Culture solution and method for transdifferentiating airway epithelial cells into lung progenitor cells

Abstract

The invention discloses a culture solution and a method for transdifferentiating airway epithelial cells into lung progenitor cells. In particular to a culture solution for transdifferentiating airway epithelial cells into lung progenitor cells. The culture solution is prepared by adding FGF10, IGF1, KGF, a Wnt agonist, retinoic acid, BMP4, a PRMT5 inhibitor, a p38 kinase inhibitor and a Smoothed agonist on the basis of a basic culture medium. A large number of SOX < 2 + > lung epithelial cells are obtained by separating mouse atmospheric duct epithelial cells and performing 3D culture amplification in vitro, and then conditions capable of obtaining far-end SOX < 9 + > epithelial cells are screened by optimizing morphogenetic factors and regulatory molecules of various signal channels in a culture system. By adopting the culture solution and the method disclosed by the invention, the near-end SOX < 2 + > epithelial cells can be efficiently transdifferentiated into the far-end SOX < 9 + > epithelial cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a culture medium and a method for transdifferentiating airway epithelial cells into lung progenitor cells. Background Art

[0002] Respiratory diseases are one of the most common causes of death worldwide and impose a huge burden on healthcare systems (Naghavi et al., 2017). Whether they are environmentally induced respiratory diseases, such as chronic obstructive pulmonary disease (COPD) or asthma, or hereditary, such as cystic fibrosis (CF) or primary ciliary dyskinesia (PCD), they are a major part of human health today (Hubbard, 2006). The lung is a non-regenerative organ. Although the lung can spontaneously repair to a certain extent after injury, it is all through the repair of airway cells produced by activating adult stem cells and the proliferation of alveoli (Liu, Z. et al., 2016; Basil, MC et al., 2020). The current treatment for end-stage lung disease is transplantation, which has the lowest survival rate of any solid organ transplant (about 50% after 5 years), partly due to the rejection and side effects of immunosuppressive therapy (Christie et al., 2008). Therefore, using autologous stem cells to generate transplant tissue or perform tissue repair is a good way to solve immune rejection. Bioengineered airways can now be used to repair patients' narrowed left main bronchus (Wu et al., 2009), that is, epithelial cells and bone marrow-derived chondrocytes from patients with bronchomalacia are cultured and then inoculated into decellularized tracheal segments. There have also been studies in which mixed populations of neonatal or fetal rat lung epithelial cells and endothelial cells were inoculated into decellularized donor lung scaffolds and transplanted into recipient rats. These transplants promoted gas exchange in a very short period of time (several hours) (Ott et al., 2010). Although these studies represent technological advances, to date, there has been no technology to cultivate large quantities of stem cells that can be used to repair lung damage. The key to solving this problem is to obtain stem cells that can differentiate into all lung epithelial cells. However, in the lungs of adults or mice, there are only adult stem cells with regional specificity and limited differentiation ability, which cannot meet the needs of repairing damage to any part of the lung.

[0003] During the proximal-distal branching development of the trachea and bronchi, SOX9+ / Id2+ cells are initially pluripotent and can simultaneously generate airway and alveolar epithelial cells, that is, before embryonic day E13.5, SOX2+ proximal airway progenitor cells are differentiated from SOX9+ epithelial progenitor cells, and the fate of SOX9+ epithelial progenitor cells is limited by the time of embryonic development. After embryonic day E16.5, SOX9+ distal precursor cells no longer have the ability to differentiate into SOX2+ proximal cells (Yang et al., 2018). In other words, the development of the embryonic lung begins with a group of SOX9+ pluripotent stem cells. This research result suggests that in situ regeneration of the lung organ is a process of transdifferentiation (or dedifferentiation) of epithelial cells, that is, transdifferentiation of proximal SOX2+ cells into distal SOX9+ epithelial cells. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to transdifferentiate airway epithelial cells into lung progenitor cells, including how to transdifferentiate proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention first provides a culture medium for transdifferentiating airway epithelial cells into lung progenitor cells, wherein the culture medium can be supplemented with FGF10, IGF1, KGF, Wnt agonist, retinoic acid, BMP4, PRMT5 inhibitor, p38 kinase inhibitor and Smoothened agonist on the basis of basal culture medium.

[0006] Furthermore, in the above culture medium, the Wnt agonist may be CHIR99021.

[0007] Furthermore, in the above culture medium, the PRMT5 inhibitor may be EPZ015666.

[0008] Furthermore, in the above culture medium, the p38 kinase inhibitor may be BIRB796.

[0009] Furthermore, in the culture medium, the concentration of FGF10 may be 5-20 ng / mL, the concentration of IGF1 may be 25-35 ng / mL, the concentration of KGF may be 5-20 ng / mL, the concentration of Wnt agonist may be 1.5-3uM, the concentration of retinoic acid may be 0.5-2.5uM, the concentration of BMP4 may be 5-20 ng / mL, the concentration of PRMT5 inhibitor may be 1-3uM, the concentration of p38 kinase inhibitor may be 0.05-0.5uM, and the concentration of Smoothened agonist may be 5-50nM.

[0010] Furthermore, in the culture medium, the concentration of FGF10 may be 10 ng / mL, the concentration of IGF1 may be 30 ng / mL, the concentration of KGF may be 10 ng / mL, the concentration of Wnt agonist may be 2 uM, the concentration of retinoic acid may be 2 uM, the concentration of BMP4 may be 10 ng / mL, the concentration of PRMT5 inhibitor may be 2 uM, the concentration of p38 kinase inhibitor may be 0.1 uM, and the concentration of Smoothenedagonist may be 10 uM.

[0011] Furthermore, in the above-mentioned culture solution, the basic culture medium may contain the following components: HEPES, L-Glutamine, NaHCO3, penicillin, streptomycin, insulin, transferrin, CT, EGF, BPE, and FBS.

[0012] Furthermore, the basic culture medium may be mTEC Plus culture medium, and the mTEC Plus culture medium may be mTEC Basic culture medium supplemented with 10 mg / mL insulin, 5 mg / mL transferrin, 0.1 mg / mL CT, 25 ng / mL EGF, 30 mg / mL BPE, and 5% FBS (v / v) at a final concentration.

[0013] The mTEC Basic culture medium can be prepared by adding 15 mM HEPES, 4 mM L-Glutamine, 3.6 mM NaHCO3, 100 U / mL penicillin, and 100 mg / mL streptomycin to DMEM / F-12 at a final concentration.

[0014] The present invention also provides the use of any of the culture solutions described herein in transdifferentiating airway epithelial cells into lung progenitor cells or in preparing a product for repairing lung damage.

[0015] The present invention also provides a method for transdifferentiating airway epithelial cells into lung progenitor cells, which may include the following steps:

[0016] A1) culturing the airway epithelial cells in 3D in vitro and harvesting SOX2+ epithelial cells;

[0017] A2) Transdifferentiating the SOX2+ epithelial cells using any of the culture media described herein, and harvesting SOX9+ epithelial cells.

[0018] In the above method, the 3D culture method can be: the airway epithelial cells are prepared into 3×104 The cell suspension was prepared at a concentration of 1 cell / mL and then mixed with an equal volume of Matrigel for culture.

[0019] The Matrigel can be a soluble basement membrane extract isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, including extracellular matrix proteins (including laminin, type IV collagen, heparan sulfate proteoglycans, entactin and multiple growth factors). Specifically, the Matrigel can be a product of Corning, with a catalog number of 354230.

[0020] Furthermore, the 3D culture method may be: using airway basal cell culture medium to disperse the airway epithelial cells into a single cell suspension to 3×10 4 cells / mL, then mixed with an equal amount of Matrigel (Corning, 354230) and dropped into a 24-well plate (100uL / well), and then placed in a 37-degree CO2 incubator. After the droplets in the 24-well plate solidified (about 1 hour), 1mL of airway basal cell culture medium was added and cultured for about 9 days to harvest a large number of cells (SOX2+ epithelial cells).

[0021] The airway basal cell culture medium can be the mTEC Plus culture medium supplemented with 1.0 μM A-83-01 (TGFβ antagonist), 0.2 μM DMH-1 (BMP4 antagonist), 0.5 μM CHIR99021 (WNT agonist), and 5 μM Y27632 (ROCK inhibitor) at a final concentration.

[0022] In the above method, the transdifferentiation method may be: performing 2D culture on the SOX2+ epithelial cells after 3D culture, then replacing the culture medium with any culture medium described herein, and continuing the culture to obtain SOX9+ epithelial cells.

[0023] Furthermore, the transdifferentiation method can be: digesting the SOX2+ epithelial cells cultured in vitro for about 10 days with 0.25% trypsin at 37 degrees for 10 minutes, adding DMEM / F12 culture medium (containing 10% FBS) to terminate the digestion, using a pipette to blow and aspirate until the airway spheroids are dissociated into single cells and there is no visible Matrigel in the liquid, discarding the supernatant after centrifugation, blowing up the precipitate with 1 mL of airway basal cell culture medium, transferring it to a cell culture dish to which airway basal cell culture medium has been added in advance, culturing at 37 degrees, and replacing the cells with freshly prepared airway basal cell culture medium the next day to obtain 2D cultured airway epithelial cells (SOX2+ epithelial cells).

[0024] The 2D cultured airway epithelial cells are subcultured into a new 6 cm culture dish, and the cell confluence is about 30%. The next day, the culture medium is replaced with any culture medium described in this article (such as conditioned culture medium 9F), about 4 mL / dish. When the cell confluence is greater than 80%, the cells can be subcultured to obtain SOX9+ epithelial cells.

[0025] The in situ regeneration of mammalian lungs is a scientific problem that needs to be solved urgently in the world's medical and scientific research fields, and there are currently no successful reports. The present invention uses mammalian mice as a model to solve the technical problem of transdifferentiation of proximal SOX2+ epithelial cells of the lung bronchus into distal SOX9+ epithelial cells, creating conditions for the ultimate in achieving in situ regeneration of lungs.

[0026] The present invention isolates mouse large airway epithelial cells and amplifies them in vitro for 3D culture to obtain a large number of SOX2+ lung epithelial cells, and then optimizes the culture system's Wnt agonists, morphogens such as BMP4 and FGF10, and regulatory molecules of multiple signaling pathways by adding small molecule compounds, and screens conditions that can obtain distal SOX9+ epithelial cells. The experimental results show that the culture medium and transdifferentiation method of the present invention can efficiently transdifferentiate proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells, and the transdifferentiation efficiency can reach 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Organoids derived from mouse lung proximal cells.

[0028] Figure 2 This is an immunofluorescence staining image of mouse lung proximal cells cultured under 3D-to-2D conditions. Figure 2 A in the middle is DAPI; Figure 2 Middle B is SOX2; Figure 2 The C in the middle stands for SOX9.

[0029] Figure 3 This is an immunofluorescence staining image of the second generation of transdifferentiated cells cultured under 9F conditions. Figure 3 A in the middle is DAPI; Figure 3 Middle B is SOX2; Figure 3 The C in the middle stands for SOX9.

[0030] Figure 4 This is an immunofluorescence staining image of the second generation of transdifferentiated cells cultured under 9F conditions. Figure 4 A in the middle is DAPI; Figure 4 Middle B is SOX9; Figure 4 The C in the middle stands for FoxP2.

[0031] Figure 5 This is an immunofluorescence staining image of the second generation of transdifferentiated cells cultured under 9F conditions. Figure 5A in the middle is DAPI; Figure 5 Middle B is ETV5; Figure 5 The C in the middle stands for SOX9.

[0032] Figure 6 This is an immunofluorescence staining image of the second generation of transdifferentiated cells cultured under 9F conditions. Figure 6 A in the middle is DAPI; Figure 6 Middle B is SOX2; Figure 6 Middle C is SOX9; Figure 6 D in the figure represents Trp63.

[0033] Figure 7 It is the transdifferentiation efficiency of cells cultured under 9F or 9F with one factor removed (9F-1) for 72 hours. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0036] The FGF10 in the following examples is a product of Novoprotein, catalog number CR11;

[0037] IGF1 is a product of Novoprotein, catalog number C023;

[0038] KGF is a product of Novoprotein, catalog number CH73;

[0039] CHIR99021 is a product of Tocris, catalog number 5989;

[0040] RA (Retinoic Acid) is a product of Sigma, catalog number R2625;

[0041] BMP4 is a product of Seer Ruicheng Company, product number PF0901;

[0042] EPZ015666 is a product of SELLECK, item number S7748;

[0043] BIRB796 is a product of Tocris, catalog number 5989;

[0044] Smoothened Agonist is a product of Selleck, item number S7779.

[0045] The DMEM / F-12 in the following examples is a product of Thermofisher, catalog number 12634010.

[0046] In this article, insulin means insulin; transferrin means transferrin; CT (Cholera Toxin) means cholera toxin; EGF means epidermal growth factor; BPE (Bovine Pituitary Extract) means bovine pituitary extract; FBS means fetal bovine serum; HEPES means HEPES buffer; L-Glutamine means L-glutamine; NaHCO3 means sodium bicarbonate; penicillin means penicillin; and streptomycin means streptomycin.

[0047] In the following embodiments, Insulin is a product of Sigma, with the catalog number I6634; transferrin is a product of Sigma, with the catalog number T1147; CT is a product of Sigma, with the catalog number C8052; EGF is a product of Coning, with the catalog number 354001; BPE is a product of Sigma, with the catalog number P1476; HEPES buffer is a product of Life Technologies, with the catalog number 31765-092; L-Glutamine is a product of Life Technologies, with the catalog number 35050-061; and penicillin / streptomycin is a product of Life Technologies, with the catalog number 15140-163.

[0048] Example 1. Obtaining a large number of SOX2+ epithelial cells

[0049] This example establishes a three-dimensional culture system for bronchial epithelial cells, and harvests a large number of SOX2+ cells (proximal SOX2+ epithelial cells) by culturing proximal lung organoids. The specific steps are as follows:

[0050] 1. Isolation of mouse large airway epithelial cells

[0051] Take mice 1-7 days after birth and place them on ice. When the mice are in a syncope, use small scissors to make a longitudinal midline incision from the clavicle to the submandibular area, then lift the trachea and use curved forceps to directly cut the esophagus from the posterior surface of the trachea. Use small scissors to cut the trachea at the bifurcation of the main bronchus, lift it gently, and put the excised trachea into ice-cold PBS buffer. It is necessary to separate the trachea of ​​3-6 mice, then use small scissors to cut each trachea longitudinally, put it into the digestive solution (1.5mg / mL Pronase, using Ham's F-12 culture medium as solvent), and digest it overnight in a refrigerator at 4 degrees.

[0052] 2. In vitro 3D culture of SOX2+ cells

[0053] Take out the trachea that has been digested overnight in a 4-degree refrigerator, transfer the trachea and the digestion solution to a 15 mL centrifuge tube, add an equal volume of DMEM / F12 culture solution (containing 10% FBS) to terminate the digestion, turn the centrifuge tube upside down 5 times, remove the trachea with tweezers and discard it, and centrifuge the remaining liquid at 1000 rpm for 3 minutes. After centrifugation, discard the liquid above, and the remaining precipitate is the digested airway epithelial cells. Use airway basal cell culture solution to blow the cells into a single cell suspension to 3×10 4 cells / mL, then mixed with an equal amount of Matrigel (Corning, 354230) and dripped into a 24-well plate (100uL / well), which was then placed in a 37-degree CO2 incubator. After the droplets in the 24-well plate solidified (about 1 hour), 1mL of airway basal cell culture medium was added and cultured for about 9 days ( Figure 1 ) can harvest a large number of cells.

[0054] The airway basal cell culture medium is mTEC Plus culture medium supplemented with a final concentration of 1.0 μM A-83-01 (TGFβ antagonist), 0.2 μM DMH-1 (BMP4 antagonist), 0.5 μM CHIR99021 (WNT agonist), and 5 μM Y27632 (ROCK inhibitor).

[0055] mTEC Plus culture medium is based on mTEC Basic culture medium and supplemented with a final concentration of 10 mg / mL insulin, 5 mg / mL transferrin, 0.1 mg / mL CT, 25 ng / mL EGF, 30 mg / mL BPE, and 5% FBS (v / v).

[0056] mTEC Basic culture medium is prepared by adding 15mM HEPES, 4mM L-Glutamine, 3.6mM NaHCO3, 100U / mL penicillin, and 100mg / mL streptomycin to DMEM / F-12.

[0057] 3. 2D culture

[0058] The upper culture medium of the airway spheroids cultured in vitro for about 10 days was discarded, and 0.25% pancreatin was blown up and transferred to a 15mL centrifuge tube, and then digested in a 37-degree water bath for 10 minutes, and DMEM / F12 culture medium (containing 10% FBS) was added to terminate the digestion, and the airway spheroids were blown up with a pipette until they were dissociated into single cells and there was no visible Matrigel in the liquid, and the centrifuge tube was centrifuged at 1000rpm for 3 minutes. After centrifugation, the supernatant was discarded, and the precipitate was blown up with 1mL of airway basal cell culture medium, and transferred to a 6cm cell culture dish with 3mL of airway basal cell culture medium added in advance, and placed in a 37-degree CO2 incubator. The cells were replaced with freshly prepared airway basal cell culture medium the next day.

[0059] Immunofluorescence detection of cultured airway basal cells ( Figure 2 ): 1. Fix cells with 4% paraformaldehyde (PFA) for 5 min at room temperature. 2. Wash cells three times with 0.2% Triton X-100 in PBS+. Incubate cells with wash buffer at room temperature for 30 seconds between each wash. 3. Cover cells with primary antibody diluted in 1% BSA in PBS+ and incubate for 2 hours at room temperature or overnight at 4°C. 4. Wash cells 4 times with 0.2% Triton X-100 in PBS+. Incubate cells with wash buffer at room temperature for 30 seconds between each wash. 5. Cover cells with fluorescently conjugated secondary antibody diluted in 1% BSA in PBS+ and incubate for 1 hour at room temperature (protected from light). 6. Wash cells 4 times with 0.2% Triton X-100 in PBS+. 7. Cover cells with enough 1μg / mL DAPI solution. After incubation for 1 minute, image cells using a Zeiss inverted fluorescence microscope.

[0060] The results are as follows Figure 2 As shown, the proximal lung airway organoid spheroids cultured in 3D conditions were digested into single cells and then cultured in a 2D culture system. It was found that the proximal epithelial cells still expressed SOX2 but not SOX9 ( Figure 2 B, C), these cells were used as experimental cells for subsequent transdifferentiation of SOX2+ cells into SOX9+ cells.

[0061] Example 2: Transdifferentiation of proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells

[0062] By adding small molecule compounds, optimizing the Wnt agonists, morphogens such as BMP4, FGF10, and regulatory molecules of multiple signaling pathways in the culture system, we screened and obtained the culture medium and method that can transdifferentiate proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells. The specific method for transdifferentiating proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells is as follows:

[0063] 1. Prepare conditioned medium 9F

[0064] The conditioned medium after screening and optimization includes 9 factors (including small molecule compounds, growth factors and morphogens), named conditioned medium 9F (also known as conditioned medium TFICKRBEPS). Conditioned medium 9F includes mTECPlus culture medium, fibroblast growth factor 10 (FGF10), insulin-like growth factor 1 (IGF1), keratinocyte growth factor (KGF, also known as fibroblast growth factor 7), Wnt agonist (Wnt-Agonist), small molecule compound retinoic acid (Retinoic Acid, RA, retinoic acid, molecular formula: C 20 H 28 O2; CAS No.: 302-79-4), bone morphogenetic protein 4 (BMP4), PRMT5 inhibitor (Prmt5-inhibitor), p38 kinase inhibitor (p38 kinase-inhibitor), small molecule compound Smoothened agonist (SAG, smooth agonist, molecular formula: C 28 H 28 ClN3OS, CAS number: 912545-86-9).

[0065] Specifically, the Wnt agonist is a small molecule compound CHIR99021 (molecular formula: C 22 H 18 C l2 N8; CAS number: 252917-06-9);

[0066] The PRMT5 inhibitor (Prmt5-inhibitor) is a small molecule compound EPZ015666 (molecular formula: C 20 H 25 N5O3; CAS No.: 1616391-65-1);

[0067] The p38 kinase inhibitor is a small molecule compound BIRB796 (molecular formula: C 31 H 37N5O3; CAS number: 285983-48-4).

[0068] The conditioned medium 9F is prepared with mTEC Plus culture medium as solvent and 9 factors as solutes, wherein the contents of the 9 factors added in the conditioned medium 9F are as follows:

[0069] F: 10 ng / mL FGF10;

[0070] I: 30 ng / mL IGF1;

[0071] K: 10 ng / mL KGF;

[0072] C: 2uM CHIR99021(Wnt-Agonist);

[0073] R: 2uM RA (Retinoic Acid);

[0074] B: 10 ng / mL BMP4;

[0075] E: 2uM EPZ015666(Prmt5-inhibitor);

[0076] P: 0.1uM BIRB796 (p38 kinase-inhibitor);

[0077] S: 10nM Smoothened Agonist.

[0078] mTEC Plus culture medium is mTEC Basic culture medium supplemented with 10 mg / mL insulin, 5 mg / mL transferrin, 0.1 mg / mL CT, 25 ng / mL EGF, 30 mg / mL BPE, and 5% FBS (v / v) at final concentrations.

[0079] mTEC Basic culture medium is: DMEM / F-12 with final concentrations of 15mM HEPES, 4mM L-Glutamine, 3.6mM NaHCO3, 100U / mL penicillin, and 100mg / mL streptomycin added.

[0080] The preparation method of conditional culture medium 9F is shown in Table 1: (based on the preparation of 50 mL culture medium)

[0081] Table 1. Preparation of Conditioned Medium 9F

[0082] Component name Stock concentration Added volume Final concentration FGF10 50ug / mL 10μL 10 ng / mL IGF1 100ug / mL 15μL 30 ng / mL CHIR99021 10mM 10μL 2uM KGF 50ug / mL 10μL 10 ng / mL RA 50mM 2μL 2uM BMP4 150ug / mL 3.33μL 10 ng / mL EPZ015666 10mM 10μL 2uM BIRB796 10mM 0.5 μL 0.1uM SAG 200uM 2.5μL 10nM

[0083] 2. Transdifferentiation of SOX2+ cells

[0084] The 2D cultured airway cells were subcultured into a new 6 cm culture dish when the cell confluence was about 30%. The next day, the culture medium was replaced with conditioned medium 9F, about 4 mL / dish. When the cell confluence was greater than 80%, the cells could be subcultured. During the cell subculture, a portion of the cells could be spread onto small discs for subsequent immunofluorescence staining identification.

[0085] Immunofluorescence detection of transdifferentiated cells ( Figure 3 ): 1. Cells need to be plated on small discs with a confluence of about 30%-50%. Fresh culture medium (9F) is replaced on the second day. On the third day, cells are fixed with 4% paraformaldehyde (PFA) at room temperature for 5 minutes. 2. Wash the small discs 3 times with 0.2% Triton X-100 in PBS. Incubate cells with wash buffer at room temperature for 5 minutes between each wash. 3. Cover cells with 1% BSA in PBS + diluted primary antibody and incubate at room temperature for 2 hours or at 4°C overnight. 4. Wash cells 3 times with 0.2% Triton X-100 in PBS. 5. Cover cells with 1% BSA in PBS + diluted fluorescent-conjugated secondary antibody and incubate at room temperature for 1 hour (protected from light). 6. Wash cells 3 times with 0.2% Triton X-100 in PBS. 7. Cover the small discs with enough 1μg / mL DAPI solution. After incubation for 10 min, the small disc was placed upside down on a slide containing mounting medium and photographed using a Leica SP8 digital slide scanning system.

[0086] The results are as follows Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown, using the conditioned medium 9F, namely TFICKRBEPS, the cells were passaged after 72 hours of action, and lung epithelial progenitor cells (i.e., distal SOX9+ epithelial cells) expressing progenitor cell markers such as Sox9, FoxP2, and ETV5 were obtained. After optimization, the components in the conditioned medium 9F of the present invention can efficiently and synergistically induce the proximal SOX2+ epithelial cells to transdifferentiate into distal SOX9+ epithelial cells. After statistically analyzing the proportion of cells with high SOX9 strong positive expression and SOX2+ negative or weak positive expression, as shown in FIG. Figure 7 , and found that the transdifferentiation efficiency can reach about 20%, which is much higher than the efficiency of cell transdifferentiation or reprogramming in other studies.

[0087] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for transdifferentiating airway epithelial cells into lung progenitor cells, characterized in that: The culture medium is prepared by adding FGF10, IGF1, KGF, Wnt agonist, retinoic acid, BMP4, PRMT5 inhibitor, p38 kinase inhibitor and Smoothened agonist to the basic culture medium.

2. The culture solution according to claim 1, characterized in that The Wnt agonist is CHIR99021.

3. The culture solution according to claim 1 or 2, characterized in that The PRMT5 inhibitor is EPZ015666.

4. The culture solution according to any one of claims 1 to 3, characterized in that The p38 kinase inhibitor is BIRB796.

5. The culture solution according to any one of claims 1 to 4, characterized in that In the culture medium, the concentration of FGF10 is 5-20ng / mL, the concentration of IGF1 is 25-35ng / mL, the concentration of KGF is 5-20ng / mL, the concentration of Wnt agonist is 1.5-3uM, the concentration of retinoic acid is 0.5-2.5uM, the concentration of BMP4 is 5-20ng / mL, the concentration of PRMT5 inhibitor is 1-3uM, the concentration of p38 kinase inhibitor is 0.05-0.5uM, and the concentration of Smoothened agonist is 5-50nM.

6. The culture solution according to any one of claims 1 to 5, characterized in that In the culture medium, the concentration of FGF10 is 10 ng / mL, the concentration of IGF1 is 30 ng / mL, the concentration of KGF is 10 ng / mL, the concentration of Wnt agonist is 2 uM, the concentration of retinoic acid is 2 uM, the concentration of BMP4 is 10 ng / mL, the concentration of PRMT5 inhibitor is 2 uM, the concentration of p38 kinase inhibitor is 0.1 uM, and the concentration of Smoothened agonist is 10 nM.

7. The culture solution according to any one of claims 1 to 6, characterized in that The basic culture medium contains the following components: HEPES, L-Glutamine, NaHCO3, penicillin, streptomycin, insulin, transferrin, CT, EGF, BPE, and FBS.

8. Use of the culture medium according to any one of claims 1 to 7 in transdifferentiating airway epithelial cells into lung progenitor cells or in preparing a product for repairing lung damage.

9. A method for transdifferentiating airway epithelial cells into lung progenitor cells, characterized in that: The method comprises the following steps: A1) culturing the airway epithelial cells in 3D in vitro and harvesting SOX2+ epithelial cells; A2) Transdifferentiating the SOX2+ epithelial cells using the culture medium described in any one of claims 1 to 7, and harvesting SOX9+ epithelial cells.

10. The method according to claim 8, characterized in that The 3D culture method is: the airway epithelial cells are prepared into 3×10 4 The cell suspension was prepared at a concentration of 1 cell / mL and then mixed with an equal volume of Matrigel for culture.

Images