed in the lung tissue of the control group mice; while the lung tissue of the small molecule treatment group mice did not express αSMA or its expression was significantly reduced; the lung tissue and alveolar structure were basically restored to normal. This indicates that the reprogramming effect of this reprogrammed nebulized inhalation reagent can promote the repair of lung damage and improve and alleviate pulmonary fibrosis.

[0062] Figure 10 The nebulized inhalation reagent 2 chemically induces the reprogramming of in situ fibroblasts into lung stem cells, promoting the repair of lung damage and improving and alleviating pulmonary fibrosis. Figure 10 Immunohistochemical staining results showed that the lung tissue of mice in the treatment group (Treat) and control group (Control) was consolidated, lacking normal alveolar structure; the marker of activated pulmonary fibroblasts, αSMA (brown staining), was highly expressed in the lung tissue of mice in the control group; while αSMA was not expressed or its expression was significantly reduced in the lung tissue of mice in the small molecule treatment group (Treat), and the lung tissue and alveolar structure were basically restored to normal. This indicates that the reprogramming effect of this reprogrammed nebulized inhalation reagent can promote the repair of lung damage and improve and alleviate pulmonary fibrosis.

[0063] Figure 11 Trial of reprogrammed lung stem cell transplantation for the treatment of lung injury and improvement and relief of pulmonary fibrosis. Figure 11 Immunohistochemical staining results of the treated group (Treat) and the control group (Control) showed that the lung tissue of mice in the control group was consolidated, lacking normal alveolar structure; the marker of activated pulmonary fibroblasts, αSMA (brown staining), was highly expressed in the lung tissue of mice in the control group; while αSMA was not expressed or its expression was significantly reduced in the lung tissue of mice in the treated group (Treat); the lung tissue and alveolar structure were basically restored to normal. This indicates that the chemically induced reprogrammed lung stem cells have the effect of transplanting to treat lung injury and improve and alleviate pulmonary fibrosis. It also shows that the cells induced by this small molecule combination to reprogram fibroblasts are lung stem cells, which have the effect of treating lung injury and improving and alleviating pulmonary fibrosis, while fibroblasts do not possess such functional roles.

[0064] Figure 12 A comparative study on the independent induction of cell transformation (morphological comparison) by each individual small molecule component (GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, TGFβ inhibitor) of chemical small molecule composition 6. Figure 12The results show that, compared with the control group MF on the left and the small molecule treatment groups on the right, when each individual small molecule component in the small molecule composition exists independently, it does not have the function of chemically inducing fibroblasts to reprogram into lung stem cells; the morphology of fibroblasts before and after induction treatment is basically unchanged, and the morphology of the cells in the treatment group is still similar to that of fibroblasts, and has no resemblance to the morphology of lung stem cells. Detailed Implementation

[0065] Through in-depth research, the inventors have revealed a method for chemically inducing the reprogramming of fibroblasts into lung stem cells using only a small chemical molecule composition as the active ingredient. This method does not introduce or use any exogenous genes / transcription factors / microRNA (miRNA) genes, their RNA, proteins, peptides, or various exogenous cytokines or growth factors during the reprogramming process. This method is primarily used to induce the reprogramming of fibroblasts into lung stem cells to prepare lung stem cells (including morphologically similar BASCs, AT2Cs, BCs, and DASCs), providing a source of lung stem cells for clinical and research purposes. Aqueous or organic solvents, physiological saline, buffers, and other carriers or excipients, or cell-based culture media, can also be added to prepare reagents or reprogramming media for inducing the reprogramming of fibroblasts into lung stem cells.

[0066] Basic mechanism

[0067] The chemically induced reprogramming technology and method of the present invention belong to the category of chemically induced lung stem cell reprogramming; it uses only chemical small molecule compositions to chemically induce fibroblasts (differentiated cells) to reprogram into lung stem cells (adult or tissue stem cells).

[0068] This invention utilizes the individual small molecule components of a chemical composition—GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compounds—each to exert their unique ability to induce and regulate specific signaling pathways or epigenetic functions, thereby producing a synergistic effect. It targets and induces the inhibition of the GSK3β signaling pathway and G9aHMT epigenetic enzyme in fibroblasts, and regulates the retinoic acid (RA) signaling pathway, causing coordinated changes in these related signaling pathways and epigenetics in fibroblasts. This results in a new overall functional effect of the chemical composition: inducing and regulating the formation of new signaling pathways and epigenetic changes in fibroblasts, leading to stem cell reprogramming of fibroblasts, transformation of their cellular gene expression profile, and induction of fibroblast reprogramming into lung stem cells. This overall function of the chemical composition in inducing fibroblast reprogramming into lung stem cells is a function that no single small molecule within the composition possesses when existing independently.

[0069] Unless otherwise stated, " / " in this invention means "or".

[0070] Characteristics of Small Molecule Regulation: Small molecules used in stem cell research are generally classified and named according to their common function of targeting and inducing specific cell signaling pathways or epigenetic enzymes (this classification and naming work is completed collaboratively by chemists and biologists). The category name is usually based on their specific function of inducing and regulating specific signaling pathways or epigenetic modifying enzymes. For example, all small molecules in the GSK3β inhibitor category share the common function of targeting and inhibiting the GSK3β signaling pathway. Differences in chemical structure, physical, or chemical properties between small molecules within the same category do not affect the performance of their common specific function. While there may be differences in effective dosage, activity level, and degree of effect among small molecule inhibitors within the same category, there is no essential difference in their specific function of inducing and regulating specific signaling pathways or epigenetic enzyme activity. The mechanism of chemically induced fibroblast reprogramming into lung stem cells in this invention utilizes the specific function of each small molecule within the small molecule combination in regulating specific cell signaling pathways or epigenetic activity. Therefore, based on the unique characteristics of small chemical molecules, small chemical molecules within the same category have essentially the same efficacy when used as individual components; and when used as components in a composition, they also have essentially the same efficacy in the overall organic composition, differing only in degree, but without any qualitative difference. This is common knowledge well-known to those skilled in the art.

[0071] Therefore, in the specific embodiments of this invention, only 2-3 representative chemical small molecules of each category are listed to form representative small molecule compositions for representative experiments. It is unnecessary to exhaustively list all chemical small molecules within each category, nor is it possible to exhaustively list all small molecule combinations within the limited space of this specification. Providing representative experimental evidence is a well-known convention and common sense among those skilled in the art.

[0072] Therefore, GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds, and optional TGFβ inhibitors, each comprising numerous small molecule compounds of the same class that induce and regulate the same specific cell signaling pathway or the same epigenetic modifying enzyme, and exert consistent functional activities, effects, and outcomes, are expected to induce fibroblast reprogramming into lung stem cells to varying degrees. Therefore, small molecule compounds of the same class that induce and regulate the same signaling pathway or the same epigenetic enzyme at the same target and exert the same functional effect, as well as the small molecule combinations that can induce and regulate fibroblast reprogramming into lung stem cells, are all within the scope of protection of this invention.

[0073] On the other hand, fibroblasts, also known as fibroblasts, are the main cellular components of loose connective tissue, derived from mesenchymal cells during the embryonic period. Based on their different functional activity states, fibroblasts can be divided into fibroblasts and fibroblasts; fibroblasts are highly active, with weakly basic cytoplasm, exhibiting significant protein synthesis and secretion activities; those in a mature or quiescent state are called fibroblasts; under certain conditions, the two can interconvert. Fibroblasts have different types and exist in various tissues or organs in the body; they have different names and characteristics in different tissues or organs, including: skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, pancreatic fibroblasts, and fibroblasts in other tissues or organs.

[0074] The fibroblasts induced by the method or small molecule composition of the present invention, i.e., the fibroblasts described above, include: human fibroblasts or mammalian fibroblasts; preferably, including but not limited to: human or mammalian fibroblasts, skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts from other human or mammalian tissues or organs. More preferably, skin fibroblasts.

[0075] Preparation of small molecule compositions for chemically induced fibroblasts as lung stem cells

[0076] It should be understood that small chemical molecules within the same category differ only in effective dosage, activity level, and effect, but their ability to induce, inhibit, or regulate specific signaling pathways or epigenetic enzymes is essentially the same. Therefore, in addition to the specific and representative small chemical molecule GSK3β inhibitors (CHIR-99021, LiCl, BIO, LY2090314) listed in the embodiments of this invention, other small GSK3β inhibitors that target and inhibit the GSK3β cell signaling pathway can also achieve the same technical effect and should also be included in this invention.

[0077] In addition to the specific and representative G9aHMT inhibitors (BIX01294, UNC0638, or UNC0642) listed in the embodiments of this invention, other small chemical molecule G9aHMT inhibitors that target and induce G9aHMT inhibition can also achieve the same technical effect and should also be included in this invention.

[0078] Similarly, other small molecule retinoic acid compounds that target and activate the RA signaling pathway and promote cell differentiation, besides the specific and representative retinoic acid compounds (RA, 13-cis-retinoic acid or 9-cis-retinoic acid) listed in the embodiments of the present invention, can also achieve the same technical effect and should also be included in the present invention.

[0079] Similarly, other small chemical molecule TGFβ inhibitors that target and inhibit the TGFβ signaling pathway, besides the specific and representative TGFβ inhibitors (SB431542, A83-01, or RepSox) listed in the embodiments of this invention, can achieve the same technical effect and should also be included in this invention.

[0080] As used herein, the terms “containing” or “including” include “comprising”, “substantially consisting of”, and “consisting of”.

[0081] As used herein, the term "consistently composed of" means that, in addition to the essential ingredients or components, the composition may contain small amounts of minor components and / or impurities that do not affect the active ingredient. For example, it may contain sweeteners to improve flavor, antioxidants to prevent oxidation, and other pharmaceutical additives, carriers, and excipients commonly used in the art. In this invention, the phrase "comprising GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds" or "composed of GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds" includes situations where it is "consistently composed of GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds," "primarily composed of GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds as active ingredients," "comprising GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds as the sole active ingredient," or "consistently composed of GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds as active ingredients."

[0082] As used herein, "parts by weight" or "number of parts by weight" are used interchangeably. A part by weight can be any fixed weight expressed in micrograms, milligrams, grams, or kilograms (e.g., 1 μg, 1 mg, 1 g, 2 g, 5 g, or kg). For example, a composition consisting of 1 part by weight of component a and 9 parts by weight of component b can be a composition consisting of 1 g of component a + 9 g of component b, or 10 g of component a + 90 g of component b, etc. In the composition, the percentage content of a component = (number of parts by weight of that component / sum of the number of parts by weight of all components) × 100%. Therefore, in a composition consisting of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.

[0083] Furthermore, in solution, the aforementioned "parts by weight" can also be converted into "moles"; "parts by weight ratio" can also be converted into "molar concentration ratio". The weight unit of the parts by weight ratio can be any weight unit such as kilogram (kg), milligram (mg), microgram (ug); the molar unit of the molar concentration ratio can be any molar concentration unit such as mol (M), millimole (mM), micromol (μM).

[0084] The chemical small molecule composition contains: GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, and TGFβ inhibitor, in a weight ratio of (5-80):(0.1-50):(0.05-20):(0.1-50); preferably (10-70):(0.5-40):(0.15-15):(0.5-40); or in solution, a molar concentration ratio of (0.1-20):(0.01-20):(0.1-20):(0.01-20); preferably (0.5-15):(0.05-10):(0.5-10):(0.05-10).

[0085] For example, the composition may include the components and their weight parts as shown in Table 1 or the molar concentration as shown in Table 2 (in solution state).

[0086] Table 1. Parts by weight (weight units: kg, mg, μg, etc.)

[0087]

[0088] Table 2. Molar concentration ratios (molar units: M, mM, μM...)

[0089]

[0090] The formulation ranges in Tables 1 and 2 can be used as a reference guide. However, it should be understood that when used to develop and prepare small molecule compositions, the effective dosage of the composition may vary depending on the mode of administration and the physical condition of the treatment subject or handler, or the severity of the disease model. Furthermore, for in vivo use, "weight / kg (body weight)" is typically used as the dosage unit; when the small molecule composition is applied to large animals and patients with liver disease, the effective dosage for large animals or humans (including dosage conversion for solid or solution states) is calculated using the appropriate professional conversion formula based on the dosage used in small animals.

[0091] It should also be understood that, due to the large number of small molecule members in each category of chemical small molecules, and the significant differences in the effective dosage and activity of each small molecule member, although the inventors have conducted extensive research on the induction and regulation of stem cell directed differentiation and cell reprogramming using chemical small molecule compositions and have carried out numerous experiments, it is impossible to list all examples in the specification. The examples are only for the induction and regulation of the combination components of small molecule compositions, and the experimental concentrations of representative small molecules of each category (e.g., for GSK3β inhibitors, representative small molecule inhibitors such as CHIR-99021, LiCl, and BIO are used as examples). Therefore, the reasonable concentration range summarized in the claims of this invention naturally includes, but is not limited to, the experimental concentration ranges of specific representative small molecules of each category in the examples. This basic and simple principle is understandable to those skilled in the art.

[0092] For example, small molecule compounds that are GSK3β inhibitors vary widely in molecular weight. In this invention, when concentration / dosage is defined using "parts by weight" and "molar concentration," the range of molar concentrations for compounds with vastly different molecular weights may vary considerably. Therefore, in a preferred embodiment of this invention, the molar concentration is calculated based on the molecular weight of the GSK3β inhibitor; μM is used for large molecular weights, and mM is used for smaller molecular weights.

[0093] As used in this invention, the GSK3β inhibitor refers to a general term for small molecule inhibitors that can target and inhibit the cellular GSK3β signaling pathway, including but not limited to: CHIR-99021, BIO, LiCl, IM-12, TWS119, Ly2090314, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, SB216763, AZD1080, and other GSK3β small molecule inhibitors or small molecule compounds that induce the same target to inhibit the GSK3β signaling pathway; preferably, the GSK3β inhibitors CHIR-99021, LiCl, BIO, and LY2090314.

[0094] As a preferred embodiment of the present invention, the GSK3β inhibitor is CHIR-99021, also known as CT99021; its molecular structure is shown in the following formula (I):

[0095]

[0096] The aforementioned small molecule G9aHMT inhibitors refer to a general term for small molecule inhibitors that can target and inhibit G9aHMT, including but not limited to: BIX01294, UNC0638, A-366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321, BRD4770, HKMTI-1-247, HKMTI-1-248, CPUY074020, DCG066, and other small molecule inhibitors or small molecule compounds that induce the same target to inhibit G9aHMT; preferably, they are G9aHMT inhibitors BIX01294, UNC0638, or UNC0642.

[0097] As a preferred embodiment of the present invention, the G9aHMT inhibitor is BIX01294 (or BIX-01294); its molecular structure is shown in the following formula (II):

[0098]

[0099] The aforementioned small molecule TGFβ inhibitors refer to a general term for small molecule inhibitors that can inhibit the TGFβ signaling pathway in cells, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388, SB505124, EW-7197, Galunisertib, and other small molecule TGFβ inhibitors or small molecule compounds that induce the same target to inhibit the TGFβ signaling pathway; preferably, the TGFβ inhibitors SB431542, A83-01, or RepSox.

[0100] As a preferred embodiment of the present invention, the chemical small molecule TGFβ inhibitor is the small molecule SB431542 (or SB-431542); its molecular structure is shown in the following formula (III):

[0101]

[0102] As used in this invention, the retinoic acid compounds are differentiation inducers capable of specifically binding to retinoic acid response elements (RAREs), thereby regulating the RA signaling pathway and the transcriptional activity of specific nuclear genes, and promoting cell differentiation; including but not limited to: retinoic acid (RA), also known as all-trans retinoic acid (ATRA); 13-cis-retinoic acid (13-CRA), 9-cis-retinoic acid (9-CRA), UAB7, UAB8, TTNPB, and 3-methyl-TTN. PB, AM80, AM580, CD437, Targretin, LGD1069, and other retinoic acid compounds that induce the regulation of the RA signaling pathway and the transcriptional activity of specific nuclear genes at the same target to promote cell differentiation; or pharmaceutical products, analogs and / or their salts, hydrates or precursors, or combinations thereof; preferably retinoic acid (RA), 13-cis-retinoic acid or 9-cis-retinoic acid.

[0103] Retinoids have the function of regulating cell proliferation, differentiation, and apoptosis. They activate corresponding retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) proteins, and through specific binding to retinoic acid response elements (RARE), induce and regulate the RA signaling pathway and the transcriptional activity of specific nuclear genes, thus promoting cell differentiation. Many retinoids and their isoform derivatives have the same or similar functions, making them an important class of differentiation inducers.

[0104] As a preferred embodiment of the present invention, the retinoic acid (RA), also known as all-trans retinoic acid (ATRA), tretinoin, vitamin A acid, vitamin formic acid, retinoic acid, all-trans retinoic acid, and tretinoic acid formic acid, has the molecular structural formula shown in the following formula (V):

[0105]

[0106] The present invention also includes compounds, pharmaceutical products, analogues and / or salts, hydrates or precursors equivalent to the above-mentioned small molecule compounds I, II, III, and IV; as well as naturally occurring and artificially synthesized compounds thereof.

[0107] Analogs of the small molecule compounds include, but are not limited to, isomers and racemates of the small molecule compounds. The compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, or cis or trans isomers.

[0108] The term "salt" includes, but is not limited to: (1) salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; and (2) salts formed with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium).

[0109] The term "precursor of the compound" refers to a compound, or a salt or solution of a compound thereof, which, when applied or treated by appropriate methods, can be converted into any of the aforementioned compounds in a culture medium, or in an animal, or in a human body.

[0110] It should be understood that small chemical molecules are classified and named according to their common function of inducing and regulating specific cell signaling pathways or epigenetic enzymes. Within the same category, small molecule inhibitors differ only in effective dosage, activity level, and degree of effect; their function of inducing and regulating specific signaling pathways or epigenetic enzyme activity remains essentially the same. Therefore, based on the above-mentioned characteristics of the method of this invention using small chemical molecules, and due to the limitations of this specification, the embodiments of this invention only cite 2-3 representative small chemical molecules of each category to form representative small molecule compositions for representative experiments. However, those skilled in the art, based on the disclosure of this specification and well-known conventions and common sense, can deduce or anticipate that any other small molecule combination of this invention will also have the same effect.

[0111] In this invention, the fibroblasts include human fibroblasts or mammalian fibroblasts; preferably, including but not limited to: skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts of other tissues or organs of humans or mammals; more preferably, skin fibroblasts.

[0112] The chemically induced fibroblast reprogramming of this invention yields Lung stem cellsThis includes: alveolar type 2 cells (AT2Cs), bronchoalveolar stem cells (BASCs), basal cells (BCs), distal airway stem cells (DASCs), or mixtures thereof (mixed cells).

[0113] Preparation of reprogrammed culture medium and reprogrammed nebulized inhalation reagent

[0114] This invention also provides small molecule compositions that can be added to organic solvents, or physiological saline, or buffer solutions, or Cell base basal culture medium Carriers / excipients / basic nutrient solutions are used to prepare reprogramming culture media or reagents for chemically inducing fibroblasts to reprogram into lung stem cells; reprogramming culture media or reprogramming nebulized inhalation reagents for inducing fibroblasts to reprogram into lung stem cells.

[0115] According to the final concentration formulation of the chemical small molecule composition provided by this invention, a small molecule composition with a specific final concentration is selected for preparation. As a preferred embodiment of this invention, different components of the specific small molecule composition are dissolved in DMSO (dimethyl sulfoxide) or other organic or aqueous solvents (ranging from 1:50 to 1:10,000) according to their different solute properties and solubilities to prepare concentrated reagents. Then, according to the required final concentration of the specific small molecule composition, the concentrated reagents of each small molecule organic solution are diluted and added to a cell basal medium (DMEM) containing 10% fetal bovine serum to obtain the reprogramming medium. No exogenous genes are used or added during this reprogramming medium or reprogramming process; nor are any exogenous cytokines or growth factors added. The percentage content of each component in the medium may fluctuate by 50%; preferably by 30%; more preferably by 20%, such as 10% or 5%. Unless otherwise stated, percentages are expressed v / v.

[0116] As a preferred embodiment of the present invention, the cell culture medium includes, but is not limited to, DMEM / F12, MEM, DMEM, F12, IMDM, RPMI1640, Neuronal basal or Fischers, all of which are commercially available products.

[0117] As a preferred embodiment of the present invention, the aforementioned reprogramming culture medium can also be prepared using serum-free culture medium if there are special requirements. The "serum-free culture medium" refers to a cell culture medium that does not contain serum but contains various nutrients (such as growth factors, tissue extracts, etc.) that support cell proliferation and biological responses. That is, a cell culture medium composed of various cytokines or growth factors, other than serum, added to a basic cell culture medium.

[0118] As a preferred embodiment of the present invention, the serum-free culture medium containing various cytokines or growth factors includes, but is not limited to, ITS, N2, B27, etc., all of which can be prepared by oneself or are commercially available products.

[0119] It should be understood that those skilled in the art are familiar with the preparation or purchase methods of the aforementioned cell-based culture media or serum-free culture media, and therefore, the cell-based culture media or serum-free culture media are not limited to those exemplified in this invention.

[0120] 1. As a preferred embodiment of the present invention, a method for preparing or formulating the aforementioned "reprogramming culture medium" is provided:

[0121] (1) Dissolve ① GSK3β inhibitors (such as CHIR99021) at a final concentration of 0.1 μM-20 μM / mM; preferably 0.5 μM-10 μM / mM; (for GSK3β inhibitors with small molecular weights such as LCI, the concentration is mM); ② G9aHMT inhibitors (such as BIX01294) at a final concentration of 0.01-20 μM; preferably 0.05-10 μM; ③ Retinoic acid compounds at a final concentration of 0.1-20 μM; preferably 0.5-10 μM. Or add ④ TGFβ inhibitors at a final concentration of 0.05-20 μM, preferably 0.5-10 μM. Dissolve each of these in DMSO (dimethyl sulfoxide) or other organic or aqueous solvents to prepare concentrated reagent solutions (ranging from 1:50 to 1:10,000). This provides a concentrated reagent of each component of the small molecule composition of the present invention for chemically inducing fibroblast reprogramming into lung stem cells;

[0122] (2) The above small molecules are prepared by dissolving and preparing concentrated reagents, diluting them in the cell basal culture medium, and mixing them to obtain the "reprogramming culture medium" for chemically induced fibroblasts to be reprogrammed into lung stem cells.

[0123] 2. As a preferred embodiment of the present invention, a method for preparing a reprogramming nebulized inhalation reagent for chemically inducing in situ reprogramming of fibroblasts into lung stem cells is also provided:

[0124] The above-mentioned small molecule combination is prepared into a 100 mg / ml concentrate according to the weight ratio of each mouse body weight. This concentrate is then diluted in 0.9% NaCl saline (50 μl / mouse) and mixed to prepare a chemically induced in situ reprogramming of fibroblasts into lung stem cells via nebulization.

[0125] As a preferred embodiment of the present invention, the dosage of each small molecule component in any of the aforementioned compositions is calculated based on body weight, and then dissolved in physiological saline (0.9% NaCl) solution to obtain a laboratory animal reprogramming nebulized inhalation reagent.

[0126] 3. As a preferred embodiment of the present invention, the present invention also discloses a method for preparing lung stem cells (BASCs, AT2Cs, BCs, DASCs) by in vitro chemical induction of fibroblasts into lung stem cells using a small chemical molecule composition, wherein the method includes the following steps:

[0127] (1) Preparation of concentrated reagent: The components of any of the compositions described in this invention are prepared into concentrated reagents according to the aforementioned method for preparing reprogrammed culture medium.

[0128] (2) Obtaining reprogramming medium: Dilute the concentrated reagent in step (1) into a cell basal medium (such as DMEM) containing 5-20% fetal bovine serum (so that the concentration of each component meets the concentration specified in the composition described above) to obtain the reprogramming medium.

[0129] (3) Inducing fibroblasts to reprogram into lung stem cells: Fibroblasts were suspended in cell basal medium (DMEM) containing 5-20% fetal bovine serum, plated, and after the cells adhered, the original medium was discarded and replaced with the reprogramming medium from step (2). The cells were cultured at 37°C and the medium was changed every 2-4 days. The cells were passaged every 3-15 days.

[0130] (4) Subculture: Discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, incubate at 37°C for 1-5 minutes to stop cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and subculture at a ratio of 1:1 to 1:3. Culture according to steps (2) and (3) of the experimental procedure, changing the medium every 2-4 days. The digestion solutions used include trypsin, EDTA, acutase, TrypleE, etc. Subculture every 3-15 days.

[0131] (5) Harvesting reprogrammed lung stem cells: After reprogramming and passage culture for 2-4 weeks following the above experimental steps (3) and (4), reprogrammed lung stem cells can be obtained. Transformed lung stem cells (Including those with basically the same or similar morphology: ciBASCs, ciAT2Cs, ciBSCs, ciDASCs); these lung stem cells can be used for related scientific research experiments.

[0132] It is important to note that the obtained Transformed lung stem cellsThis includes morphologically similar or identical lung stem cells such as BASCs, AT2Cs, BCs, and DASCs; or mixtures thereof. The predominance of any particular type of lung stem cell is correlated with the type and proportion of the small molecule combination components, as well as variations in culture time and environment. However, regardless of the specifics, the examples of this invention are unequivocally established, and the induced reprogramming yields... Lung stem cells .

[0133] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments select only 2-3 representative small molecules from each category of small chemical molecules to form representative small molecule compositions. Representative experiments are conducted according to the reprogrammed experimental methods and procedures disclosed earlier in the specification, and are used to illustrate the invention but not to limit its scope. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Short Protocols in Cell Biology* (2007) by J.S. Bonifacnon et al., translated by Zhang Jingbo, Fang Jin, Wang Haijie et al., or according to the manufacturer's recommendations.

[0134] Example 1: Preparation or formulation of a small molecule composition for inducing fibroblast reprogramming into lung stem cells (hereinafter referred to as: reprogramming small molecule composition), its reprogramming culture medium and reprogramming nebulized inhalation reagent.

[0135] Design the following small molecule composition, add it to cell basal culture medium to prepare a reprogramming culture medium, and prepare it at a molar concentration or part-of-weight concentration (the following reprogramming culture medium is at molar concentration; the reprogramming nebulizer inhalation reagent is at part-of-weight concentration); or dissolve the small molecule composition in 0.9% (w / v) NaCl to prepare a reprogramming nebulizer inhalation reagent, and administer it according to mg / kg body weight:

[0136] 1. Reprogramming the small molecule composition

[0137] (1) Reprogramming small molecule composition 1

[0138] GSK3β inhibitor CHIR-99021: final concentration 6 μM;

[0139] G9aHMT inhibitor BIX01294: final concentration 3 μM;

[0140] Retinoic acid compound RA: final concentration 5 μM;

[0141] (2) Reprogramming small molecule composition 2

[0142] GSK3β inhibitor BIO: final concentration 2μM;

[0143] G9aG9AHMT inhibitor UNC0638: final concentration 5 μM;

[0144] Retinoic acid compound RA: final concentration 2 μM;

[0145] (3) Reprogramming small molecule compositions 3

[0146] GSK3β inhibitor Ly2090314: final concentration 1 μM;

[0147] G9aHMT inhibitor UNC0642: final concentration 2.5 μM;

[0148] Retinoic acid compound RA: final concentration 3 μM;

[0149] (4) Reprogramming small molecule compositions 4

[0150] GSK3β inhibitor LiCl: final concentration 4mM;

[0151] G9aHMT inhibitor BIX01294: final concentration 2 μM;

[0152] Retinoic acid compound RA: final concentration 2 μM;

[0153] (5) Reprogramming small molecule compositions 5

[0154] GSK3β inhibitor Ly2090314: final concentration 0.5 μM;

[0155] G9aHMT inhibitor UNC0642: final concentration 5 μM;

[0156] 9-cis-retinoic acid, a retinoic acid compound: final concentration 2 μM;

[0157] TGFβ inhibitor SB431542: final concentration 2 μM;

[0158] (6) Reprogramming small molecule compositions 6

[0159] GSK3β inhibitor CHIR99021: Final concentration 3 μM;

[0160] G9aHMT inhibitor BIX01294: final concentration 2 μM;

[0161] Retinoic acid compound RA: final concentration 2.5 μM;

[0162] TGFβ inhibitor RepSox: final concentration 0.8 μM;

[0163] (7) Reprogrammed small molecule compositions 7

[0164] GSK3β inhibitor Ly2090314: final concentration 1.5 μM;

[0165] G9aHMT inhibitor BIX01294: final concentration 0.5 μM;

[0166] Retinoic acid compound RA: final concentration 5 μM;

[0167] (8) Reprogrammed small molecule composition 8 (preparation of nebulized inhalation reagent)

[0168] GSK3β inhibitor CHIR99021: 1 mg / kg;

[0169] G9aHMT inhibitor UNC0642: 0.3 mg / kg;

[0170] Retinoic acid compound RA: 1 mg / kg;

[0171] (9) Reprogrammed small molecule composition 9 (preparation of nebulized inhalation reagent)

[0172] GSK 3β inhibitor BIO: 0.5 mg / kg;

[0173] G9aHMT inhibitor BIX01294: 0.5 mg / kg;

[0174] 13-cis-retinoic acid, a retinoid compound: 0.5 mg / kg;

[0175] TGFβ inhibitor A83-01: 0.5 mg / kg;

[0176] (10) Reprogrammed small molecule compositions 10

[0177] GSK3β inhibitor LiCl: final concentration 15mM;

[0178] G9aHMT inhibitor UNC0638: final concentration 10 μM;

[0179] Retinoic acid compound RA: final concentration 5 μM.

[0180] The small molecule components of each specific small molecule composition can be prepared by dissolving them in DMSO to form a concentrated reagent, referring to the steps in the aforementioned "Preparation or Formulation Method of Reprogramming Culture Medium".

[0181] 2. Preparation of reprogrammed culture medium

[0182] The DMSO concentrate reagents of each component of the above-mentioned reprogrammed small molecule compositions 1-7 and 10 were prepared according to the steps of the aforementioned "Preparation Method of Reprogramming Culture Medium". The cell basal culture medium used was DMEM, with 10% fetal bovine serum added, to obtain reprogramming culture media 1-10 (i.e., reprogramming culture medium 1 has the same components and final concentration as composition 1, culture medium 2 has the same components and final concentration as composition 2, ..., culture medium 10 has the same components and final concentration as composition 10).

[0183] 3. Reprogramming the preparation of nebulized inhalation reagents

[0184] Dissolve the DMSO concentrate of reprogrammed small molecule compositions 8 and 9 in 0.9% NaCl solution and prepare them according to the "Preparation Method of Reprogrammed Nebulized Inhalation Reagents" (① Prepare 100 mg / ml concentrates of the small molecule compositions according to the weight ratio of each component per kilogram of body weight, then dilute and mix them in 0.9% NaCl physiological saline (50 μl / mouse)) to become "Reprogrammed Nebulized Inhalation Reagents 1 and 2 (prepared according to the same weight ratio of the components of small molecule compositions 8 and 9)".

[0185] 4. Preparation of lung stem cell suspension or formulation for transplantation

[0186] Collect lung stem cells transformed by chemically induced fibroblast reprogramming; prepare lung stem cell suspensions or preparations (0.5 × 10⁻⁶) using physiological saline. 6 (1 cell / 50ul).

[0187] Example 2: Morphological comparison experiment of lung stem cells transformed by chemically induced fibroblasts (mouse) in reprogrammed medium 1. The mouse fibroblasts used in subsequent examples were derived from C57 / BL6 mouse skin fibroblasts (MF).

[0188] Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 1. The experimental culture method for preparing lung stem cells was as described in the previous section "In vitro chemical induction of fibroblast reprogramming into lung stem cells to prepare lung stem cells (BASCs, AT2Cs, BCs, DASCs)": The concentrated reagent was diluted in DMEM containing 10% fetal bovine serum (to ensure that the concentrations of each component met the concentrations specified in the previous section "Reprogramming Small Molecule Composition 1") to obtain reprogramming medium 1. Fibroblasts (MF) were added to this medium, suspended, and plated. After cell adhesion, the medium was replaced with fresh reprogramming medium and cultured at 37°C. The medium was changed every 3 days, and the cells were passaged every 7 days. For passage culture: the original culture medium was discarded, the cells were washed once with PBS, and cell digestion solution was added to digest the cells. The digestion was stopped at 37°C for 3 minutes. The cells were centrifuged, the supernatant was discarded, and the cell pellet was resuspended and passaged at a 1:2 ratio. The medium was changed every 3 days.

[0189] After 3 weeks of culture and passage, lung stem cells were obtained. The morphology of these reprogrammed lung stem cells (ciAT2Cs) was compared with that of primary alveolar type II cells (pAT2Cs) isolated from mice and MF cells. The results are shown in [Figure 1]. Figure 1 .

[0190] Figure 1 The results showed that the lung stem cells obtained by chemically induced reprogramming had the same or similar morphology as primary alveolar type II cells (pAT2Cs, where "p" represents "primary"), but were completely different from the morphology of their inducing cells MF. Therefore, the small molecule composition of the present invention can successfully induce fibroblast reprogramming to transform lung stem cells, which belong to alveolar type II cells. The inventors refer to the lung stem cells obtained by chemically induced reprogramming as "ciAT2Cs", where "ci" represents "chemically induced transformation".

[0191] Example 3: Lung stem cells transformed from chemically induced fibroblasts (mouse) using reprogramming medium 2.

[0192] Experimental Procedure: Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 2. The experimental culture method for preparing lung stem cells was the same as in Example 2. Electron microscopy sections of the chemically induced reprogrammed lung stem cells were prepared according to conventional methods to obtain electron micrographs of the unique lamellar structure of AT2Cs. The results are shown in […]. Figure 2 .

[0193] Figure 2 The results showed that the reprogrammed lung stem cells had the lamellar structure characteristic of alveolar type II cells (AT2Cs) (electron microscopy observation results), confirming that the lung stem cells chemically induced and reprogrammed by this small molecule composition were alveolar type II cells, namely ciAT2Cs.

[0194] Example 4: Immunostaining and nuclear staining of lung stem cell-like cells transformed from chemically induced fibroblasts (mouse) using reprogrammed culture medium 3, based on the AT2Cs-specific marker SPC.

[0195] Experimental Procedure: Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 3. The experimental culture method for preparing lung stem cells was the same as in Example 2. The lung stem cell-like cells obtained by induction and transformation were subjected to routine immunofluorescence staining with SPC antibody, a specific marker of type II alveolar cells, and routine nuclear staining. Experimental results are shown in […]. Figure 3 .

[0196] Figure 3The results showed that the lung stem cells transformed from fibroblasts (mouse) induced by the small molecule composition were positive for SPC immunofluorescence staining, a specific marker of type II alveolar stem cells. The experimental results indicate that the lung stem cells transformed from fibroblasts induced by this small molecule composition are type II alveolar cells.

[0197] The lung stem cells obtained by chemically induced reprogramming according to the present invention have the same or similar morphology as pAT2Cs (Example 2). Figure 1 ), possessing the layered structure unique to AT2Cs (Example 3, Figure 2 Indicators such as ) indicate that the lung stem cells transformed by chemical induction reprogramming are alveolar type II cells (AT2Cs), namely ciAT2Cs.

[0198] Example 5: Comparative experiment on the expression of specific markers (SPC, CC10) of alveolar stem cells (BASCs) in lung stem cell-like cells transformed by chemically induced mouse fibroblast (MF) reprogramming.

[0199] Experimental Procedure: Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 4. The experimental culture method for preparing lung stem cells was the same as in Example 2. RNA was collected from the reprogrammed lung stem cell-like cells obtained from the control group (MF) and the reprogramming medium 4 treatment group, and qRT-PCR was performed to detect and compare the expression levels of specific markers (SPC, CC10) genes of alveolar stem cells (BASCs). Experimental results are shown below. Figure 4 .

[0200] Figure 4 The results showed that the expression of BASCs-specific marker genes SPC and CC10 in the lung stem cell-like cells obtained from the treatment group was upregulated; the expression of the epithelial cell-specific marker gene E-cad was also upregulated. Meanwhile, the expression of fibroblast-related genes such as Vimentin, aSMA, and Timp was downregulated. This indicates that the small molecule composition induced fibroblasts to be reprogrammed into lung stem cells, and the obtained lung stem cell-like cells were confirmed to be alveolar stem cells (BASCs) at the gene expression level. The inventors refer to this chemically induced reprogrammed alveolar stem cell as "ciBASCs".

[0201] Example 6: Immunostaining and nuclear staining experiments of specific markers (SPC, CC10) for alveolar stem cell (BASCs)-like cells transformed by chemically induced mouse fibroblast (MF) reprogramming.

[0202] Experimental Procedure: Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 5. The experimental culture method for preparing lung stem cells was the same as in Example 2. The lung stem cell-like cells obtained by induced transformation were subjected to routine immunofluorescence staining with antibodies against alveolar stem cell (BASC) specific markers (SPC, CC10) and routine immunofluorescence staining of cell nuclei. The results are shown in [Figure 1]. Figure 5 .

[0203] Figure 5 The results showed that the lung stem cells obtained through induced transformation were positive for BASCs-specific markers SPC and CC10 immunostaining. This indicates that the lung stem cells obtained through chemical induction and reprogramming of fibroblasts using this small molecule composition are alveolar stem cells (BASCs), specifically chemically inducible reprogrammed alveolar stem cells, abbreviated as "ciBASCs".

[0204] Example 7: Immunostaining and nuclear staining experiments of p63 and Krt5, markers specific to lung basal cells or lung airway stem cells, on lung stem cell-like cells transformed by chemically induced mouse fibroblast (MF) reprogramming.

[0205] Experimental Procedure: Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 6. The experimental culture method for preparing lung stem cells was the same as in Example 2. The lung stem cell-like cells obtained by induction and transformation were subjected to routine immunostaining with p63 and Krt5, markers specific to lung basal cells (BCs) or lung airway stem cells (DASCs), as well as routine nuclear staining. The experimental results are shown in […]. Figure 6 .

[0206] Figure 6 The results showed that the lung stem cell-like cells obtained through induced transformation were positive for p63 and Krt5, specific markers for lung basal cells or lung airway stem cells, respectively, using conventional immunostaining. This demonstrates that the small molecule composition induces fibroblast reprogramming into lung stem cells, which further exhibit specific marker characteristics of lung basal cells or lung airway stem cells; indicating that the lung stem cells transformed through this chemical induction reprogramming are lung basal cells (BCs) or lung airway stem cells (DASCs), abbreviated as ciBCs or ciDASCs, respectively.

[0207] Example 8: Immunostaining and nuclear staining experiments of specific markers (SPC, CC10) for alveolar stem cell (BASCs) by inducing human fibroblast (HF) reprogramming and transforming lung stem cell-like cells using a small molecule chemical composition.

[0208] Experimental Procedure: Human fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 7. The experimental culture method for preparing lung stem cells was the same as in Example 2. The lung stem cell-like cells obtained by induced transformation were subjected to routine immunostaining with antibodies against alveolar stem cell (BASC) specific markers (SPC, CC10) and routine staining of cell nuclei. The results are shown in [Figure 2]. Figure 7 .

[0209] Figure 7 The results showed that the lung stem cells obtained through induced transformation were positive for BASCs-specific markers SPC and CC10 immunostaining. This indicates that the lung stem cells obtained by inducing human fibroblast reprogramming transformation with this small molecule composition are alveolar stem cells, namely chemically induced alveolar stem cells (ciBASCs).

[0210] Example 9: Immunostaining and nuclear staining experiments of lung stem cell-like cells transformed by chemically induced human fibroblast (HF) reprogramming with p63 and Krt5, markers specific to lung basal cells or lung airway stem cells.

[0211] Experimental Procedure: Human fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 10. The experimental culture method for preparing lung stem cells was the same as in Example 2. The lung stem cell-like cells obtained by induced transformation were subjected to routine immunostaining with p63 and Krt5, specific markers for lung basal cells (BCs) or lung airway stem cells (DASCs), and routine nuclear staining. The experimental results are shown in […]. Figure 8 .

[0212] Figure 8 The results showed that the lung stem cell-like cells obtained by induced transformation were positive for p63 and Krt5, markers specific to lung basal cells (BCs) or lung airway stem cells (DASCs), using routine immunostaining. This fully demonstrates that the lung stem cell-like cells obtained by human fibroblast reprogramming transformation induced by this small molecule composition are lung basal cells or lung airway stem cells; that is, ciBCs or ciDASCs.

[0213] Example 10: Nebulized inhalation reagent 1 chemically induces in situ reprogramming of fibroblasts into lung stem cells, promoting the repair of lung damage and improving and alleviating pulmonary fibrosis.

[0214] In this embodiment, the in vivo in situ fibroblasts were chemically induced to reprogram into lung stem cells using nebulized inhalation reagent 1 (prepared with small molecule combination 8, see Example 1), and it was observed whether this promoted the repair of lung damage and improved and alleviated pulmonary fibrosis.

[0215] Experimental procedure:

[0216] 1. Establishment of a mouse model of lung injury-induced fibrosis: Eighteen male 4-5 week old C57 / BL6 mice were used to establish a mouse model of lung injury-induced pulmonary fibrosis using bleomycin (BLM).

[0217] The drug was administered via a single endotracheal intubation (nebulized inhalation reagent 1) at a dose of 5 mg / kg. Following a single BLM administration, the acute inflammatory response lasted for 8 days. On day 9, the inflammation transitioned to pulmonary fibrosis, and after 28 or 35 days, tissue matrix deposition appeared, exhibiting fibrotic changes. On day 10 of model establishment, one mouse was dissected, and lung tissue was fixed and sectioned for routine immunohistochemical staining, confirming the successful establishment of the mouse model of lung injury leading to pulmonary fibrosis.

[0218] 2. Sixteen mice with lung injury-induced pulmonary fibrosis were randomly divided into two groups: a control group and a treatment group.

[0219] 3. Take nebulized inhalation reagent 1 (prepared with small molecule combination 8) and inhale it into the treatment group animal model through a nebulizer; take the "control nebulized inhalation reagent" with the same composition but without any small molecule combination added and treat the control group animal model through a nebulizer.

[0220] 4. In the fourth week of treatment, the mouse models in the control group and the treatment group were dissected, and lung tissue was fixed and sectioned for routine immunohistochemical staining.

[0221] The experimental results showed that lung injury and pulmonary fibrosis were significantly reduced in the treated mice, and the lung tissue structure was basically restored to normal.

[0222] Immunohistochemical staining results of the most representative sections from the control group and the treatment group were compared. Results are shown below. Figure 9 .

[0223] Figure 9 The results showed that the lung tissue of control mice was consolidated, lacking normal alveolar structure; the marker of activated pulmonary fibroblasts, αSMA (brown staining), was highly expressed in the lung tissue of control mice; while the lung tissue of treatment mice did not express αSMA, or its expression was significantly reduced; the lung tissue and alveolar structure were basically restored to normal. This indicates that the reprogramming effect of this small molecule reprogrammed nebulized inhalation reagent can promote the repair of lung damage and improve and alleviate pulmonary fibrosis.

[0224] Example 11: Nebulized inhalation reagent 2 chemically induces in situ reprogramming of fibroblasts into lung stem cells, promoting the repair of lung damage and improving and alleviating pulmonary fibrosis.

[0225] In this embodiment, the in vitro fibroblast reprogramming into lung stem cells was chemically induced using nebulized inhalation reagent 2 (prepared with small molecule combination 9, see Example 1), and its effect on promoting lung damage repair and improving and alleviating pulmonary fibrosis was observed. The experimental procedure was the same as in Example 10.

[0226] Experimental results showed that, compared with the control group, the lung injury and pulmonary fibrosis in the treated group mice were significantly reduced, and the lung tissue structure was basically restored to normal. Immunohistochemical staining results of the most representative sections from both the control and treated groups were compared. Results are shown below. Figure 10 .

[0227] Figure 10 The results showed that the lung tissue of control mice underwent consolidation, losing normal alveolar structure; the marker of activated pulmonary fibroblasts, αSMA (brown staining), was highly expressed in the lung tissue of control mice; while the lung tissue of treatment mice did not express αSMA, or its expression was significantly reduced; the lung tissue and alveolar structure were basically restored to normal. This indicates that the reprogramming effect of this small molecule reprogrammed nebulized inhalation reagent can promote the repair of lung damage and improve and alleviate pulmonary fibrosis.

[0228] Example 12: Trial of reprogrammed lung stem cell transplantation for the treatment of lung injury and improvement and relief of pulmonary fibrosis

[0229] In this embodiment, lung stem cells obtained by chemically induced fibroblast reprogramming were transplanted into an animal model of lung injury-induced pulmonary fibrosis to observe their effect on improving or alleviating lung injury-induced pulmonary fibrosis.

[0230] 1. Mouse fibroblasts were chemically induced to reprogram into lung stem cells using reprogramming medium 10. The experimental culture method for preparing lung stem cells was the same as in Example 2. The reprogrammed lung stem cells were collected. A lung stem cell suspension or preparation (0.5 × 10⁻⁶) was prepared using physiological saline. 6 (50ul / cell)

[0231] 2. The establishment of a mouse model of lung injury-induced fibrosis is the same as in Example 10; 16 mice with lung injury-induced fibrosis were randomly divided into two groups: a control group and a treatment group.

[0232] 3. In the treatment group, each diseased animal underwent a single transplantation of 50 μL of transformed lung stem cell saline suspension (0.5 × 10⁻⁶) via endotracheal intubation. 6 50 μL / animal (cells); while in the control group, each diseased animal received a single intratracheal injection of 50 μL of mouse fibroblast saline suspension (0.5 × 10⁻⁶ cells / animal); 6 (50ul / cell / animal);

[0233] 4. After 30 days of treatment, the mouse models in the control group and the treatment group were dissected, and lung tissue was fixed and sectioned for routine immunohistochemical staining.

[0234] Experimental results showed that, compared with the control group, the lung injury and pulmonary fibrosis in the treated group mice were significantly reduced, and the lung tissue structure was basically restored to normal. Immunohistochemical staining results of the most representative sections from both the control and treated groups were compared, and the results are shown below. Figure 11 .

[0235] Figure 11 The results showed that the lung tissue of control mice was consolidated, lacking normal alveolar structure; the marker of activated pulmonary fibroblasts, αSMA (brown staining), was highly expressed in the lung tissue of control mice; while the lung tissue of treatment mice did not express αSMA, or its expression was significantly reduced; the lung tissue and alveolar structure of the mice were basically restored to normal. This indicates that the cells induced by the small molecule composition to reprogram fibroblasts are lung stem cells, which have therapeutic effects on lung injury and can improve and alleviate pulmonary fibrosis; while fibroblasts do not have the same therapeutic effects on lung injury and can improve and alleviate pulmonary fibrosis.

[0236] Example 13: Comparative experiment on the independent induction of cell transformation by individual small molecule components of chemical small molecule composition 6.

[0237] In this embodiment, an independent comparative assay was conducted to evaluate the induction of fibroblast transformation by each individual small molecule component (GSK3β inhibitor / G9aHMT inhibitor / retinoic acid compound / TGFβ inhibitor) of chemical small molecule composition 6. The experimental steps are as follows:

[0238] (1) Preparation of culture medium for each of the following individual small molecule components: GSK3β inhibitor CHIR99021, G9aHMT inhibitor BIX01294, TGFβ inhibitor SB431542, and retinoic acid compound RA: The culture medium for each individual small molecule component was prepared by referring to the concentration ratio of small molecule composition 6 and the reprogrammed culture medium preparation method.

[0239] (2) The method and steps for inducing fibroblasts to reprogram into lung stem cells are the same as in Example 2; the cells used are mouse fibroblasts (MF) as in Example 2.

[0240] (3) The morphology of human fibroblasts reprogrammed in culture with each single small molecule component was compared with that of fibroblasts (MF) in the control group (the only difference between the control and treatment groups was the absence of added chemical small molecule components). The experimental results are shown in […]. Figure 12 .

[0241] Figure 12 As shown in the figure, compared with the control group MF on the left and the treatment groups on the right, the individual small molecule components in the small molecule composition did not induce fibroblasts to reprogram into lung stem cells. The morphology of fibroblasts remained essentially unchanged before and after the induction treatment; the cell morphology of the treatment groups was still similar to that of fibroblasts, and showed no resemblance to lung stem cells.

[0242] Experimental results show that the individual small chemical components of the chemical small molecule composition of the present invention—GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compounds, and TGFβ inhibitor—do not have the function of inducing fibroblast reprogramming into lung stem cells when present alone.

[0243] In summary, the above embodiments selected only 3-4 representative small molecules from each category of small chemical molecules to form representative small molecule compositions. Following the reprogramming experimental methods and procedures disclosed earlier in the specification, representative experiments were conducted, and predictable representative results of inducing fibroblast reprogramming into lung stem cells were obtained. It should be noted that, based on existing experimental research, the inventors have shown that the obtained... Transformed lung stem cells This includes ciBASCs, ciAT2Cs, ciBCs, and ciDASCs with basically the same or similar shapes; which type of transformed lung tissue stem cells is dominant is related to the type and proportion of small molecule components, as well as changes in culture time and culture environment. However, regardless of the specifics, the examples of this invention are unequivocally established, and the induced reprogramming results are all... Lung stem cells .

[0244] The method for chemically inducing fibroblast reprogramming into lung stem cells using the small molecule chemical composition of the present invention has the following beneficial effects:

[0245] 1. The method of this invention involves reprogramming fibroblasts into lung stem cells without introducing or using any exogenous genes / transcription factors / microRNA (miRNA) genes, nor using any exogenous cytokines or growth factors; only using an inducing composition composed of small chemical molecules to chemically induce the reprogramming of fibroblasts into lung stem cells. The resulting transformed lung stem cells can be applied to clinical lung stem cell transplantation therapy, avoiding the carcinogenic risk of stem cell transplantation with introduced exogenous genes and overcoming the pathogenic risks of clinical transplantation of reprogrammed iPS cells.

[0246] 2. The methods for inducing fibroblasts into lung stem cells using this small molecule chemical composition include: a method for in vitro chemical induction of fibroblast reprogramming into lung stem cells using the small molecule composition; and a method for promoting the repair of lung damage and improving and alleviating pulmonary fibrosis by using the small molecule composition to chemically induce in vivo in situ fibroblast reprogramming into lung stem cells. It has multiple uses: (1) for inducing in vitro fibroblast reprogramming into lung stem cells, preparing lung stem cells, and providing lung stem cell sources for clinical and scientific research; (2) for preparing reagents or culture media for chemically inducing fibroblast reprogramming into lung stem cells; and (3) for related scientific research on the effect of small molecule chemical induction of in vivo in situ fibroblast reprogramming into lung stem cells.

[0247] 3. The method of the present invention can be derived from the patient's own fibroblasts, and the transformed lung stem cells have personalized characteristics, making them easy to enter clinical application and minimizing or avoiding the risk of immune rejection caused by allogeneic lung stem cell transplantation.

[0248] 4. The method of the present invention is simple in steps, easy to operate, low in cost, and easy to translate into clinical applications.

[0249] 5. The method of the present invention only requires conventional cultivation, has a short cycle, is suitable for mass production, and is easy to apply to industrial applications.

[0250] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A non-therapeutic method for in vitro chemical induction of fibroblast reprogramming into lung stem cells using a small chemical molecule composition, comprising: Fibroblasts were treated with a chemical small molecule composition to induce them to reprogram into lung stem cells; and during the induction and reprogramming process, no exogenous genes / transcription factors / microRNA genes, their RNA, proteins, peptides, or exogenous cytokines or growth factors were introduced or used. The small molecule chemical composition includes: GSK3β inhibitor, G9aHMT inhibitor and retinoic acid compound; or, the small molecule chemical composition consists only of GSK3β inhibitor, G9aHMT inhibitor and retinoic acid compound; The small molecule chemical composition includes: GSK3β inhibitor: 5-80 parts by weight; or final concentration in solution: 0.1-20 μM; G9aHMT inhibitor: 0.1-50 parts by weight; or final concentration in solution: 0.01-20 μM; Retinoic acid compounds: 0.05-20 parts by weight; or final concentration in solution: 0.1-20 μM; The GSK3β inhibitor is selected from CHIR-99021, BIO, Ly2090314 or LiCl; the G9aHMT inhibitor is selected from BIX01294, UNC0638 or UNC0642; and the retinoic acid compound is selected from RA, 9-cis-retinoic acid or 13-cis-retinoic acid.

2. The method as described in claim 1, characterized in that, In the chemical small molecule composition: GSK3β inhibitor: 10-70 parts by weight; or a final concentration of 0.5-15 μM in solution. G9aHMT inhibitor: 0.5-40 parts by weight; or final concentration in solution: 0.05-10 μM; Retinoic acid compounds: 0.15-15 parts by weight; or final concentration in solution: 0.5-10 μM.

3. The method as described in claim 1, characterized in that, The chemical small molecule composition also includes: TGFβ inhibitor: 0.1-50 parts by weight; or final concentration in solution: 0.01-20 μM; The TGFβ inhibitor is selected from SB431542, RepSox, or A83-01.

4. The method as described in claim 3, characterized in that, In the chemical small molecule composition: TGFβ inhibitor: 0.5-40 parts by weight; or final concentration in solution: 0.05-10 μM.

5. The method as described in claim 3, characterized in that, The chemical small molecule composition consists of GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds and TGFβ inhibitors.

6. The method as described in any one of claims 1 or 3, characterized in that, The method includes adding a cell-based culture medium, or an organic solvent or physiological saline, to a small molecule composition to obtain a reprogramming culture medium or reagent for chemically inducing fibroblasts to reprogram into lung stem cells.

7. The method as described in claim 6, characterized in that, The method includes: (1) Preparation of concentrated reagent: Concentrated reagent is prepared by dissolving each component in an organic solvent or an aqueous solvent according to any of the small molecule compositions described above. (2) Obtaining reprogramming culture medium for reprogramming lung stem cells from fibroblasts: Dilute the concentrated reagent in step (1) into the cell basal culture medium containing 5-20% fetal bovine serum to obtain the reprogramming culture medium. (3) The weight ratio of each component of the small molecule combination is prepared according to the weight ratio of kilogram body weight, and a 100mg / ml concentrate is prepared. The concentrate is then diluted in 0.9% NaCl saline and mixed to obtain a chemically induced in vivo in situ fibroblast reprogramming into lung stem cells reprogramming nebulized inhalation reagent. (4) Inducing fibroblasts to reprogram into lung stem cells: Fibroblasts were suspended in a cell basal medium containing 5-20% fetal bovine serum, plated, and after the cells adhered, the medium was replaced with the reprogramming medium from step (2), cultured at 37°C, and the medium was changed every 2-4 days; the cells were passaged every 3-15 days. (5) Passaging of induced fibroblast reprogrammed lung stem cells: Discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37°C, 1-5 minutes, stop cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and passage at 1:1-1:3 to plate; culture in the reprogramming medium of (2), change the medium every 2-4 days; the digestion solution used includes trypsin, EDTA, acutase, and TrypleE; passage every 3-15 days; (6) Harvesting transformed lung stem cells: After reprogramming lung stem cells through (4) or (5) above and passing them through culture for 2-4 weeks, fibroblasts can be reprogrammed into lung stem cells, and transformed lung stem cells can be obtained. In the above method, no exogenous genes / transcription factors / microRNA genes, or their RNA, protein, or polypeptide inducing factors are introduced or used; no exogenous cytokines or growth factors are added; only chemical small molecule compositions are used to chemically induce fibroblasts to reprogram into lung stem cells.

8. The method as described in claim 7, characterized in that, In (1), the concentrated solution is prepared in the range of 1:50 to 1:10,000.

9. The method as described in claim 7, characterized in that, In (1), the organic solvent used in the preparation of the concentrated reagent includes dimethyl sulfoxide.

10. The method as described in claim 7, characterized in that, In (1), the aqueous solvent used in the preparation of the concentrated reagent includes: water, physiological saline, and phosphate buffer.

11. The method as described in claim 7, characterized in that, In (2), the reprogramming medium for reprogramming lung stem cells from fibroblasts is obtained by diluting the concentrated reagent in step (1) into a cell basal medium containing 5-20% fetal bovine serum to obtain the reprogramming medium; wherein the percentage content of each component of the medium fluctuates by 20%.

12. The method as described in any one of claims 1 or 3, characterized in that, The applications of chemically induced fibroblast reprogramming into lung stem cells include: reprogramming fibroblasts into lung stem cells to prepare transformed lung stem cells, providing a source of lung stem cells for clinical and research purposes.

13. The method as described in any one of claims 1 or 3, characterized in that, Applications of chemically induced fibroblast reprogramming into lung stem cells include: preparing reagents or culture media for inducing fibroblast reprogramming into lung stem cells.

14. The method as described in any one of claims 1 or 3, characterized in that, The fibroblasts mentioned are selected from human or mammalian fibroblasts.

15. The method as described in claim 14, characterized in that, The fibroblasts mentioned are selected from: skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, and pancreatic fibroblasts.

16. The method as described in claim 15, characterized in that, The fibroblasts mentioned are skin fibroblasts.

17. The method as claimed in any one of claims 1 or 3, characterized in that, Lung stem cells obtained through chemically induced fibroblast reprogramming include: type II alveolar cells, bronchioloalveolar stem cells, basal cells, distal airway stem cells, or a mixture thereof.

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