A culture medium and culture method for amplifying adult human lung pluripotent stem cells

Abstract

This invention discloses a culture medium and method for expanding adult human lung pluripotent stem cells. It relates to the field of primary lung stem cell culture technology, including specific components, culture methods based on the culture medium, and applications. The culture medium of this invention can continuously and stably culture adult human lung stem cells for more than 30 generations. This invention can stably expand adult human lung pluripotent stem cells at the 2D level, and the stem cells maintain a high proliferation rate and multi-cell differentiation potential of lung epithelial cells after continuous passage. Importantly, after in vivo transplantation of the expanded adult pluripotent stem cells, the cells can achieve efficient cell integration and functional alveolar structure reconstruction in fibrotic lung tissue, and the cell transplantation safety is high. This is of great significance for advancing stem cell therapy for respiratory diseases and research on the pathogenic mechanisms of respiratory diseases, and also provides excellent seed cells for subsequent construction of functional human lung organoids and screening of drugs for respiratory diseases represented by pulmonary fibrosis.

Description

Technical Field

[0001] This invention relates to the field of primary lung stem cell culture technology, and more specifically to a culture medium and culture method for expanding adult human lung pluripotent stem cells. Background Technology

[0002] Alveoli are crucial tissue units in the lungs for oxygen and carbon dioxide exchange. Abnormal alveolar cell function can lead to impaired oxygen and carbon dioxide exchange, and severe pathological changes can cause respiratory distress or even suffocation and death in animals. Under normal physiological conditions, alveolar stem cells residing in the alveolar tissue of adult animals can be activated, proliferate, and differentiate into mature alveolar cells when the alveolar tissue is damaged, participating in alveolar reconstruction and repair to a certain extent. However, in advanced COPD, idiopathic pulmonary fibrosis, and other chronic respiratory diseases, lung homeostasis is disrupted, alveolar stem cells are lost or severely damaged, and alveolar reconstruction function is lost, resulting in extremely high mortality rates in late-stage cases. Currently, organ transplantation is the most effective treatment for advanced pulmonary fibrosis, but the biggest problem with organ transplantation is the scarcity of donors and the risk of immune rejection after transplantation. Given that stem cells residing in lung tissue can participate in lung injury repair to a certain extent, lung stem cell therapy shows great promise in the treatment of respiratory diseases. Furthermore, stem cell therapy has demonstrated good therapeutic effects in different animal models. Therefore, lung stem cell therapy is expected to become the most effective method for treating advanced respiratory diseases in humans.

[0003] Because stem cell therapy requires a large number of donor lung stem cells, and current stem cell culture systems both domestically and internationally cannot achieve large-scale in vitro culture of human lung stem cells, the lung stem cells with alveolar differentiation capacity currently obtained are mainly derived from iPS induction, and their passage culture must rely on the 3D Matrigel gel system. However, Matrigel gel contains various proteins and growth factors, the specific composition of which is unclear, batch-to-batch product variations are significant, and the price is expensive. Considering the maintenance of lung stem cell characteristics and the stability of the culture system, this system is not suitable for large-scale in vitro expansion culture. Importantly, currently, in vitro expanded human lung stem cells cannot achieve efficient alveolar structural reconstruction in severely fibrotic pulmonary tissues. Establishing a large-scale expansion system for lung stem cells that can be used for alveolar regeneration and reconstruction is a crucial problem that urgently needs to be solved in the current cell therapy for pulmonary fibrosis.

[0004] Therefore, establishing an efficient and long-term stable 2D culture system for expanding adult human lung pluripotent stem cells is an urgent problem to be solved in stem cell therapy and drug efficacy evaluation for respiratory diseases. At the same time, it is of great significance for advancing stem cell therapy for respiratory diseases, clinical drug screening, the construction of functional human lung organoids, and research on the pathogenic mechanisms of respiratory diseases. Summary of the Invention

[0005] In view of this, the present invention provides a culture medium and method for expanding adult human lung pluripotent stem cells. The culture medium system provided by the present invention can continuously and stably culture adult human lung pluripotent stem cells for more than 30 generations (adult lung stem cells cultured up to the 38th generation still maintain good cell morphology and a high proliferation rate), while maintaining strong stem cell characteristics and differentiation potential. Importantly, after in vivo transplantation, these stem cells can achieve efficient cell integration and alveolar structure reconstruction in pulmonary fibrosis tissue, and the transplantation safety is high. This is of great significance for advancing stem cell therapy for respiratory diseases and research on the pathogenic mechanisms of respiratory diseases, and also provides excellent seed cells for subsequent construction of functional human lung organoids and screening of drugs for respiratory diseases represented by pulmonary fibrosis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A culture medium for expanding adult human lung pluripotent stem cells comprises: 1x N2 supplement, 1x B27 supplement, bovine serum albumin 5-20 μg / mL, glutamine 2-4 mM, glutathione 70-150 μg / mL, superoxide dismutase 450-700 U / mL, epidermal growth factor 20-200 ng / mL, fibroblast growth factor 50-400 ng / mL, Y-27632 10-20 μM, CHIR99021 3-9 μM, A-8301 10-20 μM, SB431542 5-20 μM, DMH-1 1-10 μM, DAPT 5-20 μM, dexamethasone 0.5-2 μM, bucladesine sodium 10-20 μM, ascorbic acid 2-4 mM, and ethanolamine 0.1-0.5 μL / mL.

[0008] Preferred ingredients include: 1x N2 supplement, 1x B27 supplement, bovine serum albumin 5 μg / mL, glutamine 2 mM, glutathione 70 μg / mL, superoxide dismutase 450 U / mL, epidermal growth factor 20 ng / mL, fibroblast growth factor 50 ng / mL, Y-27632 10 μM, CHIR99021 3 μM, A-8301 10 μM, SB431542 5 μM, DMH-1 1 μM, DAPT 5 μM, dexamethasone 0.5 μM, bucladesine sodium 10 μM, ascorbic acid 2 mM, and ethanolamine 0.1 μL / mL.

[0009] Preferred: DMEM / F12 is used as the solvent.

[0010] The present invention also provides a method for preparing any of the above-mentioned culture media, comprising the following steps: (1) Bovine serum albumin, glutamine, glutathione, superoxide dismutase, epidermal growth factor, fibroblast growth factor, Bucladesine sodium and ascorbic acid were dissolved in DMEM / F12 to prepare a stock solution; Y-27632, CHIR99021, A-8301, SB431542, DMH-1, DAPT, and Dexamethasone were dissolved in DMSO to prepare stock solutions. (2) Add each stock solution from step (1) to DMEM / F12 according to the final concentration of the culture medium used, and finally add ethanolamine.

[0011] The present invention also provides a method for culturing adult human lung pluripotent stem cells, comprising the following steps: (1) culturing primary lung cells in collagen-coated culture dishes using DMEM / F12 medium containing 10% FBS; (2) Collect non-adherent lung stem cells and culture them in any of the above-mentioned culture media to obtain adult human lung pluripotent stem cells.

[0012] Preferred method: In step (2), half of the medium is replaced every other day during the culture process.

[0013] The present invention also provides the application of any of the above-mentioned culture media in the preparation of adult human lung pluripotent stem cells.

[0014] The present invention also provides the application of any of the above-mentioned culture media in the preparation of expanded lung stem cell products.

[0015] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a culture medium and culture method for expanding adult human lung pluripotent stem cells. The technical effects achieved are as follows: the culture medium of the present invention can continuously and stably culture adult human lung pluripotent stem cells for more than 30 generations. Compared with the existing culture systems in the world, a more stable 2D culture system for long-term in vitro expansion of adult human lung pluripotent stem cells has been established. After continuous passage, lung stem cells can maintain a high proliferation rate and strong stem cell properties. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1The attached figures show the cell morphology of primary, P5, P10, P20, and P30 adult human lung pluripotent stem cells provided in Example 3 of the present invention, where a represents cells obtained from the culture medium of Example 1; and b represents cells obtained from the culture medium of Example 2.

[0018] Figure 2 The attached figures show the in vitro expansion and proliferation rate of adult human lung pluripotent stem cells provided in Example 3 of the present invention and the results of P30 cell karyotype analysis, where a: in vitro expansion and proliferation rate; b: P30 cell karyotype analysis.

[0019] Figure 3 The attached figure shows an immunofluorescence assay of multiple gene expression in adult human lung pluripotent stem cells passaged to the 25th generation, as provided in Example 4 of this invention.

[0020] Figure 4 The attached figure shows the expression of ACE2 protein in adult human lung pluripotent stem cells passaged to the 25th generation, as determined by immunofluorescence.

[0021] Figure 5 The attached figure shows the identification results of qPCR identification of marker genes for high expression of lung progenitor cells after 30 generations of continuous expansion of adult lung stem cells provided in Example 5 of the present invention, wherein a: adult lung stem cells express marker genes of embryonic lung (epithelial) stem cells; b: adult lung stem cells express marker genes of adult lung (epithelial) stem cells.

[0022] Figure 6 The attached figure is a verification diagram of tumor formation after axillary transplantation of adult lung stem cells following continuous expansion for 25 generations, as provided in Example 6 of the present invention. In the figure: a: Human lung stem cells expanded and transplanted into the armpit of nude mice do not form tumors; b: Human lung cancer cells A549 transplanted into the armpit showed significant tumor formation after 14 days.

[0023] Figure 7 The attached figure is a verification diagram of the participation of expanded P15 adult lung stem cells in alveolar epithelial cell remodeling in mice with pulmonary fibrosis after in vivo transplantation, as provided in Example 7 of the present invention. In the figure, a: the integrated distribution of transplanted P15 adult lung stem cells in the fibrotic lung tissue of mice (HNA, human nuclear antigen, marking the transplanted human lung stem cells and their progeny produced by proliferation and differentiation); b: the upper figure shows that the transplanted human lung stem cells and their proliferating progeny (hGFADH) differentiate into type I alveolar epithelial cells (HOPX); the lower figure shows that the transplanted human lung stem cells and their proliferating progeny (HNA) differentiate into type II alveolar epithelial cells (SPC). Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] This invention discloses a culture medium and a culture method for expanding adult human lung pluripotent stem cells.

[0026] Example 1 A culture medium for expanding adult human lung pluripotent stem cells, comprising: DMEM / F12 as solvent, 1x N2 supplement (Gibco, 17502048), 1x B27 supplement (Gibco, 17504044), bovine serum albumin (Sigma-Aldrich, A9418) 5 μg / mL, glutamine (Gibco, 35050061) 2 mM, glutathione 70 μg / mL, superoxide dismutase 450 U / mL, and epidermal growth factor (MedChemExpress). HY-P72982A ) 20 ng / mL, fiber growth factor (MedChemExpress, HY-P70439) 50 ng / mL, Y-27632 10 μM (MedChemExpress, HY-10071), CHIR99021 3μM (MedChemExpress, HY-10182), A-8301 (MedChemExpress, HY-10432) 10 μM, SB431542 (MedChemExpress, HY-10431) 5 μM, DMH-1 (MedChemExpress, HY-12273) 1 μM, DAPT (MedChemExpress, HY-13027) 5 μM, Dexamethasone 0.5 μM, Bucladesine sodium (MedChemExpress, HY-B0764) 10 μM, ascorbic acid 2 mM ethanolamine (Sigma-Aldrich, E9508) 0.1 μL / mL.

[0027] The preparation method includes the following steps: (1) Bovine serum albumin was dissolved in DMEM / F12 to prepare a stock solution of 20 mg / mL: Glutamine was dissolved in DMEM / F12 to prepare a 200 mM stock solution. Glutathione was dissolved in DMEM / F12 to prepare a stock solution of 100 mg / ml: Superoxide dismutase was dissolved in DMEM / F12 to prepare a stock solution of 50 kU / mL: Epidermal growth factor was dissolved in DMEM / F12 to prepare a stock solution of 50 ug / ml: Fiber growth factor was dissolved in DMEM / F12 to prepare a stock solution of 50 ug / ml: Prepare a 200 mM stock solution of Bucladesine sodium DMEM / F12: Ascorbic acid was dissolved in DMEM / F12 to prepare a 2 M stock solution: Y-27632 was dissolved in DMSO to prepare a 10 mM stock solution: CHIR99021 was dissolved in DMSO to prepare a 10 mM stock solution: A-8301 was dissolved in DMSO to prepare a 10 mM stock solution: SB431542 was dissolved in DMSO to prepare a 100 mM stock solution: DMH-1 was dissolved in DMSO to prepare a 10 mM stock solution: DAPT was dissolved in DMSO to prepare a 100 mM stock solution: Dexamethasone was dissolved in DMSO to prepare a 10 mM stock solution; (2) Add the above components to DMEM / F12 according to the final concentration of the culture medium used, and finally add ethanolamine to make the final concentration 0.1 μL / mL.

[0028] Example 2 A culture medium for expanding adult human lung pluripotent stem cells comprises: DMEM / F12 as solvent, 1x N2 supplement, 1x B27 supplement, bovine serum albumin 20 μg / mL, glutamine 4 mM, glutathione 150 μg / mL, superoxide dismutase 700 U / mL, epidermal growth factor 200 ng / mL, fibroblast growth factor 400 ng / mL, Y-27632 20 μM, CHIR99021 9 μM, A-8301 20 μM, SB431542 20 μM, DMH-1 10 μM, DAPT 20 μM, dexamethasone 2 μM, bucladesine sodium 20 μM, ascorbic acid 4 mM, and ethanolamine 0.5 μL / mL.

[0029] Preparation method: First, prepare the mother liquor of each component according to the method of Example 1. Then, add each mother liquor of step (1) to DMEM / F12 according to the final concentration of this example. Finally, add ethanolamine to make the final concentration 0.5 μL / mL.

[0030] Example 3 In vitro culture method for adult human lung pluripotent stem cells A collagen coating solution with a concentration of 20 μg / mL was prepared using 0.02M glacial acetic acid and coated onto 6 cm cell culture dishes. Adult human lung stem cells were isolated and extracted from adult human lung tissue by digestion with 250 U / ml collagenase. The extracted lung cells were seeded into collagen-coated culture dishes and cultured overnight in a DMEM / F12 cell culture incubator containing 10% fetal bovine serum (FBS) to allow other cells besides lung stem cells to adhere to the culture dish.

[0031] Sixteen hours later, non-adherent lung stem cells were collected and cultured using the culture media prepared in Examples 1 and 2, respectively, with half the medium replaced every other day. After 15 days of culture, visible adult lung stem cell clones were formed, with smooth edges and dense internal cell density (see Appendix). Figure 1 P0).

[0032] When the cell clone diameter is greater than 1 mm, the stem cell clones are digested with collagenase and passaged into 6-well plates at a ratio of 1:3, with approximately 1 x 10 cells per well. 5 / well. When the cell confluence in the 6-well cell culture plate reaches approximately 80%, continue passage to the 6-well cell culture plate at a 1:10 ratio (1 x 10 cells per well). 5 / well). Adult human lung pluripotent stem cells cultured in the culture media of Examples 1 and 2 for more than 30 consecutive passages maintained a high proliferation rate and good stem cell morphological characteristics. Figure 1 The images show the morphology of cells at different passages (P0, P5, P10, P20, and P30) after cell expansion culture, demonstrating that adult human lung pluripotent stem cells, after 30 passages, still maintain a good cell state.

[0033] Depend on Figure 2 From a, we can see that the cell expansion and proliferation fold can be as high as 1437; from Figure 2 As shown in Figure b, the karyotype analysis of P30 stem cells reveals that the cells maintain a normal chromosome number and morphology. These data further demonstrate that the culture medium of Example 1 can stably expand adult human lung pluripotent stem cells for at least 30 generations.

[0034] Example 4 The maintenance of stem cell properties by the in vitro expansion system of adult human lung pluripotent stem cells To verify that adult lung stem cells cultured in the culture medium of Example 1 can still maintain pluripotent stem cell characteristics after 25 passages, lung stem cells passaged 25 times were selected for verification. When the cells grew to 80%, the culture medium was discarded, the cells were washed with PBS, and then fixed with 4% paraformaldehyde solution at room temperature for 30 minutes, followed by washing with PBS. Antibodies and proteins labeled with proximal lung epithelial progenitor cells (SOX2), distal lung epithelial progenitor cells (SOX9), type II alveolar progenitor cells (SFTPC), basal progenitor cells (KRT5), P63 progenitor cells (P63), and the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) were added to identify stem cell characteristics and whether the cells expressed the SARS-CoV-2 receptor ACE2. The antibodies and cells were incubated overnight at 4°C, and the cells were washed three times with PBS. Then, the corresponding immunofluorescent secondary antibodies were added, and the cells were incubated at room temperature for 1 hour, followed by washing with PBS three times. The cells were then incubated with DAPI staining solution for 5 minutes, washed with PBS three times, and observed under a fluorescence microscope (see Appendix). Figure 3 ).

[0035] Experimental results: SOX2 labeled proximal lung precursor cells, and SOX9 labeled distal lung precursor cells; KRT5 labeled basal precursor cells, and P63 labeled distal lung P63 precursor cells. P63+ / KRT5- labeling indicated that the cells possessed the properties of proximal lung non-basal P63 precursor cells; SFTPC and SOX9, a marker for distal lung precursor cells, co-labeled distal lung AT2 precursor cells. Depend on Figure 4 The lung distal progenitor cell marker SOX9 and angiotensin-converting enzyme 2 (ACE2) were used to jointly label and characterize cells that have the SARS-CoV-2 receptor ACE2.

[0036] The results showed that the human adult lung stem cells expanded in the culture medium of the example had high cell totipotency and expressed the SARS-CoV-2 receptor ACE2.

[0037] Example 5 Real-time quantitative PCR was used to verify that in vitro expanded adult human lung pluripotent stem cells expressed genes from lung stem cells. Cells cultured for 30 generations and cells extracted from adult lung tissue were washed with PBS, lysed thoroughly with Trizol, and centrifuged at 12,000 rpm for 15 minutes at 4°C with 1 / 2 chloroform added. The supernatant was collected and an equal volume of isopropanol was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 10 minutes, then centrifuged at 12,000 rpm for 15 minutes at 4°C. The precipitate was then washed thoroughly with 75% ethanol, air-dried at room temperature until translucent, and the RNA was dissolved in water.

[0038] RNA reverse transcription was performed using a reverse transcription kit (Promega, A5001). First, 500 ng of RNA was added to 1 μL of primer dT, and the volume was adjusted to 5 μL with ddH2O. The mixture was heated at 70°C for 5 minutes to open the helical structure of the RNA, and then immediately incubated on ice for at least 5 minutes. Next, 4 μL of 5X reaction buffer, 4 μL of MgCl2, 1 μL of PCR dNTP, 0.5 μL of RNase inhibitors, 1 μL of reverse transcriptase, and 4.5 μL of ddH2O were added to the reaction mixture. After thorough mixing, the mixture was annealed at 25°C for 5 minutes, extended at 42°C for 1 hour, and then inactivated at 70°C for 15 minutes. The expression levels of the corresponding genes were detected by real-time quantitative PCR.

[0039] The reaction system for real-time quantitative PCR is shown in Table 1, the reaction conditions are shown in Table 2, and the (conventional) primer sequences are shown in Table 3; the results are attached. Figure 5 As shown.

[0040]

[0041]

[0042] Table 3

[0043] As attached Figure 5 The adult human lung pluripotent stem cells shown were continuously expanded and cultured in vitro for 30 generations, expressing genes from embryonic lung stem cells. Figure 5 (a) and adult lung stem cell gene ( Figure 5 (b) indicates that continuously expanded lung stem cells still have high pluripotency.

[0044] Example 6 In vitro expanded adult human lung pluripotent stem cells have a high safety profile for in vivo transplantation. To verify the safety of in vivo transplantation of adult lung stem cells cultured in the culture medium of Example 1, and to verify whether tumor formation occurred in vivo, human lung stem cells passaged 24 times were injected subaxillarily into nude mice at a dose of 4 x 10⁻⁶. 5 / mouse, control group nude mice were injected with human lung cancer cells A549, injection dose was 2x10 5 / Only.

[0045] The results showed that no tumors formed in the axillae of nude mice injected with human lung stem cells within 3 months, while tumors enlarged 14 days after injection of lung cancer cells A5491 (see appendix). Figure 6(The red area marked with tumor formation) indicates that the expanded human lung stem cells have a high safety profile for in vivo transplantation.

[0046] Example 7 In vitro expanded adult human lung pluripotent stem cells were transplanted in vivo to participate in alveolar reconstruction. To verify whether adult lung stem cells cultured in the culture medium of Example 1 can participate in alveolar reconstruction in pulmonary fibrosis tissue, human lung stem cells from passage P15 were used for cell transplantation into fibrotic NOG mice, with a transplanted cell quantity of 6 x 10⁻⁶ cells. 5 Lung tissue was harvested from each cell line 60 days after transplantation, following cardiac perfusion. The tissue was dehydrated using a sucrose gradient and then frozen sectioned for immunohistochemistry. Antibodies labeled with human-derived HNA, GAPDH, type I alveolar cells (HOPX), and type II alveolar cells (SFTPC) were used to trace the lineage of transplanted human lung stem cells differentiating into alveolar cells in vivo. The cells were incubated overnight at 4°C with the antibodies, washed three times with PBS, and then incubated with the corresponding immunofluorescent secondary antibody at room temperature for 1 hour. After washing three times with PBS, the cells were incubated with DAPI staining for 5 minutes, washed three times with PBS, and observed under a fluorescence microscope.

[0047] Depend on Figure 7 The progeny cells resulting from the proliferation and differentiation of transplanted adult human lung stem cells labeled with HNA and hGAPDH are derived from... Figure 7 As can be seen from a, transplanted human lung stem cells can integrate efficiently into pulmonary fibrosis tissue; Figure 7 From b, we can know that: hGAPDH + HOPX + This indicates that the transplanted human lung stem cells differentiated into type I alveolar cells; hHNA + SFTPC + This indicates that the transplanted human lung stem cells differentiated into type II alveolar cells.

[0048] The above results indicate that after transplantation of in vitro expanded human lung stem cells into mice with pulmonary fibrosis, they can achieve large-scale integration and participate in alveolar reconstruction.

[0049] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0050] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for expanding adult human lung pluripotent stem cells, characterized in that, include: 1x N2 supplement, 1x B27 supplement, bovine serum albumin 5-20 μg / mL, glutamine 2-4 mM, glutathione 70-150 μg / mL, superoxide dismutase 450-700 U / mL, epidermal growth factor 20-200 ng / mL, fibroblast growth factor 50-400 ng / mL, Y-27632 10-20 μM, CHIR99021 3-9 μM, A-8301 10-20 μM, SB431542 5-20 μM, DMH-1 1-10 μM, DAPT 5-20 μM, dexamethasone 0.5-2 μM, Bucladesine sodium 10-20 μM, ascorbic acid 2-4 mM, ethanolamine 0.1-0.5 μL / mL.

2. The culture medium as described in claim 1, characterized in that, include: 1x N2 supplement, 1x B27 supplement, bovine serum albumin 5 μg / mL, glutamine 2 mM, glutathione 70 μg / mL, superoxide dismutase 450 U / mL, epidermal growth factor 20 ng / mL, fibroblast growth factor 50 ng / mL, Y-27632 10 μM, CHIR99021 3 μM, A-8301 10 μM, SB431542 5 μM, DMH-1 1 μM, DAPT 5 μM, dexamethasone 0.5 μM, bucladesine sodium 10 μM, ascorbic acid 2 mM, ethanolamine 0.1 μL / mL.

3. The culture medium as described in claim 1 or 2, characterized in that, DMEM / F12 was used as the solvent.

4. A method for preparing the culture medium according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Bovine serum albumin, glutamine, glutathione, superoxide dismutase, epidermal growth factor, fibroblast growth factor, Bucladesine sodium and ascorbic acid were dissolved in DMEM / F12 to prepare a stock solution; Y-27632, CHIR99021, A-8301, SB431542, DMH-1, DAPT, and Dexamethasone were dissolved in DMSO to prepare stock solutions. (2) Add each stock solution from step (1) to DMEM / F12 according to the final concentration of the culture medium used, and finally add ethanolamine.

5. A method for culturing adult human lung pluripotent stem cells, characterized in that, Includes the following steps: (1) Primary lung cells were cultured in collagen-coated culture dishes using DMEM / F12 medium containing 10% FBS; (2) Collect non-adherent lung stem cells and culture them in the culture medium described in any one of claims 1-3 to obtain adult human lung pluripotent stem cells.

6. The method for culturing adult human lung pluripotent stem cells as described in claim 5, characterized in that, Step (2) During the culture process, half of the medium should be replaced every other day.

7. The use of the culture medium described in any one of claims 1 to 3 in the preparation of adult human lung pluripotent stem cells.

8. The use of the culture medium according to any one of claims 1 to 3 in the preparation of expanded lung stem cell products.

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