Method for inducing porcine embryonic stem cells to differentiate skeletal muscle cells by using complete small molecule compound

Abstract

The invention belongs to the field of stem cells, and particularly relates to a method for inducing porcine embryonic stem cells to differentiate skeletal muscle cells by using a complete small molecule compound. The method comprises the following steps: S1, an induction stage: culturing the porcine embryonic stem cells in a culture medium for two days, and replacing with culture medium culture in the induction stage; s2, a differentiation stage: a differentiation stage culture medium is used for culture. The method is simple in operation process and high in efficiency, and large-scale production of the cell meat is facilitated while differentiation of the porcine embryonic stem cells into the skeletal muscle cells is achieved.

Description

Technical Field

[0001] The invention belongs to the field of stem cells, and in particular, relates to a method for inducing pig embryonic stem cells to differentiate into skeletal muscle cells using complete small molecule compounds. Background Art

[0002] Muscle stem cells from various animals are the seed cells for making cell-cultured meat, but muscle stem cells are very easy to lose their stemness and differentiation ability when cultured in vitro. Therefore, early muscle stem cells are often used for differentiation to make cell-cultured meat, which requires continuous separation of muscle stem cells through muscle tissue, which is not only troublesome to operate but also increases costs. Pluripotent stem cells have the ability to proliferate indefinitely and differentiate in multiple directions. There are currently many reports on the differentiation of human pluripotent stem cells into skeletal muscle cells, which is mainly achieved by overexpressing PAX7 or MYOD1, which requires genetic manipulation and has high requirements. Small molecule compounds are economical, efficient, and easy to operate. At present, some of them are induced by small molecule compounds, but they involve multiple stages of culture medium replacement and the operation process is cumbersome.

[0003] For example, CN104212762A discloses a method for culturing urine-derived multipotent stem cells by in vitro small molecule induction, including obtaining urine-derived cells from human urine samples, inducing and culturing the obtained primary cells in vitro with small molecules to obtain P1 generation hUSCs clones that adhere to the wall, picking hUSCs clones with good growth status for inoculation, and continuing to subculture to obtain hUSCs with long spindle shape and good status. The hUSCs prepared by this method can be induced to differentiate into osteoblasts, adipocytes, skeletal muscle cells, neurons, fibroblasts or smooth muscle cells. However, the operation process of this method is cumbersome and the efficiency is not high.

[0004] There are few reports on the differentiation of porcine embryonic stem cells into skeletal muscle cells. It is worth exploring how to achieve the differentiation of porcine embryonic stem cells into skeletal muscle cells while facilitating the large-scale production of cell-based meat. Summary of the invention

[0005] The purpose of the present invention is to provide a method for inducing porcine embryonic stem cells to differentiate into skeletal muscle cells using a complete small molecule compound. The method has a simple operation process and high efficiency, and is conducive to the large-scale production of cell meat while achieving porcine embryonic stem cells to differentiate into skeletal muscle cells.

[0006] To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0007] A method for inducing porcine embryonic stem cells to differentiate into skeletal muscle cells using a completely small molecule compound, wherein the method comprises the following steps:

[0008] S1, induction stage: culture porcine embryonic stem cells in culture medium for two days, then switch to induction stage culture medium;

[0009] S2, differentiation stage: change to differentiation stage culture medium.

[0010] The method of the present invention has a simple operation process and high efficiency. Compared with the method of obtaining skeletal muscle by differentiating porcine muscle satellite cells, the high efficiency of obtaining skeletal muscle by differentiating porcine embryonic stem cells by the method of the present invention is reflected in the following aspects: (1) High efficiency of skeletal muscle differentiation: Under the same cell amount, the efficiency of porcine embryonic stem cells in differentiating into skeletal muscle cells is higher than that of porcine muscle satellite cells; (2) Seed cells are easy to obtain and have strong proliferation ability, which significantly improves the efficiency of skeletal muscle production: a large number of seed cells can be obtained in the induction stage for subsequent skeletal muscle differentiation. However, porcine muscle satellite cells have the problems of being inconvenient to obtain, having a small number of passages, and having a small amount of expansion.

[0011] Further,

[0012] The induction phase culture medium includes IMDM, HS, penicillin-streptomycin, CHIR99021, SB431542, hr-EGF, Insulin, Dexamethasone, L-ascorbic acid and Y-27632;

[0013] The differentiation stage culture medium includes IMDM, KSR, IGF-1, Forskolin, SB431542, Dexamethasone and DAPT.

[0014] Furthermore, in step S1, the cells are cultured in the induction stage medium for 26 to 49 days.

[0015] Furthermore, in step S2, the cells are cultured in a differentiation stage medium for 7 to 16 days.

[0016] Furthermore, the induction stage and differentiation stage are selected from any one of the following 1)-5):

[0017] 1) Cultivate in the induction phase medium for 26 days and in the differentiation phase medium for 16 days;

[0018] 2) Cultivate in the induction phase medium for 30 days and in the differentiation phase medium for 14 days;

[0019] 3) Cultivate in the induction phase medium for 35 days and in the differentiation phase medium for 9 days;

[0020] 4) Cultivate in the induction phase medium for 38 days and in the differentiation phase medium for 9 days;

[0021] 5) Culture in the induction phase medium for 49 days and culture in the differentiation phase medium for 7 days.

[0022] Furthermore, the induction stage and the differentiation stage also include seed cell storage and seed cell recovery.

[0023] Furthermore, in step S1, the culture time in the induction stage culture medium is 26 days.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The method of the invention has a simple operation process and high efficiency, and is conducive to the large-scale production of cell meat while realizing the differentiation of porcine embryonic stem cells into skeletal muscle cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a complete flow chart of small molecule compounds inducing porcine embryonic stem cells to differentiate into skeletal muscle cells;

[0027] Figure 2 This is a diagram of cell morphology during the induction phase of pig embryonic stem cell differentiation into skeletal muscle cells induced by completely small molecule compounds;

[0028] Figure 3 This is a cell morphology diagram of the differentiation stage during the process of pig embryonic stem cells differentiating into skeletal muscle cells induced by complete small molecule compounds, where A, B, C, D, and E are cells with different proliferation days in the induction stage, and a, b, c, d, and e are cells in the corresponding differentiation stages;

[0029] Figure 4 This is the immunofluorescence result of the cells induced for 26 days and the cells differentiated for 16 days. Green is the myosin heavy chain (MyHC) that marks the myotubes, and blue is the cell nucleus.

[0030] Figure 5 This is the immunofluorescence result of the cells induced for 30 days and the cells differentiated for 14 days. Green is the myosin heavy chain (MyHC) that marks the myotubes, and blue is the cell nucleus.

[0031] Figure 6 This is the immunofluorescence result of cells induced for 35 days and 9 days of differentiation. Green is myosin heavy chain (MyHC) that marks myotubes, and blue is cell nuclei.

[0032] Figure 7This is the immunofluorescence result of cells induced for 38 days and 9 days of differentiation. Green is myosin heavy chain (MyHC) that marks myotubes, and blue is cell nuclei.

[0033] Figure 8 This is the immunofluorescence result of cells induced for 49 days and 7 days of differentiation. Green is the myosin heavy chain (MyHC) that marks the myotubes, and blue is the cell nucleus. DETAILED DESCRIPTION

[0034] The following are specific embodiments of the present invention, and the embodiments are intended to further describe the present invention rather than to limit the present invention.

[0035] The culture medium information involved in the following embodiments is as follows:

[0036] (1) Induction stage culture medium

[0037] Components Brand and item number storage solution Working concentration IMDM Gibco,31980030 1x HS Gibco,26050088 5％ penicillin-streptomycin Thermo,15140-122 100x 1x CHIR99021 Selleck,S1263 20mM 3μM SB431542 APExBIO,A8249 20mM 2μM hr-EGF PeproTech,AF-100-15 100 μg / mL 10 ng / mL Insulin Selleck,S6955 10mg / mL 10 μg / mL Dexamethasone Sigma,D4902 25mM 10μM L-Ascorbic Acid Sigma,A4544 50mg / mL 50 μg / mL Y-27632 Selleck,S1049 20mM 1μM

[0038] (2) Differentiation stage culture medium

[0039] Components Brand and item number storage solution Working concentration IMDM Gibco,31980030 1x 1x KSR Thermo,10828028 15％ IGF-1 PeproTech,250-19 10 μg / mL 10 ng / mL Forskolin Selleck,S2449 100mM 10μM SB431542 APExBIO,A8249 20mM 10μM Dexamethasone Sigma,D4902 25mM 10μM DAPT APExBIO,A8200 20mM 1μM

[0040] Example 1: Complete small molecule compounds induce porcine embryonic stem cells to differentiate into skeletal muscle cells

[0041] After culturing porcine embryonic stem cells (PESCs) in culture medium for two days, the medium was replaced with the induction stage medium. After culturing for 26 days, the cells can be frozen and stored as seed cells for subsequent revival for proliferation and skeletal muscle cell differentiation (see Figure 1 Steps 1, 2, 3, and 4) can also be used to directly differentiate into skeletal muscle cells after induction (see Figure 1 Steps 1 and 4). This embodiment provides a method for inducing porcine embryonic stem cells to directly differentiate into skeletal muscle cells using a completely small molecule compound (see Figure 1 Step 1, 4), the specific steps are as follows:

[0042] S1. Induction stage: After two days of culture in pig embryonic stem cell culture medium, pig embryonic stem cells show compact colony growth. Subsequently, when the culture medium is replaced with the induction stage culture medium, the pig embryonic stem cells become loose, gradually turn into spindle shape, and can proliferate rapidly. Figure 2 The images of cells proliferating at different time points during the induction phase are provided. During the induction phase, cells are passaged at a ratio of 1:3, approximately once every two days.

[0043] S2, differentiation stage: cells induced to proliferate for 26, 30, 35, 38, and 49 days were selected and cultured with differentiation stage medium for 16, 14, 9, 9, and 7 days, and it can be seen that the cells fused into multinucleated myotube-like cells in the differentiation medium (see Figure 3 ), and the cells can be seen to shrink spontaneously.

[0044] Example 2: Completely small molecule compounds induce porcine embryonic stem cells to differentiate into skeletal muscle cells

[0045] This embodiment provides a method for inducing porcine embryonic stem cells with a complete small molecule compound, and then differentiating them into skeletal muscle cells after seed cell storage and seed cell recovery (see Figure 1 Steps 1, 2, 3, 4), the specific steps are as follows:

[0046] S1. Induction stage: After two days of culture in the culture medium, pig embryonic stem cells show compact colony growth. Subsequently, the culture medium is replaced with the induction stage medium. The pig embryonic stem cells become loose, gradually become spindle-shaped, and can proliferate rapidly. Figure 2 The images of cells proliferating at different time points during the induction phase are provided. During the induction phase, cells are passaged at a ratio of 1:3, approximately once every two days.

[0047] S2. Seed cell storage: The cells cultured in the induction medium for 26 days are stored as seed cells;

[0048] S3. Seed cell recovery: 30 minutes in advance, add 0.1% gelatin solution to the cell culture dish for plating. Before use, discard the 0.1% gelatin solution in the culture dish and wash it with PBS for use. Take out the stored seed cells from the liquid nitrogen tank, melt them in a 37°C water bath, take the cell suspension in a 15ml centrifuge tube, centrifuge at 300rpm for 3 minutes, and discard the supernatant. Resuspend with the induction stage medium preheated at 37°C. Add the cell suspension to the culture dish that has been pre-plated with gelatin to obtain the recovered seed cells.

[0049] S4, differentiation stage: The revived seed cells are cultured in the differentiation stage medium, and it can be seen that the cells fuse into multinucleated myotube-like cells in the differentiation medium.

[0050] Example 3: Immunofluorescence identification of skeletal muscle cell differentiation induced by complete small molecule compounds

[0051] The expression of myosin heavy chain (MyHC), a marker antigen of myotubes, was detected in the differentiated cells corresponding to the cells induced for 26, 30, 35, 38, and 49 days. The specific steps include the following:

[0052] (1) Wash with PBS three times, 5 min each time, and fix with 4% paraformaldehyde for 10 min;

[0053] (2) Wash with PBS three times, 5 min each time, and permeabilize with TritionX-100 permeabilization solution for 10 min;

[0054] (3) Wash with PBS three times, 5 min each time, and add 5% goat serum to block for 1 h at room temperature;

[0055] (4) Wash with PBS three times, 5 min each time, dilute the primary antibody (Abcam, Anti-Fast Myosin Skeletal Heavy chain, ab51263) with 1% goat serum at a ratio of 1:1000, and incubate at 4°C overnight;

[0056] (5) The next day, the cells were allowed to warm up at room temperature and washed with PBS three times for 5 min each time. Secondary antibodies (CST, Anti-mouse IgG (H+L), F(ab')2Fragment (Alexa Fluor 594Conjugate), 8890S) were also diluted with 1% goat serum and incubated in the dark for 1 hour; washed with PBS 3 times, 5 minutes each time, incubated with 1 μg / mL DAPI for 5 minutes, washed with PBS 2 times, 5 minutes each time, observed under a fluorescence microscope and photographed.

[0057] The results show that cells induced at different days were treated with the induction scheme of the present invention (see Figure 1 ), after differentiation, MyHC-positive multinucleated myotubes were obtained (see Figure 4-8 ). This indicates that the induction scheme of the present invention can induce porcine embryonic stem cells to differentiate into skeletal muscle cells, and the operation is simple and convenient, and a large number of seed cells can be obtained.

Claims

1. A method for inducing porcine embryonic stem cells to differentiate into skeletal muscle cells using a completely small molecule compound, characterized in that: The method comprises the following steps: S1, induction stage: culture porcine embryonic stem cells in culture medium for two days, then switch to induction stage culture medium; S2, differentiation stage: change to differentiation stage culture medium.

2. The method according to claim 1, characterized in that The induction phase culture medium includes IMDM, HS, penicillin-streptomycin, CHIR99021, SB431542, hr-EGF, Insulin, Dexamethasone, L-ascorbic acid and Y-27632.

3. The method according to claim 1, characterized in that The differentiation stage culture medium includes IMDM, KSR, IGF-1, Forskolin, SB431542, Dexamethasone and DAPT.

4. The method according to claim 3, characterized in that: In step S1, the cells are cultured in the induction stage medium for 26 to 49 days.

5. The method according to claim 4, characterized in that In step S2, the cells are cultured in a differentiation stage medium for 7 to 16 days.

6. The method according to claim 5, characterized in that The induction stage and differentiation stage are selected from any one of the following 1)-5): 1) Cultivate in the induction phase medium for 26 days and in the differentiation phase medium for 16 days; 2) Cultivate in the induction phase medium for 30 days and in the differentiation phase medium for 14 days; 3) Cultivate in the induction phase medium for 35 days and in the differentiation phase medium for 9 days; 4) Cultivate in the induction phase medium for 38 days and in the differentiation phase medium for 9 days; 5) Culture in the induction phase medium for 49 days and culture in the differentiation phase medium for 7 days.

7. The method according to any one of claims 1 to 3, characterized in that: The induction phase and the differentiation phase also include seed cell storage and seed cell recovery.

8. The method according to claim 7, characterized in that In step S1, the culture time in the induction phase culture medium is 26 days.

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