In vitro induction of respiratory tract organoids and uses thereof

Abstract

The present invention relates to the in vitro induction of respiratory organoids, and to the use of the obtained respiratory organoids.

Description

Technical Field

[0001] This invention belongs to the field of cell differentiation and regenerative medicine, specifically relating to the in vitro culture of organoids. Background Technology

[0002] In the field of respiratory diseases, many are intractable. Due to the lack of effective treatments besides surgical lung transplantation, the demand for lung regenerative medicine research, including cell models, organ models, and cell therapy, is increasing. To date, although protocols for generating lung organoids from human pluripotent stem cells (PSCs), tissue-resident adult stem cells (ASCs), embryonic lung, embryonic stem cells, and induced pluripotent stem cells (iPSCs) have been established, improvements to in vitro culture systems for lung organoids are still needed. In particular, the current differentiation and quantity efficiency of lung progenitor cells (NKX2.1) is very low, greatly limiting the application of lung organoids in cell therapy, drug screening, and other fields. Therefore, establishing rapid, large-scale, and highly NKX2.1-expressing lung progenitor cells, and a differentiation system based on these lung progenitor cells, has significant scientific and clinical translational implications. Summary of the Invention

[0003] This invention provides a method for differentiating embryonic stem cells into foregut endoderm, dissociating collected or separated foregut endoderm globules to form lung progenitor cells, and then differentiating the lung progenitor cells into mature airway lung organoids and alveolar organoids. This method can obtain higher yields of lung progenitor cells that specifically highly express NKX2.1, and is simple and easy to implement.

[0004] In a first aspect, the present invention provides a method for inducing the formation of lung progenitor cells, the method comprising the following steps: (1) Obtain the anterior foregut endoderm cell mass, and dissociate the anterior foregut endoderm cell mass into anterior foregut endoderm cells; and (2) Inducing the anterior foregut endoderm cells to form lung progenitor cells.

[0005] In some implementations, the purity of the foregut endoderm cells induced to form lung progenitor cells is above 95%.

[0006] Preferably, the purity of the foregut endoderm cells induced to form lung progenitor cells is above 97%.

[0007] In some embodiments, the lung progenitor cells express the markers NKX2.1 and / or SOX9 and / or SOX2.

[0008] In some embodiments, the foregut endoderm cell mass is obtained by inducing stem cells to form shaped endoderm cells, and by inducing shaped endoderm cells.

[0009] In some implementations, the stem cells are embryonic stem cells or adult stem cells.

[0010] In some embodiments, the embryonic stem cells are isolated embryonic stem cells, primary embryonic stem cells, or a cell line established from a population or cell line thereof.

[0011] In some implementations, the stem cells are pluripotent stem cells, such as embryonic stem cells or mesenchymal stem cells.

[0012] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). The iPSC cells can be commercially available cell lines or induced from donor cells, including one or more of the following: chorionic villus cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic tissues (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, hepatocytes, mesenchymal stem cells, peripheral blood cells, mammary epithelial cells, adipose stem cells, umbilical cord stroma, and placenta.

[0013] In some implementations, the stem cells are human stem cells or non-human mammalian stem cells.

[0014] In some embodiments, the step of inducing embryonic stem cells to form defined endoderm cells includes culturing the embryonic stem cells in a culture medium supplemented with Activin A and GSK3β signaling pathway inhibitors.

[0015] In some embodiments, the culture medium containing Activin A and GSK3β signaling pathway inhibitors is an embryonic stem cell culture medium to which Activin A and GSK3β signaling pathway inhibitors have been added.

[0016] In some implementations, the GSK3β signaling pathway inhibitors include, but are not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, and AZD1080.

[0017] Preferably, the GSK3β signaling pathway inhibitor is CHIR99021, which is a selective inhibitor of the liver glycogen synthesis kinase (GSK3β) receptor.

[0018] In some embodiments, the concentration of CHIR99021 in the culture medium is 0.1-3 μM.

[0019] In some implementations, stem cells are induced to form fixed endoderm cells within 3 days.

[0020] In some embodiments, embryonic stem cells are cultured on day 0 (D0) using a medium supplemented with Activin A and a GSK3β signaling pathway inhibitor; on day 1 (D1), they are cultured again using a medium supplemented with Activin A and a GSK3β signaling pathway inhibitor; and on day 2 (D2), they are cultured again using a medium supplemented with Activin A. Preferably, the GSK3β signaling pathway inhibitor is CHIR99021.

[0021] In some embodiments, the concentration of Activin A in the culture medium is 80-120 ng / mL. Preferably, the concentration of Activin A in the culture medium is 100 ng / mL.

[0022] In some embodiments, on day 0 (D0), the concentration of CHIR99021 in the culture medium is 2-4 μM. Preferably, the concentration of CHIR99021 in the culture medium is 3 μM.

[0023] In some implementations, MCDB131 medium is used as the basal medium for inducing stem cells to form morphological endoderm cells.

[0024] In some embodiments, the step of inducing the formation of a foregut endoderm cell mass from the shaped endoderm cells includes culturing embryonic stem cells in a culture medium supplemented with TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, FGF4 (fibroblast growth factor 4), Sonic Hedgehog (SHH) agonists, and GSK3β signaling pathway inhibitors.

[0025] In some embodiments, the culture medium containing TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, FGF4 (fibroblast growth factor 4), Sonic Hedgehog (SHH) agonists, and GSK3β signaling pathway inhibitors is a foregut endoderm cell culture medium supplemented with TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, FGF4 (fibroblast growth factor 4), Sonic Hedgehog (SHH) agonists, and GSK3β signaling pathway inhibitors.

[0026] In some implementations, the TGFβ signaling pathway inhibitor is, for example, LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, or SB431542.

[0027] Preferably, the TGFβ signaling pathway inhibitor is SB431542.

[0028] In some embodiments, the concentration of SB431542 is 5-15 μM. Preferably, the concentration of SB431542 is 10 μM.

[0029] In some embodiments, the BMP signaling pathway inhibitor is, for example, Noggin, Dorsomorphin, DMH1, or LDN-193189.

[0030] In some implementations, the BMP signaling pathway inhibitor is Noggin.

[0031] In some embodiments, the concentration of Noggin is 150 ng / mL to 250 ng / mL. Preferably, the concentration of Noggin is 200 ng / mL.

[0032] In some embodiments, the concentration of FGF4 is 400-600 ng / mL. Preferably, the concentration of FGF4 is 500 ng / mL.

[0033] In some implementations, the GSK3β signaling pathway inhibitors include, but are not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, and AZD1080.

[0034] Preferably, the GSK3β signaling pathway inhibitor is CHIR99021.

[0035] In some embodiments, the concentration of CHIR99021 in the culture medium is 1-3 μM, preferably 2 μM.

[0036] In some embodiments, the SHH agonist is SHH or SAG. Preferably, the SHH agonist is SAG.

[0037] In some embodiments, the concentration of SAG in the culture medium is 0.5-1.5 μM, preferably 1 μM.

[0038] In some implementations, the shaped endoderm cells are induced to form an anterior foregut endoderm cell mass within 5 days.

[0039] In some implementations, DMEM / F-12 medium or modified DMEM / F-12 medium is used as the basal medium for inducing the formation of anterior foregut endoderm cell masses from shaped endoderm cells.

[0040] In some embodiments, the anterior foregut endoderm cell mass is an anterior foregut endoderm globule.

[0041] In some implementations, 3D foregut endoderm globules appear on day 3 of the AFE stage. Free-floating foregut endoderm globules will appear on days 4–7 of the AFE stage.

[0042] In some implementations, the collected anterior foregut endoderm globules are dissociated into anterior foregut endoderm single cells.

[0043] The dissociation can be achieved by conventional methods, including but not limited to enzymatic, chemical, or mechanical methods. Enzymatic dissociation can be performed using, for example, accutase, dispersase, or trypsin.

[0044] In some implementations, single cells of the foregut endoderm are cultured in an extracellular matrix or hydrogel environment.

[0045] In some implementations, single cells of the foregut endoderm are induced into lung progenitor cells in Matrigel using lung progenitor cell (LPC) culture medium.

[0046] Cell culture is performed using Matrigel matrix or protein hydrogel, and specific growth factors are added to enable cells to proliferate and differentiate in a suspended and stable environment.

[0047] In some embodiments, the lung progenitor cell culture medium contains Notch signaling pathway inhibitors, BMP4, fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), GSK3β signaling pathway inhibitors, and retinoic acid (RA).

[0048] In some embodiments, the Notch signaling pathway inhibitor is DAPT (N-(N-(3,5-difluorophenylacetyl)-L-alanyl)-S-phenylglycine t-butyl ester) or dibenzozazepine (DBZ). Preferably, the Notch signaling pathway inhibitor is DAPT.

[0049] Preferably, the concentration of DAPT in the culture medium is 15-25 μM. More preferably, the concentration of DAPT in the culture medium is 20 μM.

[0050] BMP4 refers to bone morphogenetic protein 4, which can activate BMP4 receptor signaling.

[0051] In some embodiments, the concentration of BMP4 in the lung progenitor cell culture medium is 15-25 ng / mL, preferably 20 ng / mL.

[0052] In some embodiments, the concentrations of FGF7 and / or FGF10 in the lung progenitor cell culture medium are 5-15 ng / mL, and preferably, the concentrations of FGF7 and / or FGF10 are 10 ng / mL.

[0053] In some implementations, the GSK3β signaling pathway inhibitors include, but are not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, and AZD1080.

[0054] Preferably, the GSK3β signaling pathway inhibitor is CHIR99021, which is a selective inhibitor of the liver glycogen synthesis kinase (GSK3β) receptor.

[0055] In some embodiments, the concentration of CHIR99021 in the lung progenitor cell culture medium is 1-5 μM, preferably 3 μM. In some embodiments, the concentration of RA in the lung progenitor cell culture medium is 40-60 nM, preferably, the concentration of RA in the lung progenitor cell culture medium is 50 nM.

[0056] The RA (vitamin A) mentioned is a metabolite of vitamin A. It can bind to and activate retinoic acid receptors, induce changes in gene expression, and lead to the inhibition of cell differentiation, cell proliferation, and tumorigenesis.

[0057] In some implementations, foregut endoderm cells are induced to form lung progenitor cells within 7 days.

[0058] In some implementations, DMEM / F-12 medium or modified DMEM / F-12 medium is used as the basal medium for lung progenitor cells.

[0059] In a second aspect, the present invention provides lung progenitor cells obtained by the method of the first aspect.

[0060] In some implementations, the purity of the lung progenitor cells is above 97%.

[0061] In some embodiments, the lung progenitor cells express the markers NKX2.1 and / or SOX9 and / or SOX2.

[0062] In some embodiments, the lung progenitor cells have an onion ring morphology.

[0063] In a third aspect, the present invention provides a method for inducing lung progenitor cells obtained by the method of the first aspect to form respiratory organoids, the method comprising culturing the lung progenitor cells in a culture medium containing Dexamethasone, 8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX) and FGF7.

[0064] In some embodiments, the culture medium also contains an inhibitor of the TGFβ signaling pathway.

[0065] In some implementations, the TGFβ signaling pathway inhibitor is, for example, LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, or SB431542.

[0066] Preferably, the TGFβ signaling pathway inhibitor is SB431542.

[0067] In some embodiments, the concentration of SB431542 is 5-15 μM. Preferably, the concentration of SB431542 is 10 μM.

[0068] In some embodiments, the culture medium also contains GSK3β signaling pathway inhibitors.

[0069] In some implementations, the GSK3β signaling pathway inhibitors include, but are not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, and AZD1080.

[0070] Preferably, the GSK3β signaling pathway inhibitor is CHIR99021.

[0071] In some embodiments, the concentration of CHIR99021 in the culture medium is 2-4 μM, preferably 3 μM.

[0072] In some implementations, the respiratory organoids are lung airway organoids.

[0073] In some implementations, suspension culture is used to obtain airway organoids.

[0074] In some implementations, the airway organoid is an everted airway organoid.

[0075] In some implementations, the lung airway organoids express the biomarkers TP63, SCGB1A1, CHGA, FOXJ1, and MUC5AC.

[0076] In some implementations, the respiratory organoids are alveolar organoids.

[0077] In some embodiments, the alveolar organoids express the biomarkers HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC, and / or SLC34A2.

[0078] In some implementations, lung progenitor cells are induced to form respiratory organoids within 30 days and can be maintained in culture for a long period of time.

[0079] In some implementations, DMEM / F-12 medium or modified DMEM / F-12 medium is used as the basal medium.

[0080] Fourthly, the present invention provides respiratory organoids obtained by the method of the third aspect.

[0081] In some implementations, the respiratory organoids are lung airway organoids.

[0082] In some embodiments, the lung airway organoids express the biomarkers TP63, SCGB1A1, CHGA, FOXJ1, and / or MUC5AC.

[0083] In some implementations, the respiratory organoids are alveolar organoids.

[0084] In some embodiments, the alveolar organoids express the biomarkers HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC, and / or SLC34A2.

[0085] In some implementations, the lung airway organoids are everted airway organoids.

[0086] Fifthly, the present invention provides the use of lung progenitor cells obtained by the method of the first aspect, lung progenitor cells of the second aspect, respiratory organoids obtained by the method of the third aspect, or respiratory organoids of the fourth aspect in the preparation of cells or organoids for treating lung-related diseases, or in the preparation of cell models or organoid models for studying lung-related diseases.

[0087] In a sixth aspect, the present invention provides a pharmaceutical formulation comprising lung progenitor cells obtained by the method of the first aspect, lung progenitor cells of the second aspect, respiratory organoids obtained by the method of the third aspect, or respiratory organoids of the fourth aspect.

[0088] In a seventh aspect, the present invention provides a method for screening therapeutic or preventative drugs, wherein the method includes the step of contacting a respiratory organoid obtained by the method of the third aspect or a respiratory organoid of the fourth aspect with a candidate molecule.

[0089] This invention establishes a rapid (<10 days) high-purity (98.3%) NKX2.1 lung progenitor cell differentiation system by dissociating the foregut endoderm microspheres and maintaining cell viability, and further combining the effects of signaling pathways such as BMP, FGF, and Wnt.

[0090] The obtained lung progenitor cells were further differentiated to obtain airway and alveolar organoids with mature physiological functions (including ciliary movement, mucus secretion, ion channels, viral infection, etc.).

[0091] The high-throughput differentiation method of this invention will greatly improve the translational medicine applications of lung organoids, such as disease models, drug development, and cell therapy.

[0092] Definitions: Human life begins with the fusion of sperm and egg to form a fertilized egg (E0, day 0 of the embryonic period). The fertilized egg undergoes cleavage to form a blastocyst, which implants into the mother's uterus around E7 for further development. Starting at E14, the embryo undergoes gastrulation, with large-scale directional migration of cells in the posterior part of the embryo, forming primitive streaks. These primitive streaks further differentiate into mesoderm and definitive endoderm, while cells in the anterior part of the embryo differentiate into ectoderm. Based on this, the embryo develops into a three-layered embryo with endoderm, mesoderm, and ectoderm. Through complex signaling pathway regulation, the three-layered embryo further forms the primordia of various organs, ultimately forming the various organs of all systems in our body, including the nervous, digestive, respiratory, cardiovascular, and urinary / reproductive systems.

[0093] The human respiratory tract consists of continuously branching airways and terminal alveoli. Airways are the passageways for air to enter and exit the lungs, while alveoli are the functional units for gas exchange. Airways and alveoli are covered by airway epithelium and alveolar epithelium, respectively.

[0094] In this invention, "foregut endoderm" refers to the endoderm that forms the anterior portion of the liver endoderm. "Foregut endoderm" includes, for example, the pharyngeal endoderm and other more highly differentiated endoderm cell populations, and contains multiple cell types exhibiting different expression patterns of molecular markers. "Foregut endoderm" will develop into various tissues, such as the tonsils, tympanic membrane, thyroid gland, parathyroid glands, thymus, trachea, esophagus, stomach, lungs, larynx, and / or pharynx.

[0095] "Definitive endoderm cells (DE)" refer to cells that express one or more markers of a defined endoderm lineage. These markers include, but are not limited to, CXCR4, SOX17, GATA-4, FOXA2, AFP, CER1, C-KIT, EPCAM, SNAI1, GSC, E-Cad, and / or N-Cad. The defined endoderm is functionally defined by one or more cells that further differentiate from endoderm tissues. This can include the lung, thyroid, liver, pancreas, or intestine.

[0096] "Airway epithelial cells" refers to the layer of epithelial cells that line the large airways (bronchioles) and small airways (bronchioles). Airway epithelial cells include ciliated, secretory, basal, and columnar cell types.

[0097] Lung progenitor cells have two important markers: NKX2.1 and / or SOX9 and / or SOX2.

[0098] Induced pluripotent stem cells (iPS cells) are pluripotent stem cells that have been reprogrammed into mature somatic cells by introducing a series of inducing factors, resulting in cells with characteristics similar to embryonic stem cells. iPS cells circumvent the ethical constraints of extracting stem cells from human embryos and avoid the risks of immune rejection in allogeneic transplantation. They also have significant potential value in establishing disease models for various organoids, including those of the brain, intestine, liver, pancreas, kidney, and lung, as well as in drug screening and development. iPS cells are similar to naturally occurring pluripotent stem cells. They can differentiate into various cell types, including but not limited to stereotyped endoderm cells, foregut endoderm cells, and airway epithelial cells.

[0099] "Organoids" are three-dimensional, multicellular aggregates derived from stem cells that differentiate and self-organize, encapsulating the structural features and cell-cell interactions of mature tissues. These organoids are three-dimensional aggregates of one or more cell types that mimic the surface appearance, structure, or function of tissues or organs.

[0100] "Induction" or "induce" relates to processes or behaviors that cause a specific effect on a cell's phenotype. Such effects can take the form of causing a change in phenotype, such as differentiating to another cell phenotype; or they can take the form of maintaining a cell in a specific cellular state, such as preventing dedifferentiation or promoting cell survival.

[0101] Pluripotent stem cells are multipotent cells with the ability to self-renew and self-replicate, and can differentiate into various cell types under certain conditions.

[0102] The terms "precursor cell," "progenitor cell," and "stem cell" are used interchangeably in the art, and in this invention refer to pluripotent or lineage-determined progenitor cells that have the potential to undergo an unlimited number of mitotic divisions to renew themselves or differentiate into daughter cells of a desired cell type. Compared to pluripotent stem cells, lineage-determined progenitor cells are generally considered incapable of generating numerous cell types that are phenotypically different from each other. Instead, progenitor cells will be able to generate one or possibly two lineage-determined cell types.

[0103] In this invention, "differentiation" refers to the process by which less specialized cells, such as stem cells or induced pluripotent stem cells, become more specialized cell types so that they become specific lineages, including but not limited to certain progenitor cells and more specialized somatic cells. The conditions for stem cell differentiation are well known in the art.

[0104] In this invention, "differentiation medium" refers to a cell growth medium containing or lacking certain additives, so that when cultured in the medium, stem cells, induced pluripotent cells, or incompletely differentiated cells develop into differentiated cells or cells that exhibit some or all of the characteristics of cells that are more differentiated than stem cells, induced pluripotent cells, or other similar cells.

[0105] The lung-related diseases mentioned include, but are not limited to, acute respiratory distress syndrome, tuberculosis, cough, bronchial asthma, cystic fibrosis, airway hyperresponsiveness, cough with increased airway hyperresponsiveness (cough caused by bronchitis, flu syndrome, asthma, obstructive pulmonary disease, etc.), influenza syndrome, tuberculosis, asthma (inflammatory cell infiltration of the airways, increased airway hyperresponsiveness, bronchoconstriction, excessive mucus secretion, etc.), chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, reversible airway obstruction, adult respiratory syndrome, bronchopulmonary dysplasia, airway obstruction, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis, bronchiectasis, occupational asthma, reactive airway dysfunction syndrome, interstitial lung disease, parasitic lung disease, etc.

[0106] In this application, "dissociation" refers to the breaking down of cell aggregates or clusters into smaller aggregates or single-cell suspensions. Dissociation of cell aggregates can be achieved by conventional methods, including but not limited to enzymatic, chemical, or mechanical methods. Enzymatic dissociation can be performed using, for example, accutase, dispersase, or trypsin.

[0107] The DMEM / F12 can also be replaced by one or more of the following: William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium.

[0108] The DMEM / F12 culture medium contains a 1:1 mixture of DMEM and Ham's F-12.

[0109] The DMEM / F12 comprises a modified DMEM / F-12 culture medium with its composition adjusted according to the actual application.

[0110] The DMEM / F12 modified culture medium includes, but is not limited to, DMEM-low-glucose-pyruvate-glutamine-free-phenol red-free, and DMEM / F-12-GlutaMAX. TM , DMEM / F-12-HEPES (DMEM / F-12with HEPES), DMEM-lowglucose-pyruvate-HEPES.

[0111] The DMEM / F12 is a DMEM / F12+HEPES medium containing L-glutamine, HEPES, and phenol red.

[0112] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products. Attached Figure Description

[0113] Figure 1 The flowchart of the differentiation process for inducing lung progenitor cells according to this application is shown.

[0114] Figure 2 The morphology of human embryonic stem cells differentiated into morphologically defined endoderm cells and the expression of related markers are shown. Among them, a shows the morphology of the morphologically defined endoderm on day 1, b shows the morphology of the morphologically defined endoderm on day 3, and c shows the expression of markers SOX2, FoxA2, ​​and Sox17.

[0115] Figure 3 The morphology of the 2D layer and the free-floating 3D anterior foregut endoderm globules in the AFE stage, as well as the expression of related markers, are shown. Image a shows the 2D cell layer and 3D globules of the anterior foregut endoderm on day 4, and image b shows the expression of markers SOX2 and FoxA2.

[0116] Figure 4 The morphology of lung progenitor cells and the expression of related markers are shown. Image a shows the "onion ring" morphology of lung progenitor cells on day 7, and image b shows the expression of lung progenitor cell-related markers.

[0117] Figure 5 Example 1 shows that high purity (98.3%) NKX2.1 lung progenitor cells were obtained.

[0118] Figure 6 The results show the lung cells obtained after 7 days of culture in the isolated group and the control group.

[0119] Figure 7 The morphology of lung organoids and the expression of related markers are shown. Among them, a shows the morphology of airway organoids after 30 days of induction, b shows the morphology of alveolar organoids after 30 days of induction, c shows the expression of airway organoid-related markers, and d shows the expression of alveolar organoid-related markers; the SC group is the dissociation group, and the 3D group is the control group.

[0120] Figure 8 The staining levels of airway organoids were shown.

[0121] Figure 9 The staining levels of alveolar organoids were shown.

[0122] Figure 10 The morphology and related marker expression of everted airway organoids are shown. In image a, the morphology of the everted airway organoids is shown; and in image b, the expression of related markers of everted airway organoids is shown. Detailed Implementation

[0123] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Unless otherwise specified, the techniques involved in the following embodiments are conventional techniques well-known to those skilled in the art in various fields such as cell biology, biochemistry, and molecular biology.

[0124] The reagents and instruments used in the following experiments are as follows: Instruments and equipment: Biosafety cabinet (ThermoFisher, 1389), inverted fluorescence microscope (Nikon, TS2-FL), CO2 incubator (ThermoFisher, HERAcell150i), centrifuge (Xiangyi, L600-A), cryostat (Reward, FS800), laser confocal microscope (Carl Zeiss LSM980), qPCR instrument (Bio-rad, CFX-96), PCR instrument (Bio-rad, T-100), flow cytometer (Agilent Novocyte Adcanteon), medical low-temperature freezer (Haier DW-25L262). Reagents: MCDB131 (Thermo Fisher, 10372019), Advanced DMEM / F12 (Thermo Fisher, 12634010), Accutase (Stemcell, 7920), EDTA (invitrogen, AM9261), mTeSR1 (Stemcell, 85850), Matrigel (Corning, 354277), DPBS (Thermo Fisher, 14190250), Glucose (Sigma, G7528), NaHCO3 (Sigma, S6014), BSA (Proliant, 68700), Ascorbic acid (Sigma, A4544), GlutaMAX (invitrogen, 35050079), Penicillin-Streptomycin (Thermo Fisher, 15140122), ITS-X (invitrogen, 51500056), Activin A (MCE, HY-P70311), CHIR99021 (MCE, HY-10182), FGF7 (stemcell, 78046.2), FGF4 (MCE, HY-P7014), FGF10 (MCE, HY-P70695), NOGGIN (MCE, HY-P7051A), BMP4 (MCE, H Y-P7007), SB43152 (MCE, HY-10431), SAG (MCE, HY-12848), Y27632 (MCE, HY-10583), Growth Factor Reduced (GFR) Matrigel (Corning 354230), RA Retinoic Acid (Sigma, R2625), DAPT (MCE, HY-13027), Dexamethasone (MCE, HY-14648), 8 Br-cAMP (MCE, HY-12306), IBMX (MCE, HY-12318), TB Green Premix Ex Taq (TAKARA, RR820), MaximaHMinus Reverse Transcriptase (Thermo Fisher, EP0752), Random (Thermo Fisher, SO142), Oligo (Thermo Fisher, SO132), dNTPs (Thermo Fisher, RO192), RiboLock RNase (Thermo Fisher, EO0381), RNA extraction kit RNeasy Mini Kit (AXGEN, 74106), RNase-FreeDNase Set 1 (AXGEN, 79256), 96-well qPCR plate (Bio-rad, HSP9655), 4% paraformaldehyde fixative (BBI, E672002-0500), OCT frozen section embedding medium (Biosharp, BL557A), Triton X-100 (Diamond, A110694-0500), QuickBlock™ immunostaining blocking solution (Beyotime, P0260), primary antibody dilution buffer for immunofluorescence staining (Beyotime, P0103), secondary antibody dilution buffer for immunofluorescence staining (Beyotime, P0108), Donkey serum (Abcam, ab63507), anti-quenching mounting medium (ACMEC, AS2100), SOX2 (Santa Cruz Biotechnology, sc-365823), Ki67 (Abcam, ab92742), SOX9 (Abcam, ab76997), NKX2.1 (Abcam, ab76013), P63 (Abcam, ab124762), MUC5AC (Abcam, ab3649), Acetylated Tubulin (ACTTUB) (Sigma-Aldrich, T7451), Aquaporin 5 (AQP5) (Abcam, ab92320), Sufactant Protein B (SFTPB) (SantaCruz Biotechnology, sc-133143), E-Cadherin (ECAD) (R&D Systems, AF748), Donkeyanti-Rabbit Secondary Antibody Alexa Fluor 488 (Invitrogen, A21206), Donkeyanti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor488 (Invitrogen, A21202), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 546 (Invitrogen, A10040), Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 546 (Invitrogen, A10036), Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody AlexaFluor™ 647 (Invitrogen, A21447), Hoechst 33342 (Thermo Fisher, 62249), Alexa Fluor™ 647 phalloidin (Thermo Fisher, A22287), APC anti-humanCD47 (BioLegend, 323123), PEanti-human CD26 (BioLegend, 302706). .

[0125] The cell culture conditions used in this invention are 37°C and 5% CO2, and will not be described further hereafter. All reagents and instruments used in this invention are commercially available.

[0126] Detection method: Differentiated cell samples were harvested on the last day of each differentiation stage. The expression levels of characteristic gene mRNA were detected by qPCR, the expression levels of characteristic gene protein were detected by immunofluorescence, and the purity of lung progenitor cells NKX2.1 was detected by flow cytometry.

[0127] The procedures for RNA extraction, RNA transcription, qPCR, immunofluorescence staining, and flow cytometry in this invention will be described below. All experimental methods involved in this invention are the same as those described below, and these experiments will be mentioned repeatedly; specific procedures will not be described in detail hereafter.

[0128] (I) Harvest RNA and perform qPCR a. RNA extraction: All reagents used in the RNA extraction process are included in the commercial RNA extraction kit. Discard the culture medium in the culture plate, wash once with DPBS, and discard the washing solution. Add 500 μL of RlypLE digestion buffer to each well of a 24-well plate and incubate for about 7 minutes. Under a microscope, when about 90% of the cells appear bright and transparent, add 1 mL of this stage of culture medium to stop digestion. Mix well by pipetting, collect the cells in a 1.5 mL centrifuge tube, and centrifuge at 1450 rpm for 5 minutes. Discard the supernatant, add 1 mL of DPBS, and mix gently. Centrifuge at 1450 rpm for 5 minutes and discard the supernatant. Add 600 μL of lysis buffer (Buffer RLT + β-ME), invert and mix 4-6 times, and let stand for 5 minutes.

[0129] Add an equal volume (600 μl) of 70% anhydrous ethanol to the above sample and mix thoroughly.

[0130] Transfer the liquid from the previous step into the filter column of the RNA extraction kit and centrifuge at 12,000 rpm for 1 minute. Transfer 600 μl at a time, in two separate transfers.

[0131] Discard the filtrate from the previous centrifugation step, add 350 μl of washing buffer RW1 to the filter column, and centrifuge at 12000 rpm for 1 minute.

[0132] Discard the filtrate from the previous centrifugation step, add 80 μL of DNA digestion solution (10 μL Nase I + 70 μL buffer from the RNA extraction kit) to the filter column, let stand at room temperature for 20 minutes, and digest and extract DNA from the cells.

[0133] After standing, add 350 μL of LW1 washing solution, centrifuge at 12000 rpm for 1 minute, and discard the filtrate.

[0134] Add 700 μL LW1 washing solution to the filter column, centrifuge at 12000 rpm for 1 minute, and discard the filtrate.

[0135] Add 500 μL LW2 washing solution to the filter column, centrifuge at 12000 rpm for 1 minute, and discard the filtrate.

[0136] Add 500 μL LW2 washing solution to the filter column, centrifuge at 12000 rpm for 1 minute, and discard the filtrate.

[0137] Place the filter column back into the collection tube provided with the kit and centrifuge at 12,000 rpm for 2 minutes. Transfer the filter column to a new collection tube.

[0138] Add 60 μL of LNase Free H2O to the collection tube and let it stand for 5 minutes.

[0139] Place the filter column together with the new collection tube and centrifuge at 12,000 rpm for 3 minutes. The liquid in the collection tube is the extracted RNA solution.

[0140] b. cDNA transcription Add the RNA harvested in the previous step to 0.2 mL centrifuge tubes in the order shown in Table 1 below.

[0141] Table 1 Mix gently, centrifuge briefly, and then heat at 65°C for 5 minutes on a PCR instrument. After heating, centrifuge briefly again and place on ice for 3 minutes.

[0142] Add the primers from Table 2 below to the 0.2 mL centrifuge tubes listed above in sequence (this step should be performed on ice).

[0143] Table 2 The mixed liquid was heated on a PCR instrument with the following reaction program: 25°C for 10 minutes, 50°C for 1 hour, and 85°C for 5 minutes.

[0144] The resulting product is the extracted RNA reverse transcription cDNA product.

[0145] c. qPCR The obtained cDNA was prepared on ice according to the order shown in Table 3 below: Table 3 After the reaction solution was prepared, the qPCR reaction was performed on the qPCR instrument. The reaction program was 95℃ for 30s for pre-denaturation, 95℃ for 5s, 60℃ for 30s, for 40 cycles.

[0146] The qPCR reaction system and procedure were performed in accordance with the (TAKARA, RR820) reagent instructions.

[0147] In this experiment, the data processing method for qPCR was the ▲▲CT method, and the criteria for differential analysis were: This indicates a significant difference, p < 0.05; This indicates a highly significant difference, p < 0.001; This indicates that the difference is extremely significant, p < 0.0001; This indicates that the difference is extremely significant, p < 0.00001. (II) Immunofluorescence staining experiment The main purpose of immunofluorescence staining is to reveal characteristic proteins through fluorescence by means of specific antigen-antibody binding.

[0148] The specific steps for fluorescent staining are as follows: 1. Collect organoids by natural sedimentation or low-speed centrifugation (1450 rpm, 5 minutes), add 2 mL of DPBS, mix gently, rinse once, let stand for 5 minutes, and discard the supernatant. Add 2 mL of 4% PFA (paraformaldehyde fixative), and fix at 4°C for 30 minutes. After standing, discard the supernatant, add 2 mL of DPBS to the sample, and store at 4°C for later use.

[0149] 2. Discard the DPBS in the sample, add 2 mL of immunostaining blocking solution, incubate at room temperature for 30 minutes, allow to settle naturally or centrifuge at low speed (1450 rpm, 5 minutes) and discard the supernatant. Incubate with primary antibody overnight at 4°C.

[0150] 3. Remove primary antibody by natural sedimentation or low-speed centrifugation (1450 rpm, 5 minutes). Wash 1-2 times with DPBS, 5 minutes each time. Add secondary antibody. Incubate with secondary antibody at room temperature in the dark for 1 hour. After incubation, wash 3 times with DPBS, 5 minutes each time. Immunofluorescence staining experiment completed.

[0151] (III) Flow cytometry experiment The main purpose of flow cytometry experiments is to verify the purity of LPC stage lung progenitor cells NKX2.1.

[0152] The specific steps of flow cytometry experiments are as follows: 1. Collect LPCd7-d10 organoids and allow them to settle naturally or centrifuge at low speed (1450 rpm, 5 minutes). Add 2 mL of PBS, mix well by pipetting, rinse once, let stand for 5 minutes, and discard the supernatant. Add Accutase and digest at 37°C for 10 minutes.

[0153] 2. Observe under a microscope to digest the sample into single cells. If single cells are not formed, the digestion time can be extended.

[0154] 3. After digesting the organoids into single cells, add 3-5 mL of DPBS to stop the digestion, and centrifuge at low speed (1450 rpm, 5 minutes) to remove the digestion fluid.

[0155] 4. Resuspend the cells in 2 mL of DPBS, divide them into a negative control group and a CD26+CD47 staining group, and prepare the antibody according to the antibody instructions.

[0156] 5. Centrifuge at low speed (1450 rpm, 5 minutes) to remove DPBS. Add DPBS to the control group to resuspend the cells, and add the prepared antibody to the staining group. Incubate at 4°C for 30 minutes.

[0157] 6. Wash the cells after antibody incubation with DPBS, centrifuge at 1450 rpm for 5 min and discard the supernatant.

[0158] 7. Resuspend the cells in a small amount of DPBS (500μL-1mL), pass through a flow cytometry tube, and complete the flow cytometry experiment.

[0159] Example 1: Differentiation of human embryonic stem cells into lung progenitor cells and lung organoids This embodiment describes a method for differentiating human embryonic stem cells (hereinafter referred to as stem cells) into lung progenitor cells and lung organoids. Before stem cells enter differentiation, the basic stem cell culture process, such as stem cell passaging and pre-differentiation platelet placement, is first described, followed by a description of stem cell differentiation. The human embryonic stem cells are isolated or obtained from human embryos that have not developed in vivo within 14 days of fertilization.

[0160] Unless otherwise specified, all 6-well and 24-well plates used for stem cell culture in this embodiment require pre-incubation with Matrigel before use. The specific procedure is as follows: Mix commercial Matrigel with DMEM F12 according to the manufacturer's dilution factor, then incubate the plates at 37°C for 2 hours using 1 mL of the mixture per well in 6-well plates and 300 μL per well in 24-well plates. Passaging: Stem cells are cultured in 6-well plates using mTesR1 as the medium, with 2 mL of medium per well, and the medium is changed every 24 hours. When the cell density in the 6-well plates reaches 80-90%, discard the culture medium, wash once with DPBS, discard the washing solution, add 1 mL of 0.5 mM EDTA digestion solution to each well, and digest at room temperature for 5-10 minutes. Under a microscope, when 80-90% of the cells are shiny, discard the digestion solution, gently wash the cells off with mTesR1, and seed them evenly into the next 6-well plate at a 1:10 ratio. This completes the stem cell passage. After 4-5 days, the stem cell density reaches 80-90%, and the next passage or plating can be carried out.

[0161] Plate Formation: Stem cells were cultured in 6-well plates using mTesR1 as the medium, 2 mL per well, and the medium was changed every 24 hours. When the stem cell density reached approximately 80-90% confluence in the 6-well plates, the culture medium was discarded, and the cells were washed once with DPBS, then the washing solution was discarded. 500 μL of Accutase was added to each well, and the cells were incubated at 37°C for 3-5 minutes. Under a microscope, when 80-90% of the cells appeared shiny, 2 mL of DPBS was added to each well to stop the digestion. The cells were then pipetted and collected in 15 mL centrifuge tubes and centrifuged at 1450 rpm for 5 minutes. The supernatant was discarded, and 1 mL of mTesR1 (containing 10 μM Y27632) was added and the cells were pipetted and counted. Cells were cultured at a density of 0.8 × 10⁶ cells / well. 5 Each cell was evenly seeded into a 24-well plate, and after 48-72 hours, the cells grew to about 90% and entered the differentiation stage.

[0162] See Figure 1 The cells differentiate from human embryonic stem cells into a large number of lung progenitor cells that highly express NKX2.1. The specific implementation steps are as follows: 1) Obtain endoderm spheres from the anterior foregut 1.1) Human embryonic stem cells differentiate into well-defined endoderm cells DE culture medium differentiates human embryonic stem cells into defined endoderm cells.

[0163] The DE medium consisted of: MCDB131 medium as a base, 5 mg / mL BSA, 10-20 mM glucose, 1.5 mg / mL NaHCO3, 0.25 mM Vitamin C, 1% GlutaMAX, 1% penicillin-streptomycin, 100 ng / mL Activin A, 0.1-3 μM ChIR99021, and insulin-transferrin-seleno-aminoethanol (ITS-X) (1:50000). The cells were cultured in DE medium for 3 days, with the medium changed every 24 hours. The morphology of human embryonic stem cells differentiating into well-defined endoderm cells is as follows... Figure 2 As shown in ab, the relevant marker expressions are as follows: Figure 2 As shown in c.

[0164] 1.2) The final endoderm cells differentiate into anterior foregut endoderm globules. The contents of AFE medium are: Using Advanced DMEM / F12 medium as the base, 10 mM HEPES, N2 (1:100), B27 (1:50), 1% GlutaMAX, 1% penicillin and streptomycin, 10 μM SB431542, 200 ng / mL Noggin, 1 μM SAG, 50-500 ng / mL FGF4, and 2 μM ChIR99021, the medium was completely changed every 24 hours, and the spheroids were observed once according to the following method. The morphology of the 2D layer and the free-floating 3D anterior foregut endoderm spheroids in the AFE stage is as follows. Figure 3 As shown in a, the expression of markers related to 3D anterior foregut endodermal globules is as follows: Figure 3 As shown in b.

[0165] a) Observe the monolayer under a microscope. 3D anterior foregut endoderm globules may be visible as early as day 2-3 of the AFE stage. Free-floating anterior foregut endoderm globules will appear on days 4-7 of the AFE stage.

[0166] b) Using a 1 mL pipette, collect the culture supernatant and the floating 3D foregut endoderm microspheres into a 15 mL centrifuge tube.

[0167] Compared with existing technologies that directly perform 2D enzymatic digestion or directly inoculate 3D microspheres after 7 days of induction with DE and AFE, this application first collects these suspended or floating foregut endoderm microspheres and then performs enzymatic digestion, resulting in higher yield and higher purity of the lung progenitor cells obtained in the final culture.

[0168] 2) Obtain lung progenitor cells 2.1) Obtain single cells of endoderm globules from the foregut. Control group: No dissociation, direct inoculation with anterior foregut endoderm globules. Rinse the collected pellets from step 1.2)b) with DPBS, allow them to settle naturally for 5-10 minutes, and carefully aspirate the supernatant. Add an appropriate volume of GFR Matrigel using a pre-cooled (-20℃) pipette tip and gently pipette to mix.

[0169] Dissociation group: Dissociation of endoderm globules in the anterior foregut Rinse the collected microspheres from step 1.2)b) with DPBS, digest with Accutase at 37°C for 5 minutes, gently pipette to dissociate the microspheres into single cells, stop digestion with 3 mL of DPBS, and gently mix. Centrifuge at 1450 rpm for 5 minutes and carefully aspirate the supernatant. Using a pre-cooled (-20°C) pipette tip, add an appropriate volume of GFR Matrigel and gently pipette to mix.

[0170] 2.2) Inoculate single cells of anterior foregut endoderm globules Using a pre-cooled (-20℃) pipette tip, gently and evenly distribute GFR Matrigel into the center of each well of a 24-well culture plate, with each droplet volume preferably between 25μL and 30μL. Incubate at 37℃ for 10 minutes to solidify the GFR Matrigel, then add LPC medium containing 10μM Y27632.

[0171] The contents of LPC medium are: Based on Advanced DMEM / F12 medium, the following solutions were added: 10 mM HEPES, N2 (1:100), B27 (1:50), 1% GlutaMAX, 1% penicillin and streptomycin, 20 μM DAPT, 20 ng / mL BMP4, 10 ng / mL FGF7, 10 ng / mL FGF10, 3 μM hIR99021, and 50 nM RA. The culture medium was changed every 2-3 days for a total of 7 days. The morphology of lung progenitor cells after 7 days of dissociation was as follows: Figure 4 As shown in a, the expression of the relevant markers is as follows: Figure 4 As shown in b. The above method yielded high-purity (98.3%) NKX2.1 lung progenitor cells (see Figure b). Figure 5 ).

[0172] according to Figure 6 The results showed that after 7 days of culture, the lung cell structure with an "onion ring" shape in the dissociation group was nearly 5 times larger than that in the control group and the dissociation group. This indicates that the expansion rate of lung progenitor cells was significantly increased after dissociation.

[0173] 3) Obtaining lung organoids 3.1) Lung progenitor cells were induced into lung airway organoids using airway organoid (HAWO) culture medium. The contents of HAWO medium are as follows: Based on Advanced DMEM / F12 medium, the following solutions were added: 2.5 mg / mL BSA, 10 mM HEPES, B27 (1:100), 1% GlutaMAX, 1% penicillin-streptomycin, insulin-transferrin-selenoethanolamine (ITS-X) (1:1000), 50 nM Mexamethasone, 100 nM 8Br-cAMP, 100 nM 3-isobutyl-1-methylxanthine, and 10 ng / mL FGF7. The culture medium was changed every 48-72 hours for a total of 30 days, and the culture could be maintained long-term. The morphology of the airway organoids after 30 days of induction is shown below. Figure 7 As shown in a, the expression of markers detected by qPCR is as follows: Figure 7 As shown in c, staining level identification is as follows: Figure 8 As shown.

[0174] 3.2) Lung progenitor cells were induced into alveolar organoids using alveolar organoid (HALO) medium. The contents of HALO medium are: Based on Advanced DMEM / F12 medium, the following solutions were added: 2.5 mg / mL BSA, 10 mM HEPES, B27 (1:100), 1% GlutaMAX, 1% penicillin-streptomycin, insulin-transferrin-selenoethanolamine (ITS-X) (1:1000), 50 nM Mexamethasone, 100 nM 8Br-cAMP, 100 nM 3-isobutyl-1-methylxanthine, 10 ng / mL FGF7, 3 μM ChIR99021, and 10 μM SB431542. The culture medium was changed every 48-72 hours for a total of 30 days, and long-term culture was possible. The morphology of alveolar organoids after 30 days of induction was compared with... Figure 7 As shown in b, the expression of markers detected by qPCR is as follows: Figure 7 As shown in d, staining level identification is as follows: Figure 9 As shown.

[0175] according to Figure 7 The results also show that the dissociation group has superiority in the relevant gene levels during the later stages of organoid induced differentiation and maturation.

[0176] Example 2: Everted Airway Organoid The normal airway organoid culture method is as follows: lung progenitor cells are induced into lung airway organoids using HAWO medium.

[0177] The contents of HAWO medium are as follows: Using Advanced DMEM / F12 medium as the base, 2.5 mg / mL BSA, 10 mM HEPES, B27 (1:100), 1% GlutaMAX, 1% penicillin-streptomycin, insulin-transferrin-selenoethanolamine (ITS-X) (1:1000), 50 nM Mexamethasone, 100 nM 8Br-cAMP, 100 nM 3-isobutyl-1-methylxanthine, and 10 ng / mL FGF7, the culture medium was changed every 48-72 hours for a total of 30 days, and the culture could be maintained for a long time.

[0178] Culture method for everted airway organoids: Change the culture medium every 48-72 hours for a total of two weeks. Afterward, detach the organoids from Matrigel droplets, wash once with DPBS, and suspend them in low-adhesion six-well plates. Continue culture using HAWO medium, changing the culture medium every 48-72 hours for long-term maintenance. The only difference is whether the organoids are cultured in Matrigel droplets or in suspension; otherwise, it is the same as in Example 1. Morphology and marker expression of everted airway organoids are as follows: Figure 10 As shown.

[0179] In this embodiment, airway organoids can spontaneously evert after suspension culture to form everted airway organoids. The method of this application can obtain airway organoids with everted airway epithelium, which can be used to construct in vitro three-dimensional models of lung organoids. This is of great significance for pathogen infection modeling, high-throughput drug screening, research on lung diseases such as chronic obstructive pulmonary disease, interstitial lung disease, inflammatory lung disease, asthma, and emphysema, as well as lung function studies, including functional studies of alveoli, airways, and lung lobes.

Claims

1. A method for inducing the formation of lung progenitor cells, the method comprising the following steps: (1) Obtaining anterior foregut endoderm globules: Inducing stem cells to form fixed endoderm cells; Inducing fixed endoderm cells to differentiate into anterior foregut endoderm globules; (2) Obtaining lung progenitor cells: collect floating anterior foregut endoderm globules, dissociate the collected anterior foregut endoderm globules into anterior foregut endoderm single cells, and induce the formation of lung progenitor cells; in, Inducing embryonic stem cells to form defined endoderm cells involves culturing them in a medium supplemented with Activin A and ChIR99021; The process of inducing the formation of foregut endoderm globules from the shaped endoderm cells includes culturing them in a culture medium supplemented with SB431542, Noggin, FGF4, SAG, and CHIR99021. The induction of lung progenitor cells includes culturing single cells of the foregut endoderm under extracellular matrix or hydrogel conditions, and inducing them into lung progenitor cells using a lung progenitor cell culture medium containing DAPT, BMP4, fibroblast growth factor 7, fibroblast growth factor 10, CHIR99021, and retinoic acid. Wherein, the stem cells are embryonic stem cells and / or induced pluripotent stem cells; The embryonic stem cells are isolated or obtained from human embryos that have not developed in vivo and are less than 14 days old after fertilization.

2. The method according to claim 1, wherein the purity of the lung progenitor cells induced from the anterior foregut endoderm is above 95%.

3. The method according to claim 1, wherein the purity of the lung progenitor cells induced from the anterior foregut endoderm is above 97%.

4. The method according to claim 1, wherein the expression markers of the lung progenitor cells are NKX2.1 and / or SOX9 and / or SOX2.

5. The method according to claim 1, wherein the concentration of CHIR99021 in the culture medium for inducing embryonic stem cells to form morphological endoderm cells is 0.1-3 μM.

6. The method according to claim 1, wherein the concentration of Activin A in the culture medium for inducing embryonic stem cells to form morphological endoderm cells is 80-120 ng / mL.

7. The method according to claim 1, wherein MCDB131 medium is used as the basal medium for inducing embryonic stem cells to form morphological endoderm cells.

8. The method of claim 1, wherein embryonic stem cells are induced to form fixed endoderm cells within 3 days.

9. The method of claim 1, wherein the step of inducing embryonic stem cells to form defined endoderm cells comprises: On day 0 (D0), embryonic stem cells were cultured in a medium supplemented with Activin A and ChIR99021. On day 1 (D1), the culture was continued using a medium supplemented with Activin A and ChIR99021; On day 2 (D2), the culture was continued using a medium supplemented with Activin A.

10. The method according to claim 1, wherein the concentration of SB431542 in the culture medium for inducing the shaped endoderm cells to form anterior foregut endoderm globules is 5-15 μM.

11. The method according to claim 1, wherein the concentration of Noggin in the culture medium for inducing the shaped endoderm cells to form anterior foregut endoderm globules is 150 ng / mL to 250 ng / mL.

12. The method according to claim 1, wherein the concentration of FGF4 in the culture medium for inducing the shaped endoderm cells to form anterior foregut endoderm globules is 400-600 ng / mL.

13. The method according to claim 1, wherein the concentration of CHIR99021 in the culture medium for inducing the shaped endoderm cells to form anterior foregut endoderm globules is 1-3 μM.

14. The method according to claim 1, wherein the concentration of SAG in the culture medium for inducing the shaped endoderm cells to form anterior foregut endoderm globules is 0.5-1.5 μM.

15. The method according to claim 1, wherein DMEM / F-12 medium or DMEM / F-12 modified medium is used as the basal medium for inducing the formation of foregut endoderm globules from shaped endoderm cells.

16. The method of claim 1, wherein the shaped endoderm cells are induced to form anterior foregut endoderm globules within 5 days.

17. The method according to claim 1, wherein the anterior foregut endoderm globules appear on day 3 of the AFE stage, and the floating anterior foregut endoderm globules will appear on days 4-7 of the AFE stage.

18. The method of claim 1, wherein a single cell from the foregut endoderm is induced into lung progenitor cells in Matrigel using lung progenitor cell (LPC) culture medium.

19. The method of claim 18, wherein cell culture is performed using a Matrigel matrix or a protein hydrogel, and growth factors are added to enable the cells to proliferate and differentiate in a suspended and stable environment.

20. The method according to claim 1, wherein the concentration of DAPT in the lung progenitor cell culture medium is 15-25 μM.

21. The method according to claim 1, wherein the concentration of BMP4 in the lung progenitor cell culture medium is 15-25 ng / mL.

22. The method according to claim 1, wherein the concentrations of FGF7 and / or FGF10 in the lung progenitor cell culture medium are 5-15 ng / mL, respectively.

23. The method according to claim 1, wherein the concentration of CHIR99021 in the lung progenitor cell culture medium is 1-5 μM.

24. The method according to claim 1, wherein the concentration of RA in the lung progenitor cell culture medium is 40-60 nM.

25. The method according to claim 1, wherein DMEM / F-12 medium or DMEM / F-12 modified medium is used as the basal medium for lung progenitor cells.

26. The method of claim 1, wherein the foregut endoderm cells are induced to form lung progenitor cells within 7 days.

27. Lung progenitor cells obtained by the method of any one of claims 1-26.

28. The lung progenitor cells according to claim 27, wherein the purity of the lung progenitor cells is above 97%.

29. The lung progenitor cells according to claim 27, wherein the expression markers of the lung progenitor cells are NKX2.1 and / or SOX9 and / or SOX2.

30. The lung progenitor cells of claim 27, wherein the lung progenitor cells have an onion ring morphology.

31. A method for inducing lung progenitor cells obtained by the method of any one of claims 1-26 to form lung airway organoids and / or alveolar organoids, said method comprising culturing said lung progenitor cells in a culture medium containing Dexamethasone, 8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX) and FGF7.

32. The method of claim 31, wherein the culture medium further comprises SB431542 and / or CHIR99021.

33. The method according to claim 32, wherein the concentration of SB431542 in the culture medium is 5-15 μM.

34. The method according to claim 32, wherein the concentration of CHIR99021 in the culture medium is 2-4 μM.

35. The method of claim 31, wherein DMEM / F-12 medium or DMEM / F-12 modified medium is used as the basal medium.

36. The method of claim 31, wherein lung progenitor cells are induced to form lung airway organoids and / or alveolar organoids within 30 days.

37. The method of claim 31, wherein the expression markers of the lung airway organoids are TP63, SCGB1A1, CHGA, FOXJ1 and / or MUC5AC.

38. The method of claim 31, wherein the expression markers of the alveolar organoids are HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC and / or SLC34A2.

39. The method of claim 31, wherein suspension culture is used to obtain lung airway organoids.

40. The method of claim 31, wherein the lung airway organoid is an everted airway organoid.

41. Lung airway organoids and / or alveolar organoids obtained by the method of any one of claims 31-40.

42. The lung airway organoids and / or alveolar organoids according to claim 41, wherein the expression markers of the lung airway organoids are TP63, SCGB1A1, CHGA, FOXJ1 and / or MUC5AC.

43. The lung airway organoids and / or alveolar organoids according to claim 41, wherein the expression markers of the alveolar organoids are HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC and / or SLC34A2.

44. The lung airway organoids and / or alveolar organoids according to claim 41, wherein the lung airway organoids are everted airway organoids.

45. The lung progenitor cells obtained by the method of any one of claims 1-26, the lung progenitor cells of any one of claims 27-30, the lung airway organoids and / or alveolar organoids obtained by the method of any one of claims 31-40, or the lung airway organoids and / or alveolar organoids of any one of claims 41-44, in the preparation of cell models or organoid models for studying lung-related diseases.

46. ​​A pharmaceutical formulation comprising lung progenitor cells obtained by the method of any one of claims 1-26 or lung progenitor cells as described in any one of claims 27-30.

47. A method for screening therapeutic or preventative drugs, wherein the method includes: The step of contacting the lung airway organoids and / or alveolar organoids obtained by the method of any one of claims 31-40, or the lung airway organoids and / or alveolar organoids of any one of claims 41-44, with the candidate molecules.

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