Composition and method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells

Abstract

The invention discloses a composition and a method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells. The composition is prepared from mTeSRTMPlus, PneumaCultTM-Ex Plus Medium, an SFD basal culture medium and an induction compound combination, wherein the mTeSRTMPlus, the PneumaCultTM-Ex Plus Medium and the SFD basal culture medium are used for culturing the airway basal stem cells to differentiate into the respiratory alveolar stem cells; wherein the inducing compound combination is prepared from DAPT (Diamantadine Phosphate), PF573228, JNKIN8 and CA3 (Carbonic Acid 3). By developing the specific differential culture medium, a signal channel in a lung microenvironment can be simulated, so that the differentiation process of the basal stem cells to the RASCs is accurately controlled, the RASCs with functionality and regeneration capacity can be efficiently generated, and research on a mechanism of lung diseases and development of a new cell therapy are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a composition and method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells. Background Art

[0002] Respiratory Alveolar Stem Cells (RASCs) are a type of lung stem cell with bidirectional differentiation ability, which can play a key role in lung injury repair and tissue regeneration. These stem cells are mainly located in the respiratory bronchi and can differentiate into two key lung cells: Club cells in the bronchial epithelium and type II alveolar cells (AT2 cells) in the alveoli. Club cells have repair functions and protect lung tissue by secreting proteins, playing an important role in the repair and immune response of bronchial epithelium. AT2 cells are responsible for secreting surfactant, maintaining the stability of the alveoli, and participating in alveolar repair by transforming into type I alveolar cells (AT1) when the lungs are injured. Studies have shown that RASCs can self-renew and differentiate into these two types of cells under inflammatory and damaged conditions to achieve lung repair function, depending on the local microenvironment of the lungs. RASCs have broad application prospects in cell therapy, especially in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and acute lung injury (ALI), by promoting the regeneration of damaged tissues and hopefully restoring normal lung function.

[0003] Therefore, mastering the technology of effectively inducing basal stem cells to differentiate into RASCs in an in vitro culture environment can not only provide a basic platform for studying the biological characteristics and regulatory mechanisms of RASCs, but also provide possibilities for their application in regenerative medicine and cell therapy. By developing specific differentiation culture media, researchers can simulate the signaling pathways in the lung microenvironment, thereby precisely controlling the differentiation process of basal stem cells (BCs) into RASCs. Such a culture system can efficiently generate functional and regenerative RASCs for studying the mechanisms of lung diseases and developing new cell therapies. In addition, in the treatment of lung diseases, this type of culture medium can provide a stable source of RASCs for personalized cell therapy, avoiding direct reliance on the limited number of stem cells in the patient's body, and promoting the advancement of lung regenerative therapy. Therefore, the development of efficient in vitro differentiation culture media is a key step in promoting RASCs research and clinical application. Summary of the invention

[0004] The purpose of the present invention is to provide a composition and method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect is to provide a composition for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, including mTeSR TM Plus, PneumaCult TM -ExPlusMedium, SFD basal medium and an induction compound combination; wherein the induction compound combination includes DAPT, PF573228, JNKIN8 and CA3.

[0007] Furthermore, the inducing compound combination includes 2-20 μM DAPT, 2-20 μM PF573228, 0.1-2 μM JNKIN8 and 1-10 μM CA3.

[0008] Furthermore, the SFD basal medium comprises:

[0009] Iscove's Modification of DMEM;

[0010] B27 supplement (serum-free): 1%-2%;

[0011] N2 supplement: 1%-2%;

[0012] L-ascorbic acid: 50-200 μg / ml;

[0013] 1-Thioglycerol: 0.2-1 μM;

[0014] Penicillin-streptomycin mixture: 100-200 U / ml and 100-200 μg / ml;

[0015] GlutaMAX glutamine replacement: 1-4mM;

[0016] MEM non-essential amino acids: 1-2%;

[0017] ROCK inhibitor Y-27632: 10-250 μM;

[0018] MTG monothioglycerol: 0.1-1 μM;

[0019] FGF10: 50-200 ng / ml;

[0020] FGF7: 5-20 ng / ml;

[0021] Dexamethasone: 50-100 ng / ml;

[0022] 8-Bromo-cAMP: 0.1-1 mM;

[0023] IBMX: 0.1-1mM;

[0024] Retinoic acid RA: 0.1-1 μM.

[0025] The second aspect is to provide a method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, using the above composition, comprising the following steps:

[0026] Step 1: Dilute the matrix gel with DMEM:F12 at a ratio of 1:50-1:200 and plate it into the desired culture dish, preheating it;

[0027] Step 2: Plate 10,000 to 50,000 basal stem cells per square centimeter in the desired culture dish using PneumaCult TM -Ex Plus Medium; change the medium every 48 hours until the basal stem cells grow to 80%-95% confluence;

[0028] Step 3: Replace the first stage induction medium with SFD basal medium and mTeSR TM Plus is mixed in a 1:1 ratio, and the solution is changed every 48 hours. The first stage is 6-8 days;

[0029] Step 4: Replace the second-stage induction medium. The medium formula is a combination of 100% SFD basal medium and induction compounds. The medium is changed every 48 hours. The second stage is 8-10 days.

[0030] Furthermore, in step 1, the preheating temperature is 36-38° C. and the time is 0.5-1 hour.

[0031] Furthermore, in step 2, the volume of the replacement solution is 0.2 mL / cm 2 .

[0032] Furthermore, in step 3, the volume of the replacement solution was 0.2 mL / cm 2 .

[0033] Furthermore, in step 4, the volume of the replacement solution is 0.2 mL / cm 2 .

[0034] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0035] The present invention can simulate the signal pathways in the lung microenvironment by developing a specific differentiation culture medium, thereby accurately controlling the differentiation process of basal stem cells into RASCs, and can efficiently generate RASCs with functional and regenerative abilities, which is beneficial for studying the mechanisms of lung diseases and developing new cell therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The morphological characteristics of RASCs are shown (10×).

[0037] Figure 2 Shown are the results of immunofluorescence staining of RASCs.

[0038] Figure 3 The results of flow cytometry analysis of RASCs after digestion are shown.

[0039] Figure 4 The results of cell immunofluorescence staining after induction with SFD basal medium in Comparative Example 1 are shown. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0041] Example 1

[0042] This embodiment provides a composition for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, including mTeSR TM Plus, PneumaCult TM -Ex Plus Medium, SFD basal medium and an inducing compound combination; wherein the inducing compound combination includes 10 μM DAPT (γ-secretase inhibitor, CAS No.: 208255-80-5), 10 μM PF573228 (FAK inhibitor, CAS No.: 869288-64-2), 1 μM JNKIN8 (JNK inhibitor, CAS No.: 1410880-22-6) and 10 μM CA3 (YAP inhibitor, CAS: 300802-28-2).

[0043] The SFD basal medium comprises:

[0044] Iscove's Modification of DMEM;

[0045] B27 supplement (serum-free): 2%;

[0046] N2 supplement: 2%;

[0047] L-ascorbic acid: 150 μg / ml;

[0048] 1-Thioglycerol: 0.8 μM;

[0049] Penicillin-streptomycin mixture: 150 U / ml and 150 μg / ml;

[0050] GlutaMAX glutamine replacement: 3mM;

[0051] MEM non-essential amino acids: 1.5%;

[0052] ROCK inhibitor Y-27632: 200 μM;

[0053] MTG monothioglycerol: 0.8 μM;

[0054] FGF10: 150 ng / ml;

[0055] FGF7: 15 ng / ml;

[0056] Dexamethasone: 80ng / ml;

[0057] 8-Bromo-cAMP: 0.8 mM;

[0058] IBMX: 0.9 mM;

[0059] Retinoic acid RA: 0.7 μM.

[0060] Example 2

[0061] This embodiment provides a composition for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, including mTeSR TM Plus, PneumaCult TM -Ex Plus Medium, SFD basal medium and an inducing compound combination; wherein the inducing compound combination includes 10 μM DAPT (γ-secretase inhibitor, CAS No.: 208255-80-5), 10 μM PF573228 (FAK inhibitor, CAS No.: 869288-64-2), 1 μM JNKIN8 (JNK inhibitor, CAS No.: 1410880-22-6) and 10 μM CA3 (YAP inhibitor, CAS: 300802-28-2).

[0062] The SFD basal medium comprises:

[0063] Iscove's Modification of DMEM;

[0064] B27 supplement (serum-free): 1%;

[0065] N2 supplement: 1%;

[0066] L-ascorbic acid: 100 μg / ml;

[0067] 1-Thioglycerol: 0.5 μM;

[0068] Penicillin-streptomycin mixture: 100 U / ml and 100 μg / ml;

[0069] GlutaMAX glutamine replacement: 2mM;

[0070] MEM non-essential amino acids: 1%;

[0071] ROCK inhibitor Y-27632: 100 μM;

[0072] MTG monothioglycerol: 0.3 μM;

[0073] FGF10: 80 ng / ml;

[0074] FGF7: 10 ng / ml;

[0075] Dexamethasone: 60ng / ml;

[0076] 8-Bromo-cAMP: 0.5 mM;

[0077] IBMX: 0.6 mM;

[0078] Retinoic acid RA: 0.6 μM.

[0079] Example 3

[0080] This embodiment provides a method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, using the composition provided in Example 1, comprising the following steps:

[0081] Step 1: Dilute the matrix gel with DMEM:F12 at a ratio of 1:100 and plate it into the required culture dish, and preheat it at 37°C for more than half an hour;

[0082] Step 2: Plate 40,000 to 50,000 basal stem cells per square centimeter in the desired culture dish using PneumaCult TM -Ex Plus Medium; change the medium every 48 hours at a volume of 0.2 mL / cm 2 , until the basal stem cells grow to 80%-95% confluence, about 4-6 days;

[0083] Step 3: Replace the first stage induction medium with SFD basal medium and mTeSR TM Plus in a 1:1 ratio, change the solution every 48 hours, and the replacement volume is 0.2mL / cm 2 , the first stage is 6-8 days;

[0084] Step 4: Replace the second stage induction medium. The medium formula is 100% SFD basal medium and induction compound combination. The medium is changed every 48 hours, and the replacement volume is 0.2mL / cm 2 , the second stage is 8-10 days;

[0085] Step 5: Follow-up tryple Express (Gibco), 37°C, 10 min, and then perform detection and subsequent experiments after digestion.

[0086] Comparative Example 1

[0087] The control condition was to use only SFD basic induction medium for directed differentiation without adding any compound for induction assistance. Immunofluorescence staining was performed 14 days after induction, and the results (refer to Figure 4 ) indicates that the differentiated cells only express SCGB3A2 but not SFTPB, indicating that this condition cannot induce BCs to differentiate into RASCs.

[0088] Verification Example 1

[0089] 1. Cell Immunofluorescence Staining Protocol

[0090] The cultured cells were fixed with 4% PFA for 5 min at room temperature, rinsed in PBS, washed 3 times (5 min / time) with PBS + 0.2% TritonX-100, incubated with primary antibody for 2 hours at room temperature, or diluted in PBS + 1% BSA at 4 ° C overnight (> 16 hours). After incubation, rinsed 4 times with PBS + 0.2% TritonX-100 and placed with secondary antibody at room temperature for 1-2 hours. After rinsing 4 times with PBS + 0.2% TritonX-100, add enough DAPI solution to cover the cells for 3 minutes for DAPI staining. Then, remove the DAPI solution before imaging and replace it with PBS.

[0091] 2. Cell flow cytometry

[0092] 1) Cell counting and washing:

[0093] Take a portion of the digested cells and count them to ensure the number of cells required for the experiment (usually 10^5 to 10^6 cells);

[0094] The cells were centrifuged at 300-500 g for 5 min and the supernatant was removed;

[0095] The cells were resuspended in flow buffer (PBS+1% BSA or PBS+2% FBS) and washed again by centrifugation.

[0096] 2) Antibody staining:

[0097] Resuspend the washed cells in an appropriate amount of flow buffer, usually adding about 10^5 to 10^6 cells per 100 μL;

[0098] According to the experimental requirements, add an appropriate amount of fluorescent labeled antibody (according to the recommended concentration in the antibody manual, generally 1-10 μL), and you can choose cell surface marker antibody or intracellular staining antibody;

[0099] After mixing the cells with the antibodies, incubate them at 4°C in the dark for 30 minutes;

[0100] After the incubation, the cells were washed twice with flow cytometry buffer (centrifugation at 300-500 g for 5 min) to remove unbound antibodies;

[0101] If multiple staining is desired, the staining step can be repeated after washing, adding additional antibodies.

[0102] 3) Cell resuspension and preparation for flow cytometry analysis

[0103] Finally, resuspend the stained cells in 300-500 μL flow cytometry buffer to ensure that the cell concentration is suitable for flow cytometry analysis;

[0104] To prevent cell aggregation, filter the cell suspension using a 40 μm filter to ensure single cell suspension and avoid clogging of the tube.

[0105] 4) Flow cytometry analysis using NOVOexpress

[0106] Start the NOVOexpress flow cytometer and perform system calibration (usually including instrument performance check, fluorescence compensation setting, etc.);

[0107] Create a new experiment in NOVOexpress software, select the appropriate fluorescence channel settings, and adjust the corresponding parameters according to the staining protocol;

[0108] Load sample tubes, run sample collection, and observe scatter plots and fluorescence signals in real time to ensure the accuracy of data collection;

[0109] According to experimental requirements, set the gating strategy (such as forward scatter, side scatter, fluorescence signal, etc.) to distinguish positive and negative cell populations;

[0110] After completing sample collection, save the data file for subsequent analysis (such as FCS file format).

[0111] 5) Data analysis

[0112] The collected data were analyzed using NOVOexpress software to generate scatter plots, histograms, and perform quantitative analysis of parameters such as fluorescence intensity and positive rate.

[0113] Based on the different antibody stainings, calculate the proportions of different cell populations and interpret the results based on the experimental goals.

[0114] Experimental Results

[0115] 1. Take photos of cells after 14 days of differentiation to record morphological changes. Figure 1 As shown in Figure 2, the cells showed a dense colony structure, with cells arranged closely and in polygonal or irregular shapes, growing in clusters with clear boundaries, especially at the edges of the colonies. These cells showed strong refractive index, indicating that they had high proliferation activity.

[0116] 2. Immunofluorescence staining results ( Figure 2 ) showed that induced differentiated RASCs expressed both SFTPB and SCGB3A2 markers.

[0117] 3. After digestion, RASCs were analyzed by flow cytometry. The results showed that about 86% of the cells were SCGB3A2 positive, 24.66% of the cells were SFTPB positive, and 21.79% of the cells were double positive for SFTPB and SCGB3A2 ( Figure 3 ).

[0118] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, characterized in that: Including mTeSR TM Plus, PneumaCult TM -Ex Plus Medium, SFD basal medium and an inducing compound combination; wherein the inducing compound combination includes DAPT, PF573228, JNKIN8 and CA3.

2. The composition for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells according to claim 1, characterized in that: The inducing compound combination includes 2-20 μM DAPT, 2-20 μM PF573228, 0.1-2 μM JNKIN8 and 1-10 μM CA3.

3. The composition for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells according to claim 1, characterized in that: The SFD basal medium comprises: Iscove's Modification of DMEM; B27 supplement (serum-free): 1%-2%; N2 supplement: 1%-2%; L-ascorbic acid: 50-200 μg / ml; 1-Thioglycerol: 0.2-1 μM; Penicillin-streptomycin mixture: 100-200 U / ml and 100-200 μg / ml; GlutaMAX glutamine replacement: 1-4mM; MEM non-essential amino acids: 1-2%; ROCK inhibitor Y-27632: 10-250 μM; MTG monothioglycerol: 0.1-1 μM; FGF10: 50-200 ng / ml; FGF7: 5-20 ng / ml; Dexamethasone: 50-100 ng / ml; 8-Bromo-cAMP: 0.1-1 mM; IBMX: 0.1-1mM; Retinoic acid RA: 0.1-1 μM.

4. A method for culturing airway basal stem cells to differentiate into respiratory alveolar stem cells, using the composition according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: Dilute the matrix gel with DMEM:F12 at a ratio of 1:50-1:200 and plate it into the desired culture dish, preheating it; Step 2: Plate 10,000 to 50,000 basal stem cells per square centimeter in the desired culture dish using PneumaCult TM -Ex Plus Medium; change the medium every 48 hours until the basal stem cells grow to 80%-95% confluence; Step 3: Replace the first stage induction medium with SFD basal medium and mTeSR TM Plus is mixed in a 1:1 ratio, and the solution is changed every 48 hours. The first stage is 6-8 days; Step 4: Replace the second-stage induction medium. The medium formula is a combination of 100% SFD basal medium and induction compounds. The medium is changed every 48 hours. The second stage is 8-10 days.

5. The culture method according to claim 4, characterized in that In step 1, the preheating temperature is 36-38° C. and the time is 0.5-1 hour.

6. The culture method according to claim 4, characterized in that In step 2, the volume of the replacement solution is 0.2 mL / cm 2 .

7. The culture method according to claim 4, characterized in that: In step 3, the volume of the replacement solution is 0.2 mL / cm 2 .

8. The culture method according to claim 4, characterized in that: In step 4, the volume of the replacement solution is 0.2 mL / cm 2 .

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