Culture medium and method for airway epithelial cell transdifferentiation into lung progenitor cells

Abstract

The application discloses a culture solution and a method for transdifferentiation of airway epithelial cells into lung progenitor cells. Specifically disclosed is a culture solution for transdifferentiation of airway epithelial cells into lung progenitor cells, which is based on a basic culture medium and addition of FGF10, IGF1, KGF, a Wnt agonist, retinoic acid, BMP4, a PRMT5 inhibitor, a p38 kinase inhibitor and a Smoothened agonist. The application obtains a large amount of SOX2+ lung epithelial cells by separating mouse large airway epithelial cells, carrying out 3D culture expansion in vitro, and then screening conditions capable of obtaining distal SOX9+ epithelial cells by optimizing morphogens and various signal pathway regulating molecules in the culture system. The culture solution and the method can efficiently transdifferentiate proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the culture medium and method for transdifferentiating airway epithelial cells into lung progenitor cells. Background Technology

[0002] Respiratory diseases are among the leading causes of death worldwide and place a significant burden on healthcare systems (Naghavi et al., 2017). Whether environmentally induced respiratory diseases, such as chronic obstructive pulmonary disease (COPD) or asthma, or genetic ones, such as cystic fibrosis (CF) or primary ciliary dyskinesia (PCD), they constitute a major part of human health today (Hubbard, 2006). The lungs are non-regenerative organs. Although they can spontaneously repair themselves to some extent after injury, this repair is primarily achieved through the activation of airway cells generated from adult stem cells and alveolar proliferation (Liu, Z. et al., 2016; Basil, MC et al., 2020). Currently, transplantation is the treatment for end-stage lung disease, but its survival rate is the lowest among all solid organ transplants (approximately 50% after 5 years), partly due to rejection and side effects of immunosuppressive therapy (Christie et al., 2008). Therefore, using autologous stem cells to generate transplant tissue or for tissue repair is an excellent way to address immune rejection. Currently, bioengineered airways can be used to repair narrowed left main bronchuses in patients (Wu et al., 2009). This involves culturing epithelial cells and bone marrow-derived chondrocytes from patients with bronchomalacia and then seeding them into decellularized tracheal segments. Studies have also shown that a mixed population of neonatal or fetal rat lung epithelial cells and endothelial cells has been seeded into decellularized donor lung scaffolds and transplanted into recipient rats; these grafts promoted gas exchange within a very short time (a few hours) (Ott et al., 2010). Despite these advancements, large-scale culture techniques for stem cells suitable for lung injury repair have not yet been achieved. The key to solving this problem is obtaining stem cells capable of differentiating into all types of lung epithelial cells. However, adult or mouse lungs only contain region-specific adult stem cells with limited differentiation capacity, which are insufficient for repairing damage in any part of the lung.

[0003] During the proximal-distal branching development of the trachea and bronchi, SOX9+ / Id2+ cells are initially pluripotent, capable of simultaneously producing airway and alveolar epithelial cells. Specifically, before embryonic day 13.5 (E1), SOX2+ proximal airway progenitor cells differentiate from SOX9+ epithelial progenitor cells. Furthermore, the fate of SOX9+ epithelial progenitor cells is limited by the time frame of embryonic development; after embryonic day 16.5 (E1), SOX9+ distal progenitor cells no longer possess the ability to differentiate into SOX2+ proximal cells (Yang et al., 2018). In other words, embryonic lung development begins with a population of SOX9+ pluripotent stem cells. This finding suggests that in situ lung regeneration is a process of epithelial cell transdifferentiation (or dedifferentiation), specifically the transdifferentiation of proximal SOX2+ cells into distal SOX9+ epithelial cells. Summary of the Invention

[0004] The technical problem to be solved by this 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; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, this invention first provides a culture medium for transdifferentiating airway epithelial cells into lung progenitor cells. The culture medium can be a basal culture medium supplemented with FGF10, IGF1, KGF, Wnt agonist, retinoic acid, BMP4, PRMT5 inhibitor, p38 kinase inhibitor, and smoothed agonist.

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

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

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

[0012] Furthermore, the basal culture medium can be mTEC Plus culture medium, which can be mTEC Basic culture medium 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).

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

[0014] The present invention also provides the use of any of the culture media described herein in transdifferentiating airway epithelial cells into lung progenitor cells or in the preparation of products for lung injury repair.

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

[0016] A1) The airway epithelial cells were cultured in vitro in 3D to harvest SOX2+ epithelial cells;

[0017] A2) The SOX2+ epithelial cells were transdifferentiated using any of the culture media described herein to harvest SOX9+ epithelial cells.

[0018] In the above method, the 3D culture method can be as follows: the airway epithelial cells are prepared into 3×10⁻⁶ cells / years.4 Cell suspension of 1 cell / mL was prepared and then mixed with an equal volume of Matrigel for incubation.

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

[0020] Furthermore, the 3D culture method may be as follows: the airway epithelial cells are aspirated into a single-cell suspension using airway basal cell culture medium to a concentration of 3 × 10⁻⁶ cells / mL. 4 Calculate 100 μL / well of cells, then mix it with an equal volume of Matrigel (Corning, 354230) and drop it into a 24-well plate (100 μL / well). Place the plate in a 37°C CO2 incubator and wait for the droplets in the 24-well plate to solidify (about 1 hour). Then add 1 mL of airway basal cell culture medium and culture for about 9 days to harvest a large number of cells (SOX2+ epithelial cells).

[0021] The airway basal cell culture medium can be prepared by adding the following to the mTEC Plus culture medium: 1.0 μM A-83-01 (TGFβ antagonist), 0.2 μM DMH-1 (BMP4 antagonist), 0.5 μM CHIR99021 (WNT antagonist), and 5 μM Y27632 (ROCK inhibitor).

[0022] In the above method, the transdifferentiation method can be as follows: 3D cultured SOX2+ epithelial cells are cultured for 2D, and then the culture medium is replaced with any of the culture mediums described in this article, and culture is continued to obtain SOX9+ epithelial cells.

[0023] Further, the transdifferentiation method can be as follows: SOX2+ epithelial cells cultured in vitro for about 10 days are digested with 0.25% trypsin at 37 degrees Celsius for 10 minutes, DMEM / F12 culture medium (containing 10% FBS) is added to terminate the digestion, and the cells are pipetted until the airway spheroids dissociate into single cells and no Matrigel is visible to the naked eye in the liquid. After centrifugation, the supernatant is discarded, and the precipitate is agitated with 1 mL of airway basal cell culture medium. The precipitate is then transferred to a cell culture dish that has been pre-filled with airway basal cell culture medium and cultured at 37 degrees Celsius. The next day, the cells are replaced with freshly prepared airway basal cell culture medium to obtain 2D cultured airway epithelial cells (SOX2+ epithelial cells).

[0024] The 2D cultured airway epithelial cells were passaged into new 6cm culture dishes with a cell confluence of approximately 30%. The next day, the culture medium was replaced with any of the culture media described herein (such as Conditioned Culture Medium 9F), approximately 4mL / dish. Once the cell confluence was greater than 80%, the cells could be passaged to obtain SOX9+ epithelial cells.

[0025] In situ regeneration of mammalian lungs is a pressing scientific problem in the global medical and scientific research fields, with no successful reports to date. This invention, using the mouse as a mammalian model, solves the technical problem of transdifferentiating proximal SOX2+ epithelial cells of the lung bronchi into distal SOX9+ epithelial cells, thus creating conditions for ultimately achieving in situ regeneration of lung organs.

[0026] This invention involves isolating mouse large airway epithelial cells and expanding them in vitro using 3D culture to obtain a large number of SOX2+ lung epithelial cells. Then, by adding small molecule compounds and optimizing the culture system with morphogenetic agents such as Wnt agonists, BMP4, and FGF10, as well as regulatory molecules of various signaling pathways, conditions for obtaining distal SOX9+ epithelial cells are screened. Experimental results show that using the culture medium and transdifferentiation method of this invention, proximal SOX2+ epithelial cells can be efficiently transdifferentiated into distal SOX9+ epithelial cells, with a transdifferentiation efficiency of up to 20%. Attached Figure Description

[0027] Figure 1 It is an organoid derived from proximal cells of mouse lung.

[0028] Figure 2 Immunofluorescence staining of proximal lung cells of mice cultured under 3D-to-2D conditions. Figure 2 In the text, A stands for DAPI; Figure 2 B in the middle is SOX2; Figure 2 C in the middle stands for SOX9.

[0029] Figure 3 Immunofluorescence staining image of transdifferentiated second-generation cells cultured under 9F conditions. Figure 3 In the text, A stands for DAPI; Figure 3 B in the middle is SOX2; Figure 3 C in the middle stands for SOX9.

[0030] Figure 4 Immunofluorescence staining image of transdifferentiated second-generation cells cultured under 9F conditions. Figure 4 In the text, A stands for DAPI; Figure 4 B in the middle is SOX9; Figure 4 C stands for FoxP2.

[0031] Figure 5 Immunofluorescence staining image of transdifferentiated second-generation cells cultured under 9F conditions. Figure 5In the text, A stands for DAPI; Figure 5 B in the middle is ETV5; Figure 5 C in the middle stands for SOX9.

[0032] Figure 6 Immunofluorescence staining image of transdifferentiated second-generation cells cultured under 9F conditions. Figure 6 In the text, A stands for DAPI; Figure 6 B in the middle is SOX2; Figure 6 C in the middle stands for SOX9; Figure 6 D in the middle is Trp63.

[0033] Figure 7 The transdifferentiation efficiency of cultured cells under 9F or 9F-1 conditions (with one factor removed) after 72 hours. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] 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, part number 5989;

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

[0041] BMP4 is a product of Serrui Technology Co., Ltd., with part number PF0901;

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

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

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

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

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

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

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

[0049] This embodiment establishes a three-dimensional culture system for bronchial epithelial cells, harvesting 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 aged 1-7 days after birth and place them on ice. When the mice are in a syncope state, make a longitudinal midline incision from the clavicle to the submandibular region using small scissors. Then lift the trachea and use curved forceps to cut the esophagus directly from the posterior surface of the trachea. Cut the trachea at the bifurcation of the main bronchus with small scissors, gently lift it, and place the excised trachea into ice-cold PBS buffer. Separate the trachea of ​​3-6 mice, then longitudinally cut each trachea with small scissors and place it in digestion solution (1.5 mg / mL Pronase, using Ham's F-12 culture medium as solvent) and digest overnight at 4 degrees Celsius.

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

[0053] Remove the digested trachea from the refrigerator at 4°C. Transfer the trachea and digestion solution to a 15mL centrifuge tube. Add an equal volume of DMEM / F12 culture medium (containing 10% FBS) to terminate digestion. Invert the centrifuge tube 5 times. Remove and discard the trachea with forceps. Centrifuge the remaining liquid at 1000 rpm for 3 minutes. After centrifugation, discard the supernatant; the remaining precipitate is the digested airway epithelial cells. Disperse the cells into a single-cell suspension using airway basal cell culture medium to a concentration of 3 × 10⁶ cells / mL. 4 Cells / mL were mixed with an equal volume of Matrigel (Corning, 354230) and dropped into 24-well plates (100 μL / well). The plates were then placed in a 37°C CO2 incubator. After the droplets solidified (approximately 1 hour), 1 mL of airway basal cell culture medium was added, and the plates were cultured for approximately 9 days. Figure 1 A large number of cells can be harvested.

[0054] The airway basal cell culture medium was prepared by adding the following to mTEC Plus culture medium: 1.0 μM A-83-01 (TGFβ antagonist), 0.2 μM DMH-1 (BMP4 antagonist), 0.5 μM CHIR99021 (WNT antagonist), and 5 μM Y27632 (ROCK inhibitor).

[0055] mTEC Plus culture medium is mTEC Basic culture medium 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 15 mM HEPES, 4 mM L-Glutamine, 3.6 mM NaHCO3, 100 U / mL penicillin, and 100 mg / mL streptomycin to DMEM / F-12 solution.

[0057] 3. 2D culture

[0058] Discard the supernatant from the airway spheroids cultured in vitro for approximately 10 days. Transfer the supernatant to a 15 mL centrifuge tube using 0.25% trypsin. Digest the tube in a 37°C water bath for 10 minutes. Add DMEM / F12 medium (containing 10% FBS) to stop the digestion. Pipette the tube until the airway spheroids dissociate into single cells and no visible Matrigels remain in the liquid. Centrifuge the tube at 1000 rpm for 3 minutes. Discard the supernatant. Transfer the pellet to a 6 cm cell culture dish containing 3 mL of pre-added airway basal cell culture medium. Incubate the dish at 37°C CO2. Replace the medium with freshly prepared airway basal cell culture medium the following day.

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

[0060] The results are as follows Figure 2 As shown, after digesting lung proximal airway organoids cultured in 3D conditions into single cells and culturing them in a 2D culture system, it was found that the proximal epithelial cells still expressed SOX2 but not SOX9. Figure 2 B and C) 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 and optimizing the culture system with Wnt agonists, BMP4, FGF10, and other morphogenetic elements and regulatory molecules of multiple signaling pathways, a culture medium and method capable of transdifferentiating proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells were screened to obtain such cells. The specific method for transdifferentiating proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells is as follows:

[0063] 1. Prepare 9F conditioning medium

[0064] The optimized conditioned medium contains nine factors (including small molecule compounds, growth factors, and morphogenetic elements), named Conditioned Culture Medium 9F (also known as Conditioned Culture Medium TFICKRBEPS). Conditioned Culture Medium 9F includes mTECPlus 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, and the small molecule compound retinoic acid (RA, molecular formula: C). 20 H 28 O2; CAS No.: 302-79-4), bone morphogenetic protein 4 (BMP4), PRMT5 inhibitor, p38 kinase inhibitor, and the small molecule compound Smoothened agonist (SAG, molecular formula: C 28 H 28 ClN3OS, CAS No.: 912545-86-9).

[0065] Specifically, the Wnt agonist is the small molecule compound CHIR99021 (molecular formula: C 22 H 18 C l2 N8; CAS No.: 252917-06-9);

[0066] 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 No.: 285983-48-4).

[0068] This conditioned medium 9F is prepared using mTEC Plus culture medium as solvent and nine factors as solutes. The specific concentrations of the nine added factors in 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 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).

[0079] The mTEC Basic culture medium is 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.

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

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

[0082] FGF10 50ug / mL 10μL 10 ng / mL IGF1 100ug / mL 15μL 30ng / 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. SOX2+ cell transdifferentiation

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

[0085] Immunofluorescence detection of transdifferentiated cells ( Figure 3 Instructions: 1. Spread cells onto small discs with a confluence of approximately 30%-50%. Replace with fresh culture medium the next day (9F). On the third day, fix cells with 4% paraformaldehyde (PFA) at room temperature for 5 minutes. 2. Wash the discs three times with 0.2% Triton X-100 PBS. Incubate cells with wash buffer for 5 minutes at room temperature between each wash. 3. Cover cells with 1% BSA in PBS + diluted primary antibody and incubate at room temperature for 2 hours or overnight at 4°C. 4. Wash cells three times with 0.2% Triton X-100 PBS. 5. Cover cells with 1% BSA in PBS + diluted fluorescently conjugated secondary antibody and incubate at room temperature for 1 hour (protected from light). 6. Wash cells three times with 0.2% Triton X-100 PBS. 7. Cover the discs with sufficient 1 μg / mL DAPI solution. After incubating for 10 minutes, the small discs were inverted onto a glass slide containing the 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 conditioned medium 9F (TFICKRBEPS), after 72 hours of treatment and passage, lung epithelial progenitor cells (i.e., distal SOX9+ epithelial cells) expressing progenitor cell markers such as Sox9, FoxP2, and ETV5 can be obtained. The components of the conditioned medium 9F of this invention, after optimization, can efficiently and synergistically induce the transdifferentiation of proximal SOX2+ epithelial cells into distal SOX9+ epithelial cells. After statistical analysis of the proportion of cells with high SOX9 expression (strongly positive) and SOX2+ negative or weakly positive, such as… Figure 7 They found that the transdifferentiation efficiency could reach about 20%, which is far higher than the efficiency of cell transdifferentiation or reprogramming in other studies.

[0087] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A culture medium for transdifferentiating mouse airway epithelial cells into mouse 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 smoothed agonist to the basic culture medium. The Wnt agonist is CHIR99021; The PRMT5 inhibitor is EPZ015666; The p38 kinase inhibitor is BIRB796; In the culture medium, the concentrations of FGF10, IGF1, KGF, Wnt agonist, BMP4, PRMT5 inhibitor, p38 kinase inhibitor, and smoothed agonist were 5-50 nM. The concentrations of FGF10, IGF1, KGF, Wnt agonist, PRMT5 inhibitor, p38 kinase inhibitor, and smoothed agonist were 5-20 nM. The basic culture medium is mTEC Plus culture medium, which is mTEC Basic culture medium supplemented with a final concentration of 10 mg / mL insulin, 5 mg / mL transferrin, 0.1 mg / mL CT, 25 ng / mLEGF, 30 mg / mL BPE, and 5% FBS (v / v). The mTEC Basic culture medium was 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.

2. The culture medium according to claim 1, characterized in that, In the culture medium, the concentrations of FGF10, IGF1, KGF, Wnt agonist, retinoic acid, BMP4, PRMT5 inhibitor, p38 kinase inhibitor, and smoothed agonist were 10 nM.

3. The culture medium according to claim 1 or 2, when used to inoculate the proximal airway of mice with SOX2 + Epithelial cells transdifferentiate into mouse distal SOX9 + Application in lung progenitor cells.

4. A method for transdifferentiating mouse airway epithelial cells into mouse SOX9 cells. + The method for distal lung progenitor cells is characterized by, The method includes the following steps: A1) The mouse airway epithelial cells were cultured in vitro for 3D and SOX2 was harvested. + Epithelial cells; A2) Using the culture medium according to claim 1 or 2 to prepare the SOX2 + Epithelial cells undergo transdifferentiation to harvest SOX9. + Epithelial cells.

5. The method according to claim 4, characterized in that, The 3D culture method is as follows: the airway epithelial cells are prepared into 3×10⁶ cells / years. 4 Cell suspension of 1 cell / mL was prepared and then mixed with an equal volume of Matrigel for incubation.

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