Method for transdifferentiating tail fin cells of erythroculter ilishaeformis into fat and muscle cells and application

Abstract

The invention discloses a method for transdifferentiating tail fin cells of erythroculter ilishaeformis into fat and muscle cells and application, the method is based on 5-Azacytidine (azacytidine) to promote mesenchymal stem cells to be differentiated into muscle cells, and other small molecule compounds are added on the basis of adding the 5-Azacytidine to transdifferentiate the tail fin cells of erythroculter ilishaeformis into muscles. The method is realized through an in-vitro cell reprogramming technology, fish individuals do not need to be sacrificed, and the destructive influence of traditional muscle or fat sampling on fish bodies is avoided; a single-step induction system does not need multi-factor sequential treatment, a high-purity target cell population can be obtained only in 5-7 days at least, the standardization degree of the operation process is high, the stability among batches is excellent, specific cells can be continuously and stably produced, and a safe and reliable technical basis is provided for sustainable cell culture fish meat production.

Description

Technical Field

[0001] The invention belongs to the technical field of fish cell biology, and in particular relates to a method for transdifferentiating caudal fin cells of Erythroculter ilishaeformis into fat and muscle cells and an application thereof. Background Art

[0002] With social development and population growth, the demand for animal meat continues to increase. Alternative proteins, or "artificial meat," are considered a key solution to these problems. Since the advent of cultured beef, cellular agriculture, represented by cell-based meat, has provided a more advantageous solution for alternative protein production. Compared to other alternative proteins, such as plant-based proteins, cell-based meat, created through cell and tissue engineering technologies, is highly similar to natural meat in appearance, structure, nutritional value, and flavor, making it more readily accepted by consumers.

[0003] As the largest group of vertebrates, fish are not only ideal models for exploring the laws of life and disease mechanisms but also a vital source of high-quality protein for humans. Nearly a thousand fish cell lines have been successfully established worldwide, widely used in fields such as cell biology, genetics, immunology, and ecotoxicology. However, compared to mammals, fish cell culture technology is still relatively underdeveloped, lacking demand for translational applications. This has given rise to a new research area in fish cell culture translation: "cellular aquaculture."

[0004] The production of cell-cultured meat still faces many challenges, including the source and expansion of seed cells, low-cost serum-free culture systems, large-scale expansion, and efficient directed differentiation. Among them, the selection of seed cells is particularly critical, and their source directly determines the formulation of subsequent operational strategies. Muscle-derived MuSCs and adipose-derived ADSCs are highly favored due to their ability to differentiate into myogenic and adipose lineages in vitro. A typical example is large yellow croaker cell-cultured meat that integrates the two cell types. However, due to the rich diversity of fish, the in vitro survival and expansion of cells from different fish species require species-specific conditions to be optimized. Practice has shown that most fish muscle satellite cells (MuSCs) and adipose-derived mesenchymal stem cells (ADSCs) are difficult to passage for a long time and maintain stable proliferation and differentiation capabilities. For example, rainbow trout muscle satellite cells are prone to spontaneous differentiation in in vitro culture, making it difficult to establish a stable cell line. Although pluripotent stem cells (PSCs) have unlimited proliferation and excellent differentiation potential, their dependence on highly specialized culture media and complex multi-stage differentiation processes also restricts the large-scale production of cultured meat.

[0005] Fish fin ray tissue has a strong regenerative capacity, and the fibroblasts derived from it exhibit excellent proliferation in vitro. Research has shown that these cells can be induced to differentiate into various cell types, including muscle cells, fat cells, and neurons. Importantly, fin ray samples can be easily obtained without sacrificing the fish. Therefore, fin ray fibroblasts also have the potential to serve as seed cells for fish culture. Erythroculter chub (Erythroculter ilishaeformis) is an important commercial fish species widely distributed throughout the main water bodies of the middle and lower reaches of the Yangtze River in my country. It is highly sought after by consumers for its large size, rapid growth, tender and delicious meat, and rich nutrition. Its artificial breeding technology is mature, and a multi-billion yuan aquaculture industry has been established. However, the well-developed intermuscular spines of carp fish seriously affect their edible quality and economic value. Although gene editing technology can create intermuscular spineless strains, establishing a stable population still requires more than a decade. Therefore, producing boneless fish meat through cell culture may be a more ideal solution. Summary of the Invention

[0006] The present invention aims to address the instability of artificial fish meat seed cells and the problem of a relatively single source, and to provide a method and application for obtaining a novel seed cell source.

[0007] To solve the above technical problems, the technical solution proposed by the present invention is achieved through the following technical contents:

[0008] A method for inducing the differentiation of caudal fin cells of Erythroculter ilishaeformis into fat and myotubes comprises the following steps:

[0009] (1) The caudal fin cells of Erythroculter ilishaeformis were continuously cultured with forskolin at a concentration of 0-1 μM to obtain multipotent caudal fin cells of Erythroculter ilishaeformis;

[0010] (2) adding the cells obtained by culture in step (1) to an adipocyte differentiation medium to induce differentiation, thereby obtaining adipocytes;

[0011] (3) adding myoblast induction medium to the cells cultured in step (1) to obtain myoblasts;

[0012] (4) Adding myogenic differentiation medium to the cells induced in step (3) to induce differentiation, and obtaining myotubes.

[0013] Specifically, the components of the fat differentiation medium in step (2) include basic medium F12, insulin, 3-isobutyl-1-methylxanthine and dexamethasone.

[0014] Specifically, the concentration of the insulin is 0-20 μg / mL;

[0015] Specifically, the concentration of the 3-isobutyl-1-methylxanthine is 0-1 μM; specifically, the concentration of the dexamethasone is 0-0.5 μM.

[0016] Specifically, the components of the myoblast induction medium in step (3) include basal medium DMEM / F12, 5-azacytidine, forskolin, LY411575, Repsox and CHIR99021; the concentration of the 5-azacytidine is 0-20 μM; the concentration of the forskolin is 0-20 μM; the concentration of the LY411575 is 0-20 nM; the concentration of the Repsox is 0-10 μM; and the concentration of the CHIR99021 is 0-5 μM.

[0017] Specifically, the components of the myogenic differentiation medium in step (4) include basal medium F12 and horse serum with a concentration of 0-8%.

[0018] The present invention also provides a method for transdifferentiating Erythroculter ilishaeformis caudal fin cells into fat and muscle cells and its application in cell cultured meat.

[0019] The present invention includes the following beneficial effects:

[0020] The directed differentiation method of the present invention utilizes an induction culture system with a specific combination of regulatory factors to efficiently and directionally differentiate the caudal fin cells of Erythroculter ilicheri into adipocytes and myotubes without producing other cell types, providing a novel seed cell source for artificial fish meat. This method is implemented through in vitro cell reprogramming technology, eliminating the need to sacrifice individual fish and avoiding the destructive effects of traditional muscle or fat extraction on the fish body. The single-step induction system eliminates the need for sequential multi-factor processing and can produce a high-purity target cell population in as little as 5-7 days. The operational process is highly standardized, with excellent batch-to-batch stability, enabling the continuous and stable production of specific cells, providing a safe and reliable technical foundation for sustainable cell culture fish meat production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The 3D spheroids formed after treating Erythroculter ilishaeformis caudal fin cells with 5 μM Forskolin for 15 days according to the present invention and the HE staining image of the spheroids;

[0023] Figure 2 3D spheroids myh1 immunofluorescence staining and lipid droplet histological staining;

[0024] Figure 3The bright field and oil red staining images of lipid accumulation in the caudal fin cells of Erythroculter ilishaeformis treated with 0.5 μM Forskolin for a long time and added with adipose differentiation medium for 3 days;

[0025] Figure 4 The caudal fin cells of Erythroculter ilishaeformis treated with 0.5 μM Forskolin for a long time according to the present invention were supplemented with 15 μM 5-Azacytidine (azacytidine) and then added with 10 μM Forskolin, 10 nM LY411575, 5 μM Repsox and 2 μM CHIR99021 for 3 days. The expression of Myf5 was detected by qRT-PCR. After the addition of the above-mentioned small molecule compounds, the cells were switched to low serum differentiation medium for 2 days, and the expression of Myha was detected by qRT-PCR. The standard deviation was ns P>=0.05, **P<0.005, ****P<0.00005. DETAILED DESCRIPTION

[0026] Exemplary embodiments are described in detail herein, with examples illustrated in the accompanying drawings. When the following description refers to the drawings, identical numerals in different drawings represent identical or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all embodiments consistent with this application.

[0027] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. As used in this application and the appended claims, the singular forms "a," "an," "the," and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0028] It should be understood that although the terms first, second, third, etc. may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0029] In order to more clearly illustrate the technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to embodiments and in conjunction with the accompanying drawings. Note that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention. Unless otherwise specified, the various raw materials, reagents, instruments, and equipment used in the present invention can be purchased commercially or prepared by existing methods.

[0030] The examples are as follows:

[0031] 1. Protocol for differentiating Erythroculter ilishaeformis caudal fin cells into adipocytes:

[0032] 1.1 Caudal fin cells of Erythroculter ilishaeformis were cultured for a long time in DMEM / F12 medium containing 10% fetal bovine serum, 5 ng / mL bFGF, 0.5 μM forskolin, and 1× PS.

[0033] 1.2 After treatment with DEME / F12 medium containing 10% horse serum, 10 μg / mL insulin, 0.5 μM 3-isobutyl-1-methylxanthine (IBMX), and 0.25 μM dexamethasone for 3 days, a similar lipid accumulation phenomenon occurred (e.g. Figure 1 shown);

[0034] 1.3 To visualize lipid droplets in adipocytes, the cells were fixed with 4% PFA for 30 minutes at room temperature, washed twice with PBS for 5 minutes each, and then immersed in 60% isopropanol for 2 minutes. The cells were stained with Oil Red O to determine the accumulation of lipids (e.g. Figure 1 shown).

[0035] 2. Improvement of myogenesis induction protocol:

[0036] 2.1 Caudal fin cells of Erythroculter ilishaeformis were cultured for a long time in DMEM / F12 medium containing 10% fetal bovine serum, 5 ng / mL bFGF, 0.5 μM Forskolin, and 1× PS.

[0037] 2.2 In DMEM / F12 containing 15 μM 5-Azacytidine (a myogenic induction factor), 5 μM Forskolin, 10 nM LY411575, 5 μM Repsox, and 2 μM CHIR99021 were added and cultured for 3 days;

[0038] 2.3 Remove the culture medium, rinse once with 1 mL of PBS, add 1 mL of RNAiso Plus to lyse the cells, let stand for 10 minutes, and collect the RNAiso Plus into a 1 mL centrifuge tube;

[0039] 2.4 Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 10 minutes, centrifuge at 12,000 g at 4°C for 10 minutes, remove the supernatant, add an equal volume of isopropanol, let stand at room temperature for 10 minutes, centrifuge at 12,000 g at 4°C for 10 minutes, remove the supernatant, rinse once with 75% alcohol, centrifuge at 12,000 g at 4°C for 5 minutes, remove the alcohol, dry for 5 minutes, and dissolve the RNA in DEPC water;

[0040] 2.5 Using PrimeScript TM The RNA was reverse transcribed into first-strand cDNA using II 1st Strand cDNA Synthesis Kit, and qRT-PCR was performed using CFX ConnectTM Optics Module (BIO-RAD) and TB green, with β-actin as the internal control and 2 -△△CT The relative expression of Myf5 gene was calculated by the method, and the primers are shown in Table 1. The results showed that the above small molecule compounds can increase the expression of Myf5 (such as Figure 2 As shown), it can promote the generation of muscle lineage cells;

[0041] 2.6 After 3 days of culture as in 5.2, cells were treated with F12 medium containing 2% horse serum for 2 days. RNA extraction, cDNA synthesis, and qRT-PCR were performed according to steps 5.3-5.5. The primers are shown in Table 1. The results showed that the treatment with the above-mentioned small molecule compounds could promote the expression of Myha (e.g. Figure 3 It can promote the formation of muscle fibers.

[0042] Table 1

[0043]

[0044] 3. Protocol for differentiation of Erythroculter ilishaeformis caudal fin cells into myotubes:

[0045] 3.1 Caudal fin cells of Erythroculter ilishaeformis were cultured for a long time in DMEM / F12 medium containing 10% fetal bovine serum, 5 ng / mL bFGF, 0.5 μM Forskolin, and 1× PS.

[0046] 3.2 The cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 15 μM 5-Azacytidine, 5 μM Forskolin, 10 nM LY411575, 5 μM Repsox, 2 μM CHIR99021, and 1× PS for 3 days;

[0047] 3.3 Change to F12 culture medium containing 2% horse serum and 1× PS for 2 days;

[0048] 3.4 Remove the culture medium and fix the cells with 4% paraformaldehyde for 10 minutes, permeabilize with 0.3% Triton X-100 for 10 minutes, and then block with PBS containing 5% goat serum, 1% bovine serum albumin, and 0.3% Tween-20 for 60 minutes.

[0049] 3.5 Use anti-desmin (1:200 dilution) as the primary antibody to label myofibers and incubate in blocking solution at room temperature for 2 hours or at 4°C overnight;

[0050] 3.5 Secondary antibody was Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution) incubated at room temperature for 1 hour, and finally counterstained with 1 μg / ml DAPI. The results showed that multinucleated myotubes were formed (e.g. Figure 4 shown).

[0051] The above results show that the differentiation scheme of the caudal fin cells of Erythroculter ilicheri into fat and muscle described in the present invention can successfully differentiate the caudal fin cells into muscle and fat, which is a new seed cell source for artificial fish meat and has important significance for the sustainable development of artificial fish meat.

[0052] The components of the culture medium used in the present invention were purchased from:

[0053] 100× penicillin-streptomycin double antibody was purchased from Macklin, catalog number P917928;

[0054] D-Hanks buffer was purchased from Meilunbio, product number MA0039;

[0055] Fetal bovine serum was purchased from Gibco, catalog number 10099141C;

[0056] DMEM / F12 was purchased from Gibco, catalog number C11330500BT;

[0057] bFGF was purchased from Beyotime, product number P5453;

[0058] Forskolin was purchased from Targetmol, product number T2939;

[0059] Anhydrous ethanol was purchased from SCR, product number 10009218;

[0060] PBS buffer was purchased from Sangon Biotech, catalog number B548117-0500;

[0061] 4% paraformaldehyde was purchased from Biosharp, product number BL539A;

[0062] Triton X-100 was purchased from Sangon Biotech, catalog number A600198-0500;

[0063] Goat serum was purchased from Beyotime, product number C0265;

[0064] Bovine serum albumin was purchased from Solarbio, product number A8020;

[0065] DAPI was purchased from Solarbio, catalog number C0060;

[0066] Horse serum was purchased from Biosharp, catalog number BL209A;

[0067] Insulin was purchased from Targetmol, catalog number I189675;

[0068] IBMX (3-isobutyl-1-methylxanthine) was purchased from Targetmol, product number T1713;

[0069] Dexamethasone was purchased from Targetmol, catalog number T0947L;

[0070] The lipid mixture was purchased from Peprotech, product number LM-200;

[0071] Oil Red O was purchased from Aladdin, product number O104972;

[0072] 5-Azacytidine was purchased from MCE, product number HY-10586;

[0073] LY411575 was purchased from MCE, catalog number HY-50752;

[0074] RepSox was purchased from Targetmol, product number T6337;

[0075] CHIR99021 was purchased from Targetmol, catalog number T2310;

[0076] RNAiso plus was purchased from Takara, catalog number D9108A;

[0077] Chloroform was purchased from SCR, product number 10006818;

[0078] Isopropyl alcohol was purchased from SCR, product number 80109218;

[0079] DEPC water was purchased from Sangon Biotech, catalog number B501005-0500;

[0080] PrimeScript TMII 1st Strand cDNA Synthesis Kit was purchased from TaKaRa, catalog number 6210A;

[0081] TB green was purchased from TaKaRa, item number RR420A.

[0082] Desmin Rabbit pAb was purchased from Bioss, catalog number bs-1026R;

[0083] Alexa Fluor 488-labeled goat anti-rabbit IgG was purchased from Beyotime, catalog number A0423.

[0084] Those skilled in the art will readily conceive of other embodiments of the present application after considering the specification and practicing the contents disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present application that follow the general principles of this application and include common knowledge or customary techniques in the art that are not disclosed in this application.

[0085] It will be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. A method for inducing the differentiation of caudal fin cells of Erythroculter ilishaeformis into fat and myotubes, characterized in that: The following steps are involved: (1) The caudal fin cells of Erythroculter ilishaeformis were continuously cultured with forskolin at a concentration of 0-1 μM to obtain multipotent caudal fin cells of Erythroculter ilishaeformis; (2) adding the cells obtained by culture in step (1) to an adipocyte differentiation medium to induce differentiation, thereby obtaining adipocytes; (3) adding myoblast induction medium to the cells cultured in step (1) to obtain myoblasts; (4) Adding myogenic differentiation medium to the cells induced in step (3) to induce differentiation, and obtaining myotubes.

2. The method according to claim 1, characterized in that The components of the fat differentiation medium in step (2) include basic medium F12, insulin, 3-isobutyl-1-methylxanthine and dexamethasone.

3. The method according to claim 3, characterized in that The concentration of the insulin is 0-20 μg / mL.

4. The method according to claim 3, characterized in that The concentration of the 3-isobutyl-1-methylxanthine is 0-1 μM.

5. The method according to claim 3, characterized in that The concentration of dexamethasone was 0-0.5 μM.

6. The method according to claim 1, characterized in that The components of the myoblast induction medium in step (3) include basal medium DMEM / F12, 5-azacytidine, forskolin, LY411575, Repsox and CHIR99021; the concentration of the 5-azacytidine is 0-20 μM; the concentration of the forskolin is 0-20 μM; the concentration of the LY411575 is 0-20 nM; the concentration of the Repsox is 0-10 μM; and the concentration of the CHIR99021 is 0-5 μM.

7. The method according to claim 1, characterized in that The components of the myogenic differentiation medium in step (4) include basal medium F12 and horse serum with a concentration of 0-8%.

8. A method for transdifferentiating caudal fin cells of Erythroculter ilishaeformis into fat and muscle cells and its application in cell cultured meat.

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