In-vitro culture method of chicken pluripotent stem cells

Abstract

The application discloses an in-vitro culture method of chicken pluripotent stem cells, and belongs to the field of animal husbandry and biotechnology. By adding FGF2, IWR-1 and XAV-93 and taking STO cells as a feeder layer, a FIX culture system is established, fresh egg EGK.X stage blastoderm clear zone cells are separated, and the separated cells are cultured in the FIX culture system in a 37 DEG C incubator with 5% CO2 and saturated humidity, the culture solution is replaced every day, and the cells are subcultured every 2-3 days to carry out in-vitro culture of chicken pluripotent stem cells PSCs. The method provided by the application can make chicken PSCs proliferate rapidly in a short period of time and maintain pluripotency and differentiation potential of the cells. The method provides materials for research on biological characteristics of chicken PSCs, and lays an experimental foundation for new methods of breeding, gene modification, poultry molecular design breeding and germplasm resource cryopreservation of good breeds of poultry.

Description

Technical Field

[0001] This invention relates to a method for in vitro culture of chicken pluripotent stem cells, belonging to the fields of animal husbandry and biotechnology. Background Technology

[0002] Embryonic stem cells (PSCs) are undifferentiated cells obtained by isolating inner cell mass cells or primordial germ cells from early embryos and inhibiting differentiation under suitable in vitro and in vivo conditions. These cells possess unlimited proliferative capacity, self-renewal ability, and the potential to differentiate into various cell types, including germline cells. They have been widely used in tissue engineering, regenerative medicine, drug screening, toxicity testing, and gene function research. However, due to the unique genetic characteristics of different species, PSC culture systems vary significantly. A stable and efficient chicken PSC culture system has not yet been established. Therefore, this invention explored various culture media, feeder layers, conditioned media, growth factors, serum, serum substitutes, and small molecule compounds in chicken PSC culture, ultimately finding an efficient and widely applicable chicken PSC culture system. After in vitro culture, chicken PSCs retain their pluripotency and differentiation potential. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a method for in vitro culture of chicken pluripotent stem cells, offering a new approach for the efficient culture and maintenance of pluripotency of chicken PSCs.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a method for in vitro culture of chicken pluripotent stem cells, the method comprising the following steps:

[0005] Step 1: Prepare chicken KOSR culture medium;

[0006] Step 2, prepare FIX medium: Add FGF2, IWR-1 and XAV-939 to chicken KOSR medium to prepare chicken pluripotent stem cell (PSC) medium FIX;

[0007] Step 3, Preparation of STO feeder cells: When the STO cells grow to 80% confluence, they are treated with mitomycin C and then frozen in liquid nitrogen for later use;

[0008] Step 4: Thaw the inactivated STO feeder cells and seed them onto a culture dish;

[0009] Step 5, Isolation and culture of chicken PSCs: Isolate chicken embryonic disc cells from the bright area and culture and expand them in a culture dish with STO feeder cells and FIX medium.

[0010] Furthermore, in step 1, the chicken KOSR culture medium consists of KO-DMEM, 15% KnockOut serum substitute, 0.2% chicken serum, 1×B-27 supplement, 1×N-2 supplement, 1×GlutaMax, 1×NEAA, 0.1mM β-mercaptoethanol, 1×EmbryoMax nucleoside, 1mM sodium pyruvate and 1×penicillin / streptomycin.

[0011] Furthermore, in step 2, the concentration of FGF2 was 4.0 ng / ml, the concentration of IWR-1 was 2 μM, and the concentration of XAV-939 was 2.5 μM.

[0012] Further, step 3 specifically involves treating STO cells with 10 μg / ml mitomycin C for 2 hours when the cells reach 80% confluence, and then freezing them in liquid nitrogen for later use.

[0013] Furthermore, in step 4, the inactivated STO feeder cells were thawed the day before the chicken PSCs culture and cultured at 2×10⁻⁶. 4 pcs / cm 2 Inoculate onto culture dishes coated with 0.1% gelatin.

[0014] Further, step 5 specifically involves: collecting fresh hatching eggs and disinfecting them with 70% ethanol; cracking open the eggshell with tweezers to expose the germinal disc; placing a disinfected and dried filter paper ring on the germinal disc, then cutting along the outer edge of the filter paper ring with scissors to separate the germinal disc from the yolk; holding the filter paper with tweezers and turning it over along with the germinal disc to separate it from the yolk; washing the yolk with PBS in a culture dish to remove it, and then using an injection needle under a microscope to separate the BCs from the visible area of ​​the germinal disc; gently blowing the BCs to disperse them into a cell suspension, then centrifuging at 1500 rpm for 5 minutes to precipitate the cells; adding FIX medium, and inoculating the BCs of each embryo into a culture dish with an inactivated STO feeder layer, changing the medium daily; after 2-3 days, PSC clones are formed, and the formed PSC clones are digested with Accutase enzyme for 5-7 minutes, then inoculated into a new culture dish with STO cells for passage.

[0015] The present invention has the following technical effects: (1) The chicken PSCs culture system and in vitro culture method provided by the present invention can promote the formation of PSCs-like clones by chicken embryonic disc cells and enable them to proliferate rapidly to meet the needs of PSCs. After in vitro culture, chicken PSCs can still maintain their pluripotency and differentiation potential, which is beneficial to subsequent experiments and research.

[0016] (2) The FIX culture system used in this invention contains clearly defined additives such as cytokines, small molecule compounds, and serum substitutes, and the STO cells used are standardized production cell lines. Therefore, this culture system has significant potential for promotion and application in other laboratories. The culture system and method provided by this invention offer reliable materials and means for studying the biological characteristics of chicken PSCs. This method provides a new approach and experimental basis for the propagation of superior chicken breeds, gene modification, and germplasm resource preservation, and has important application potential. Attached Figure Description

[0017] Figure 1 These are morphological images of PSCs cultured in the FIX system during passage.

[0018] Figure 2 AKP staining and PAS staining were used to detect alkaline phosphatase and glycogen granules in PSCs;

[0019] Figure 3 The expression of pluripotency genes in PSCs was detected by RT-PCR.

[0020] Figure 4 It is an indirect immunofluorescence staining method to detect the expression of PSC-specific proteins;

[0021] Figure 5 These are images of embryoid bodies formed by the self-differentiation of PSCs;

[0022] Figure 6 The expression of genes in the three germ layers and pluripotency genes before and after PSCs form an embryoid body was detected by RT-PCR.

[0023] Figure 7 It is an indirect immunofluorescence staining method to detect the expression of three germ layer marker proteins in cells differentiated from PSCs. Detailed Implementation

[0024] The content and accompanying drawings of the present invention will be described in detail below. This embodiment is implemented under the premise of the technical solution of the present invention, and involves detailed implementation schemes and operation processes. However, the protection scope of the present invention is not limited to the following specific embodiments. The terminology used in the present invention is only for the purpose of describing specific embodiments and is not intended to limit the present invention.

[0025] Example 1:

[0026] A method for in vitro culture of chicken pluripotent stem cells, comprising the following steps:

[0027] 1. Prepare chicken KOSR medium: Add 15% KnockOut serum substitute, 0.2% chicken serum, 1×B-27 supplement, 1×N-2 supplement, 1×GlutaMax, 1×NEAA, 0.1mM β-mercaptoethanol, 1×EmbryoMax nucleoside, 1mM sodium pyruvate and 1×penicillin / streptomycin to KO-DMEM.

[0028] 2. Prepare FIX medium: Add 4.0 ng / ml FGF2, 2 μM IWR-1 and 2.5 μM MXAV-939 to chicken KOSR medium.

[0029] 3. Preparation of STO feeder cells: When STO cells reached 80% confluence, they were treated with 10 μg / ml mitomycin C for 2 h, and then frozen in liquid nitrogen for later use. The day before culturing chicken PSCs, the inactivated STO cells were thawed and cultured at 2 × 10⁻⁶ cells / mL. 4 pcs / cm 2 Inoculate onto culture dishes coated with 0.1% gelatin.

[0030] 4. Isolation and Culture of Chicken PSCs: Collect fresh chicken eggs and sterilize them with 70% ethanol. Crack the eggshell with tweezers to expose the germinal disc. Place a sterilized and dried filter paper ring on the germinal disc, then cut along the outer edge of the ring with scissors to separate the germinal disc from the yolk. Hold the filter paper with tweezers and invert it along with the germinal disc to separate it from the yolk. Wash the egg with PBS in a culture dish to remove the yolk, then isolate the BCs under a microscope using an injection needle. Disperse the BCs by gently pipetting to form a cell suspension, then centrifuge at 1500 rpm for 5 minutes to pellet the cells. Add FIX medium and inoculate the BCs of each embryo into a culture dish with an inactivated STO feeder layer, changing the medium daily. After 2-3 days, PSC clones will form. Digest the formed PSC clones with Accutase enzyme for 5-7 minutes, then inoculate them into a new culture dish with STO cells for passage (e.g., ...). Figure 1 (As shown).

[0031] Example 2: Identification of Chicken PSCs

[0032] 1. AKP staining

[0033] When PSCs were passaged to passages 1 and 6, the PSC clones were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Then, the fixed cells were stained with AKP staining working solution for 30 min at room temperature. The staining working solution contained 100 mM Tris buffer (pH 8.2-8.4), 200 mg / ml naphthol AS-MX phosphate, and 1 mg / ml solid red TR salt. After staining, the cells were washed again with PBS to stop the staining reaction. Finally, the cells were observed and photographed under a microscope. The results showed that the PSCs were AKP positive after culture, indicating that the cultured PSCs still maintained alkaline phosphatase expression (e.g., ...). Figure 2 (As shown in A).

[0034] 2. PAS staining

[0035] When PSCs were passaged to generations 1 and 6, PAS staining was performed using a Solarbio G1360 staining kit according to the manufacturer's instructions. After washing the PSC clones with PBS, fixation solution was added for 30 min, followed by two washes with water. Oxidizing agent was then added and oxidized at room temperature for 15-20 min. The slides were then rinsed four times with distilled water and air-dried. Shiff reagent was added, and the slides were placed in a humidified chamber and incubated for 8-15 min. The slides were removed from the humidified chamber and washed twice with sodium sulfite solution for 10 s each time. Then, they were rinsed with tap water for 2 min, and Mayer's hemoglobin staining solution was added for 10-30 s. The slides were rinsed with water and then observed and photographed under a microscope. The results showed that the cultured PSCs were PAS-positive, indicating that the cultured PSCs still possessed glycogen granules (such as...). Figure 2 (as shown in B).

[0036] 3. RT-PCR detection

[0037] PSCs were collected, with BCs used as a positive control and CEFs as a negative control. mRNA was extracted from each sample and reverse transcribed into cDNA. Pluripotency genes (POUV, SOX2, and NANOG) and trigerm layer genes (PAX6, PPARA, and HNF1A) were amplified by PCR, with GAPDH as an internal control. The PCR products were analyzed by agarose gel electrophoresis to detect the expression of each gene. The results showed that these cells could still express the pluripotency genes POUV, SOX2, and NANOG after culture. Figure 3 (as shown); however, after differentiation, the expression of pluripotency genes decreased, while the expression of the three germ layer genes PAX6, PPARA, and HNF1A increased (as shown). Figure 6 (As shown).

[0038] 4. Indirect immunofluorescence

[0039] PSCs were seeded on coverslips, and after cloning, they were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and washed twice with PBS. Next, they were permeabilized with PBS containing 0.5% Triton X-100 for 15 min at room temperature and incubated in blocking buffer (PBS containing 1.0% BSA) at room temperature for 1 h. Then, PSCs were incubated overnight with primary antibody. Antibodies used for detecting pluripotency genes included SSEA-1, MEA-1, SOX2, and C-KIT. Antibodies used for detecting trigerm layer genes included AFP, B4, and PAX6. After three PBS washes, PSCs were incubated with secondary antibody at room temperature for 1 h. Next, PSCs were stained with DAPI and washed three times with PBS. After adding 10 μl of anti-fluorescence quencher, the cells were mounted and observed and photographed using a fluorescence microscope. The results showed that these cells could still express pluripotency proteins SSEA-1, MEA-1, SOX2, and C-KIT (e.g., ...) after culture. Figure 4 (as shown); after differentiation, it can express the three germ layer-related proteins AFP, B4, and PAX6 (as shown). Figure 7 (As shown).

[0040] 5. Self-differentiation ability test

[0041] Chicken PSCs were digested with Accutase and collected in 15 ml centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The collected cells were then seeded into culture dishes and allowed to adhere for 1 h. The supernatant and non-adhered PSCs were then collected (this step was to remove feeder cells), and centrifuged again at 1000 rpm for 5 min. The cells were then resuspended in chicken KOSR medium and cultured in ultra-low adsorption culture dishes (CLS3473, Corning) at 37°C in a 5% CO2 incubator for 3-5 days. Subsequently, the resulting EBs were seeded in 1% gelatin-coated culture dishes and cultured at 37°C in a 5% CO2 incubator for 5 days to observe their differentiation ability. During the process, the expression of pluripotency and trigerm layer genes and proteins was detected by RT-PCR and indirect immunofluorescence assays. The results showed that after PSC differentiation, the expression of pluripotency genes decreased, while the expression of trigerm layer genes PAX6, PPARA, and HNF1A increased, and trigerm layer-related proteins AFP, B4, and PAX6 (e.g., ...) were expressed. Figure 5-7 (As shown).

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A method for in vitro culture of chicken pluripotent stem cells, characterized in that, The method includes the following steps: Step 1: Prepare chicken KOSR medium, which consists of KO-DMEM, 15% KnockOut serum substitute, 0.2% chicken serum, 1× B-27 supplement, 1× N-2 supplement, 1× GlutaMax, 1× NEAA, 0.1 mM β-mercaptoethanol, 1× EmbryoMax nucleoside, 1 mM sodium pyruvate and 1× penicillin / streptomycin; Step 2, prepare FIX medium: Add FGF2, IWR-1 and XAV-939 to chicken KOSR medium to prepare chicken pluripotent stem cell (PSC) medium FIX; Step 3, Preparation of STO feeder cells: When the STO cells grow to 80% confluence, they are treated with mitomycin C and then frozen in liquid nitrogen for later use; Step 4: Thaw the inactivated STO feeder cells and seed them onto a culture dish; Step 5, Isolation and culture of chicken PSCs: Isolate the cells in the bright area of ​​the chicken embryonic disc and culture and expand them in a culture dish with STO feeder layer cells and FIX medium.

2. The method for in vitro culture of chicken pluripotent stem cells according to claim 1, characterized in that, In step 2, the concentration of FGF2 was 4.0 ng / ml, the concentration of IWR-1 was 2 μM, and the concentration of XAV-939 was 2.5 μM.

3. The method for in vitro culture of chicken pluripotent stem cells according to claim 1, characterized in that, Step 3 is as follows: When STO cells grow to 80% confluence, treat them with 10 μg / ml mitomycin C for 2 h, and then freeze them in liquid nitrogen for later use.

4. The method for in vitro culture of chicken pluripotent stem cells according to claim 1, characterized in that, In step 4, the day before culturing chicken PSCs, the inactivated STO feeder cells are thawed and cultured at 2×10⁻⁶. 4 pcs / cm 2 Inoculate onto culture dishes coated with 0.1% gelatin.

5. The method for in vitro culture of chicken pluripotent stem cells according to claim 1, characterized in that, Step 5 involves the following steps: Collect fresh hatching eggs and sterilize them with 70% ethanol; use tweezers to crack open the eggshell to expose the germinal disc; place a sterilized and dried filter paper ring on the germinal disc, then use scissors to cut along the outer edge of the filter paper ring to separate the germinal disc from the yolk; use tweezers to hold the filter paper and flip it over along with the germinal disc to separate it from the yolk; wash the yolk with PBS in a culture dish to remove it, and then use an injection needle under a microscope to separate the blastocyst cells (BCs) from the visible area of ​​the germinal disc; The BCs were dispersed by gentle pipetting to form a cell suspension, and then centrifuged at 1500 rpm for 5 min to precipitate the cells. FIX medium was added, and the BCs of each embryo were seeded into a culture dish with an inactivated STO feeder layer. The medium was changed daily. After 2-3 days, PSC clones were formed, and the formed PSC clones were digested with Accutase enzyme for 5-7 min. Then, they were seeded into a new culture dish with STO cells for passage.

Images