Myogenic differentiation method of porcine pluripotent stem cells and application of muscle-derived differentiation method in preparation of cell culture meat

Abstract

Provided is a method for inducing muscle-derived differentiation of porcine pluripotent stem cells, which does not involve transgenosis and is free of serum addition in the whole course, and also provided is a method for preparing cell cultured meat (CM) based on the muscle-derived differentiation induction method.

Description

A method for myogenic differentiation of porcine pluripotent stem cells and its application in cell culture meat preparation

[0001] This application is based on the application with CN application number 202310840978.8 and application date July 10, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of biotechnology, and specifically, to a method for inducing myogenic differentiation of porcine pluripotent stem cells, wherein the method does not involve genetic modification and is serum-free throughout the entire process. In addition, the present invention also relates to a method for preparing cell-cultured meat (CM) based on the differentiation induction method. Background Art

[0003] Synthetic biology, guided by engineering principles, redesigns and transforms natural biological systems, designing and synthesizing new biological components, assemblies, and systems to produce targeted byproducts through microbial fermentation. Cultured meat (CM), a disruptive future food production technology and a forerunner of modern synthetic biology and cellular agriculture, is a key development in the production of edible meat tissue through the large-scale cultivation of poultry or livestock cells. The acquisition of seed cells is a major bottleneck in its development, aiming to expand the replicative capacity of skeletal muscle cells for industrial-scale expansion. For over 40 years, differentiation-competent skeletal muscle cell lines have served as model systems for skeletal muscle biology research. These cell lines are isolated from mice or humans and spontaneously generate corresponding muscle cells through serial passage. However, there is a lack of cell lines from major livestock species suitable for producing meat for human consumption. Although myoblast cell lines such as muscle satellite (stem) cells (SCs) have been isolated from livestock (such as pigs and cattle), the ability of this cell line to form mature muscle fibers is limited by the weakening differentiation ability with increasing generations and the inability to expand on a large scale in vitro, which further restricts the progress of cell-cultured meat research and development.

[0004] Unlike such adult cells, pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have unlimited renewal capacity because their early commitment to specific tissue lineages is suppressed, giving them the potential to differentiate into any somatic cell.

[0005] Skeletal muscle cells are an important part of the research and development of cell-cultured meat. How to achieve serum-free differentiation of skeletal muscle cells is another technical difficulty faced in the field. In order to realize the commercialization of CM, the heavy dependence on animal-derived components must be overcome. At present, muscle satellite cells need to be proliferated in culture medium containing different concentrations of fetal bovine serum (FBS). The addition of FBS not only provides an effective growth rate, but also can achieve myogenic differentiation by reducing serum concentration. This process is also called "serum starvation" and is also a common method for SCs myogenic differentiation. However, the use of FBS brings contamination risks and undefined substances, violates the ethical principle of using fewer animals, and is not in line with the concept of sustainable development of CM. Although SCs can undergo terminal myogenic differentiation in vitro without relying on horse serum (Messmer, T., et al. (2022). A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation. Nature Food 3, 74-+.), no large number of multinucleated myotubes were formed, and the proliferation stage of SCs in the early stage still depended on the addition of serum (Ding, S., et al. (2017). Characterization and isolation of highly purified porcine satellite cells. Cell Death Discov 3, 17003.), which shows that the current serum-free induced differentiation technology system for muscle stem cells is still immature. In addition, muscle stem cells cannot be stably passaged for a long time in vitro (Guan, X., et al. (2022). Bioprocessing technology of muscle stem cells: implications for cultured meat. Trends Biotechnol 40, 721-734.), and cannot meet the cell number required for large-scale production of CM. Therefore, it is necessary to develop a serum-free myogenic differentiation induction technology system. The development of a culture medium that allows myogenic differentiation in the absence of serum and transgenics is an important step in achieving CM production.

[0006] To date, pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells) have been used as starting cells to maintain a system that does not require the addition of serum. However, most studies on the myogenic differentiation of pluripotent stem cells have focused on human and mouse models, providing theoretical basis for human myogenic diseases. Few reports have been published on the myogenic differentiation of pluripotent stem cells from livestock. Whether these technical procedures can be applied to myogenic differentiation in other species remains unknown, and there is still a lack of a comprehensive technical system to obtain progenitor cells or mature cell types of the corresponding differentiation lineages required for CM development. Therefore, it is urgent to establish a technical system for the myogenic differentiation of livestock stem cells to provide a new technical approach for CM development.

[0007] Summary of the Invention

[0008] At present, there is no research on using pluripotent stem cells as the starting cells for CM development at home and abroad.

[0009] In this application, the inventors used porcine pluripotent stem cells (PSCs) that were stably passaged in vitro for a long time, and not only successfully achieved stable myogenic differentiation in a transgenic-free and serum-free manner, but also completed the preparation of CM derived from PSCs based on the myogenic differentiated cells combined with a 3D edible scaffold. This not only provides a new seed cell and serum-free and transgenic-free induced differentiation technology system for the research and development of CM, but also provides a new technical approach for the development of cellular agriculture and sustainable animal husbandry.

[0010] Therefore, in one aspect, the present application provides a method for inducing myogenic differentiation of porcine pluripotent stem cells, comprising:

[0011] (1) Providing porcine pluripotent stem cells;

[0012] (2) culturing the pluripotent stem cells using a first-stage differentiation medium, wherein the first-stage differentiation medium contains B27 supplement, CHIR99021, and SB431542;

[0013] (3) culturing the cells obtained in step (2) using a second-stage differentiation medium, wherein the second-stage differentiation medium contains CHIR99021, LDN193189, and FGF2;

[0014] (4) culturing the cells obtained in step (3) using a third-stage differentiation medium, wherein the third-stage differentiation medium contains HGF, IGF-1, FGF2, and LDN193189;

[0015] (5) culturing the cells obtained in step (4) using a fourth stage differentiation medium containing IGF-1; and

[0016] (6) culturing the cells obtained in step (5) using a fifth stage differentiation medium containing IGF-1 and HGF;

[0017] Thus, myoblasts are obtained.

[0018] In certain embodiments, the method further comprises step (7): culturing the cells obtained in step (6) using a sixth stage differentiation medium containing N2 supplement; thereby obtaining muscle cells having mature skeletal muscle fibers.

[0019] In certain embodiments, the method further comprises, before step (2), a step of proliferating the porcine pluripotent stem cells.

[0020] In certain embodiments, the methods are performed in the absence of feeder cells.

[0021] In certain embodiments, the first stage differentiation medium is a basal medium containing B27 supplement, CHIR99021 and SB431542.

[0022] In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.In certain embodiments, the basal culture medium is selected from DMEM / F12, IMDM.

[0023] Preferably, the first stage differentiation medium has one or more characteristics selected from the following:

[0024] (i) the first stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), and ascorbic acid;

[0025] (ii) the first stage differentiation medium does not contain serum;

[0026] (iii) in the first stage differentiation medium, the volume fraction of B27 supplement is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0027] (iv) In the first stage differentiation medium, the concentration of CHIR99021 is 0.05-20 μM (e.g., 0.05-15 μM, 0.05-10 μM, 0.1-20 μM, 0.1-15 μM, 0.1-10 μM, 1-20 μM, 1-15 μM, 1-10 μM, 3.5-20 μM, 3.5-15 μM, 3.5 -10μM, 5-20μM, 5-15μM, 5-10μM, 10μM, 1-3μM, 1-5μM, 1-8μM, 2-3μM, 2-5μM, 2-8μ M, 2-10μM, 2.5-3μM, 2.5-5μM, 2.5-8μM, 2.5-10μM, 3-5μM, 3-8μM, 3-10μM, 3μM);

[0028] (v) in the first stage differentiation medium, the concentration of SB431542 is 0.05-20 μM (e.g., 1-5 μM, 0.05-15 μM, 0.05-10 μM, 0.1-20 μM, 0.1-15 μM, 0.1-10 μM, 1-20 μM, 1-15 μM, 1-10 μM, 2.5-20 μM, 2.5-15 μM, 2.5-10 μM, 5-20 μM, 5-15 μM, 5-10 μM, 10 μM, 1-2 μM, 1-3 μM, 1-4 μM, 1.5-2 μM, 1.5-3 μM, 1.5-4 μM, 1.5-5 μM, 2-3 μM, 2-4 μM, 2-5 μM, 2 μM);

[0029] (vi) the volume fraction of non-essential amino acids (NEAA) in the first stage differentiation medium is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0030] (vi) in the first stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.3%, 0.05%-0.4%, 0.08%-0.1%, 0.08%-0.15%, 0.08%-0.2%, 0.08%-0.3%, 0.08%-0.4%, 0.08%-0.5%, 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%);

[0031] (vii) in the first stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0032] (viii) in the first stage differentiation medium, the volume fraction of KOSR (KnockOut Serum Replacement) is 5%-30% (e.g., 5%-15%, 5%-20%, 5%-25%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 15%);

[0033] (ix) In the first stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (e.g., 100-200 μM, 100-250 μM, 100-300 μM, 100-400 μM, 150-200 μM, 150-250 μM, 150-300 μM, 150-400 μM, 150-500 μM, 200-250 μM, 200-300 μM, 200-400 μM, 200-500 μM).

[0034] In certain embodiments, the second stage differentiation medium is a basal medium containing CHIR99021, LDN193189 and FGF2.

[0035] In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.In certain embodiments, the basal culture medium is selected from DMEM / F12, IMDM.

[0036] In certain embodiments, the second stage differentiation medium has one or more characteristics selected from the group consisting of:

[0037] (i) the second stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), and ascorbic acid;

[0038] (ii) the second stage differentiation medium does not contain serum;

[0039] (iii) In the second stage differentiation medium, the concentration of CHIR99021 is 0.05-20 μM (e.g., 0.05-15 μM, 0.05-10 μM, 0.1-20 μM, 0.1-15 μM, 0.1-10 μM, 1-20 μM, 1-15 μM, 1-10 μM, 3.5-20 μM, 3.5-15 μM, 3. 5-10μM, 5-20μM, 5-15μM, 5-10μM, 10μM, 1-3μM, 1-5μM, 1-8μM, 2-3μM, 2-5μM, 2-8 μM, 2-10μM, 2.5-3μM, 2.5-5μM, 2.5-8μM, 2.5-10μM, 3-5μM, 3-8μM, 3-10μM, 3μM);

[0040] (iv) In the second stage differentiation medium, the concentration of LDN193189 is 0.01-3 μM (e.g., 0.1-3 μM, 0.01-1 μM, 0.01-0.5 μM, 0.01-0.4 μM, 0.05-3 μM, 0.05-1 μM, 0.05-0.5 μM, 0.05-0.4 μM, 0.1-3 μM, 0.1-1 μM, 0.1-0.5 μM, 0.1-0.4 μM, 0.1 μM, 0.1-0.5 μM). ,0.1-0.8μM, 0.1-1μM, 0.1-1.5μM, 0.1-2μM, 0.1-2.5μM, 0.3-0.5μM, 0.3-0.8μM, 0.3-1μM, 0.3-1.5μM, 0 .3-2μM, 0.3-2.5μM, 0.3-3μM, 0.5-0.8μM, 0.5-1μM, 0.5-1.5μM, 0.5-2μM, 0.5-2.5μM, 0.5-3μM, 0.5μM);

[0041] (v) In the second stage differentiation medium, the concentration of FGF2 is 0.5-200 ng / mL (e.g., 5-50 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 30-200 ng / mL, 30-150 ng / mL, 30-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100ng / mL, 5-20ng / mL, 5-25ng / mL, 5-30ng / mL, 5-35ng / mL, 5-40ng / mL, 5-50ng / mL, 10-20ng / mL, 10-25ng / mL, 10-30ng / mL, 10-35ng / mL, 10-40ng / mL, 15- 20ng / mL, 15-25ng / mL, 15-30ng / mL, 15-35ng / mL, 15-40ng / mL, 15-50ng / mL, 20-25ng / mL, 20-30ng / mL, 20-35ng / mL, 20-40ng / mL, 20-50ng / mL, 20ng / mL);

[0042] (vi) the FGF2 is human FGF2 (e.g., recombinant human FGF2);

[0043] (vii) the volume fraction of non-essential amino acids (NEAA) in the second stage differentiation medium is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0044] (viii) in the second stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.3%, 0.05%-0.4%, 0.08%-0.1%, 0.08%-0.15%, 0.08%-0.2%, 0.08%-0.3%, 0.08%-0.4%, 0.08%-0.5%, 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%);

[0045] (ix) in the second stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0046] (x) in the second stage differentiation medium, the volume fraction of KOSR (KnockOut Serum Replacement) is 5%-30% (e.g., 5%-15%, 5%-20%, 5%-25%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 15%);

[0047] (xi) In the second stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (e.g., 100-200 μM, 100-250 μM, 100-300 μM, 100-400 μM, 150-200 μM, 150-250 μM, 150-300 μM, 150-400 μM, 150-500 μM, 200-250 μM, 200-300 μM, 200-400 μM, 200-500 μM).

[0048] In certain embodiments, the third stage differentiation medium is a basal medium containing HGF, IGF-1, FGF2 and LDN193189.

[0049] In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.In certain embodiments, the basal culture medium is selected from DMEM / F12, IMDM.

[0050] In certain embodiments, the third stage differentiation medium has one or more characteristics selected from the group consisting of:

[0051] (i) the third stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), and ascorbic acid;

[0052] (ii) the third stage differentiation medium does not contain serum;

[0053] (iii) In the third stage differentiation medium, the concentration of HGF is 0.5-200 ng / mL (e.g., 2-15 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL); ,20-100ng / mL, 50-200ng / mL, 50-150ng / mL, 50-100ng / mL, 100ng / mL, 2-10ng / mL, 2-12ng / mL, 5-10ng / mL, 5-12ng / mL, 5-15ng / mL, 8-10ng / mL, 8-12ng / mL, 8-15ng / mL, 10-12ng / mL, 10-15ng / mL, 10ng / mL);

[0054] (iv) In the third stage differentiation medium, the concentration of IGF-1 is 0.5-200 ng / mL (e.g., 1-20 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL). L, 50-100ng / mL, 100ng / mL, 1-10ng / mL, 1-12ng / mL, 1-15ng / mL, 1-18ng / mL, 5-10ng / mL, 5-12ng / mL, 5-15ng / mL, 5-18ng / mL, 5-20ng / mL, 8-10ng / mL, 8-12ng / mL, 8-15ng / mL, 8-20ng / mL, 10-12ng / mL, 10-15ng / mL, 10-18ng / mL, 10-20ng / mL, 10ng / mL);

[0055] (v) In the third stage differentiation medium, the concentration of FGF2 is 0.5-200 ng / mL (e.g., 5-50 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 30-200 ng / mL, 30-150 ng / mL, 30-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100ng / mL, 5-20ng / mL, 5-25ng / mL, 5-30ng / mL, 5-35ng / mL, 5-40ng / mL, 5-50ng / mL, 10-20ng / mL, 10-25ng / mL, 10-30ng / mL, 10-35ng / mL, 10-40ng / mL, 15- 20ng / mL, 15-25ng / mL, 15-30ng / mL, 15-35ng / mL, 15-40ng / mL, 15-50ng / mL, 20-25ng / mL, 20-30ng / mL, 20-35ng / mL, 20-40ng / mL, 20-50ng / mL, 20ng / mL);

[0056] (vi) In the third stage differentiation medium, the concentration of LDN193189 is 0.01-3 μM (e.g., 0.1-3 μM, 0.01-1 μM, 0.01-0.5 μM, 0.01-0.4 μM, 0.05-3 μM, 0.05-1 μM, 0.05-0.5 μM, 0.05-0.4 μM, 0.1-3 μM, 0.1-1 μM, 0.1-0.5 μM, 0.1-0.4 μM, 0.1 μM, 0.1-0.5 μM). ,0.1-0.8μM, 0.1-1μM, 0.1-1.5μM, 0.1-2μM, 0.1-2.5μM, 0.3-0.5μM, 0.3-0.8μM, 0.3-1μM, 0.3-1.5μM, 0 .3-2μM, 0.3-2.5μM, 0.3-3μM, 0.5-0.8μM, 0.5-1μM, 0.5-1.5μM, 0.5-2μM, 0.5-2.5μM, 0.5-3μM, 0.5μM);

[0057] (vii) the HGF is human HGF (e.g., recombinant human HGF);

[0058] (viii) the IGF-1 is human IGF-1 (e.g., recombinant human IGF-1);

[0059] (ix) the FGF2 is human FGF2 (e.g., recombinant human FGF2);

[0060] (x) the volume fraction of non-essential amino acids (NEAA) in the third stage differentiation medium is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0061] (xi) in the third stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.3%, 0.05%-0.4%, 0.08%-0.1%, 0.08%-0.15%, 0.08%-0.2%, 0.08%-0.3%, 0.08%-0.4%, 0.08%-0.5%, 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%);

[0062] (xii) in the third stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0063] (xiii) in the third stage differentiation medium, the volume fraction of KOSR (KnockOut Serum Replacement) is 5%-30% (e.g., 5%-15%, 5%-20%, 5%-25%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 15%);

[0064] (xiv) In the third stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (e.g., 100-200 μM, 100-250 μM, 100-300 μM, 100-400 μM, 150-200 μM, 150-250 μM, 150-300 μM, 150-400 μM, 150-500 μM, 200-250 μM, 200-300 μM, 200-400 μM, 200-500 μM).

[0065] In certain embodiments, the fourth stage differentiation medium is a basal medium containing IGF-1.

[0066] In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.In certain embodiments, the basal culture medium is selected from DMEM / F12, IMDM.

[0067] In certain embodiments, the fourth stage differentiation medium has one or more characteristics selected from the group consisting of:

[0068] (i) the fourth stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), and ascorbic acid;

[0069] (ii) the fourth stage differentiation medium does not contain serum;

[0070] (iii) In the fourth stage differentiation medium, the concentration of IGF-1 is 0.5-200 ng / mL (e.g., 1-20 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL). L, 50-100ng / mL, 100ng / mL, 1-10ng / mL, 1-12ng / mL, 1-15ng / mL, 1-18ng / mL, 5-10ng / mL, 5-12ng / mL, 5-15ng / mL, 5-18ng / mL, 5-20ng / mL, 8-10ng / mL, 8-12ng / mL, 8-15ng / mL, 8-20ng / mL, 10-12ng / mL, 10-15ng / mL, 10-18ng / mL, 10-20ng / mL, 10ng / mL);

[0071] (iv) the IGF-1 is human IGF-1 (e.g., recombinant human IGF-1);

[0072] (v) the volume fraction of non-essential amino acids (NEAA) in the fourth stage differentiation medium is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0073] (vi) in the fourth stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.3%, 0.05%-0.4%, 0.08%-0.1%, 0.08%-0.15%, 0.08%-0.2%, 0.08%-0.3%, 0.08%-0.4%, 0.08%-0.5%, 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%);

[0074] (vii) in the fourth stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0075] (viii) in the fourth stage differentiation medium, the volume fraction of KOSR (KnockOut Serum Replacement) is 5%-30% (e.g., 5%-15%, 5%-20%, 5%-25%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 15%);

[0076] (ix) In the fourth stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (e.g., 100-200 μM, 100-250 μM, 100-300 μM, 100-400 μM, 150-200 μM, 150-250 μM, 150-300 μM, 150-400 μM, 150-500 μM, 200-250 μM, 200-300 μM, 200-400 μM, 200-500 μM).

[0077] In certain embodiments, the fifth stage differentiation medium is a basal medium containing IGF-1 and HGF.

[0078] In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.In certain embodiments, the basal culture medium is selected from DMEM / F12, IMDM.

[0079] In certain embodiments, the fifth stage differentiation medium has one or more characteristics selected from the group consisting of:

[0080] (i) the fifth stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), and ascorbic acid;

[0081] (ii) the fifth stage differentiation medium does not contain serum;

[0082] (iii) In the fifth stage differentiation medium, the concentration of HGF is 0.5-200 ng / mL (e.g., 2-15 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL). ,20-100ng / mL, 50-200ng / mL, 50-150ng / mL, 50-100ng / mL, 100ng / mL, 2-10ng / mL, 2-12ng / mL, 5-10ng / mL, 5-12ng / mL, 5-15ng / mL, 8-10ng / mL, 8-12ng / mL, 8-15ng / mL, 10-12ng / mL, 10-15ng / mL, 10ng / mL);

[0083] (iv) In the fifth stage differentiation medium, the concentration of IGF-1 is 0.5-200 ng / mL (e.g., 1-20 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL). L, 50-100ng / mL, 100ng / mL, 1-10ng / mL, 1-12ng / mL, 1-15ng / mL, 1-18ng / mL, 5-10ng / mL, 5-12ng / mL, 5-15ng / mL, 5-18ng / mL, 5-20ng / mL, 8-10ng / mL, 8-12ng / mL, 8-15ng / mL, 8-20ng / mL, 10-12ng / mL, 10-15ng / mL, 10-18ng / mL, 10-20ng / mL, 10ng / mL);

[0084] (v) the HGF is human HGF (e.g., recombinant human HGF);

[0085] (vi) the IGF-1 is human IGF-1 (e.g., recombinant human IGF-1);

[0086] (vii) the volume fraction of non-essential amino acids (NEAA) in the fifth stage differentiation medium is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0087] (viii) in the fifth stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.3%, 0.05%-0.4%, 0.08%-0.1%, 0.08%-0.15%, 0.08%-0.2%, 0.08%-0.3%, 0.08%-0.4%, 0.08%-0.5%, 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%);

[0088] (ix) in the fifth stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0089] (x) in the fifth stage differentiation medium, the volume fraction of KOSR (KnockOut Serum Replacement) is 5%-30% (e.g., 5%-15%, 5%-20%, 5%-25%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 15%);

[0090] (xi) In the fifth stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (e.g., 100-200 μM, 100-250 μM, 100-300 μM, 100-400 μM, 150-200 μM, 150-250 μM, 150-300 μM, 150-400 μM, 150-500 μM, 200-250 μM, 200-300 μM, 200-400 μM, 200-500 μM).

[0091] In certain embodiments, the sixth stage differentiation medium is a basal medium containing N2 supplement;

[0092] In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.In certain embodiments, the basal culture medium is selected from DMEM / F12, IMDM.

[0093] In certain embodiments, the sixth stage differentiation medium has one or more of the following characteristics:

[0094] (i) the sixth stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), and ascorbic acid;

[0095] (ii) the sixth stage differentiation medium does not contain serum;

[0096] (iii) in the sixth stage differentiation medium, the volume fraction of N2 supplement is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.2%, 0.5%-1.5%, 0.5%-2%, 0.5%-2.5%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.2%, 0.8%-1.5%, 0.8%-2%, 0.8%-2.5%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.2%, 1%-1.5%, 1%-1.8%, 1%-2%, 1%-2.5%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0097] (iv) the volume fraction of non-essential amino acids (NEAA) in the stage VI differentiation medium is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0098] (v) in the sixth stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.3%, 0.05%-0.4%, 0.08%-0.1%, 0.08%-0.15%, 0.08%-0.2%, 0.08%-0.3%, 0.08%-0.4%, 0.08%-0.5%, 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%);

[0099] (vi) in the sixth stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (e.g., 0.5%-1%, 0.5%-1.5%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.8%-1%, 0.8%-1.5%, 0.8%-2%, 0.8%-3%, 0.8%-4%, 0.8%-5%, 1%-1.5%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%);

[0100] (vii) the volume fraction of KOSR (KnockOut Serum Replacement) in the sixth stage differentiation medium is 5%-30% (e.g., 5%-15%, 5%-20%, 5%-25%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 15%);

[0101] (viii) In the sixth stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (e.g., 100-200 μM, 100-250 μM, 100-300 μM, 100-400 μM, 150-200 μM, 150-250 μM, 150-300 μM, 150-400 μM, 150-500 μM, 200-250 μM, 200-300 μM, 200-400 μM, 200-500 μM).

[0102] The culture medium additives described in this application (e.g., B27 supplement, CHIR99021, SB431542, LDN193189, FGF2 (fibroblast growth factor 2), HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor-1), N2 supplement, non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement), ascorbic acid) have the meanings commonly understood by those skilled in the art.

[0103] In certain embodiments, the CAS RN of exemplary additives of the present application is as follows:

[0104] The CAS RN of CHIR99021 is 252917-06-9, the CAS RN of SB431542 is 301836-41-9, and the CAS RN of LDN193189 is 1062368-24-4.

[0105] In certain embodiments, the non-essential amino acids (NEAA) comprise L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine.

[0106] In certain embodiments, the method comprises one or more of the following features:

[0107] (i) in step (2), the cell culture time is 1-5 days (e.g., 1-3 days, 1-3.5 days, 1-4 days, 2-3 days, 2-3.5 days, 2-4 days, 2-5 days, 2.5-3 days, 2.5-3.5 days, 2.5-4 days, 2.5-5 days, 3-3.5 days, 3-4 days, 3-5 days, 3 days);

[0108] (ii) in step (3), the cell culture time is 1-5 days (e.g., 1-3 days, 1-3.5 days, 1-4 days, 2-3 days, 2-3.5 days, 2-4 days, 2-5 days, 2.5-3 days, 2.5-3.5 days, 2.5-4 days, 2.5-5 days, 3-3.5 days, 3-4 days, 3-5 days, 3 days);

[0109] (iii) in step (4), the cell culture time is 1-4 days (e.g., 1-2 days, 1-2.5 days, 1-3 days, 1.5-2 days, 1.5-2.5 days, 1.5-3 days, 1.5-4 days, 2-2.5 days, 2-3 days, 2-4 days, 2 days);

[0110] (iv) in step (5), the cell culture time is 1-8 days (e.g., 1-4 days, 1-5 days, 1-6 days, 1-7 days, 2-4 days, 2-5 days, 2-6 days, 2-7 days, 2-8 days, 3-4 days, 3-5 days, 3-6 days, 3-7 days, 3-8 days, 4-5 days, 4-6 days, 4-7 days, 4-8 days, 4 days);

[0111] (v) In step (6), the cell culture time is 5-120 days (e.g., 5-20 days, 5-25 days, 5-30 days, 5-40 days, 5-50 days, 5-80 days, 5-100 days, 10-20 days, 10-25 days, 10-30 days, 10-40 days, 10-50 days, 10-80 days, 10-100 days, 10-120 days, 15-20 days, 15-25 days) , 15-30 days, 15-40 days, 15-50 days, 15-80 days, 15-100 days, 15-120 days, 20-25 days, 20-30 days, 20-40 days, 20-50 days, 20-80 days, 20-100 days, 10-120 days, 25-30 days, 25-40 days, 25-50 days, 25-80 days, 25-100 days, 25-120 days);

[0112] (vi) in step (7), the cell culture time is 2-15 days (e.g., 2-5 days, 2-7 days, 2-10 days, 2-12 days, 5-7 days, 5-10 days, 5-12 days, 5-15 days, 7-10 days, 7-12 days, 7-15 days);

[0113] (vii) in step (3), the cells obtained in step (2) are dissociated into single cells and then inoculated into the second stage differentiation medium for culture;

[0114] (viii) in step (4), replacing the culture medium of the cells obtained in step (3) with the third stage differentiation medium and performing cell culture;

[0115] (ix) in step (5), replacing the culture medium of the cells obtained in step (4) with the fourth stage differentiation medium and performing cell culture;

[0116] (x) in step (6), replacing the culture medium of the cells obtained in step (5) with the fifth stage differentiation medium and performing cell culture;

[0117] (xi) In step (7), the culture medium of the cells obtained in step (6) is replaced with the sixth stage differentiation medium, and the cells are cultured.

[0118] In certain embodiments, the porcine pluripotent stem cells are free of exogenous genetic modification.

[0119] In certain embodiments, the porcine pluripotent stem cells are selected from porcine pgEpiSCs and induced pluripotent stem cells (iPSCs).

[0120] In certain embodiments, the porcine pluripotent stem cells are porcine pgEpiSCs.

[0121] In certain embodiments, the porcine pgEpiSCs are porcine Pre-gastrulation epiblast stem cells (pre-gastrulation epiblast stem cells) that can be stably passaged, also known as porcine stable epiblast stem cells.

[0122] In certain embodiments, the porcine pluripotent stem cells have the pluripotency of porcine pre-gastrulation embryonic epiblast cells, express one or more pluripotency markers and one or more epiblast markers, and are capable of being stably passaged.

[0123] In certain embodiments, the pluripotent stem cells are of porcine embryonic origin.

[0124] The pluripotent stem cells of the present invention express one or more pluripotency markers and one or more epiblast markers.

[0125] In certain embodiments, the one or more pluripotency markers are selected from POU5F1, NANOG, SOX2, SSEA1, SSEA4, TRA-1-81, TRA-1-60, and any combination thereof.

[0126] In certain embodiments, the pluripotent stem cells express one or more (eg, at least one, at least two, or all) of POU5F1, NANOG, and SOX2.

[0127] In certain embodiments, the pluripotent stem cells express one or more (eg, at least one, at least two, at least three, or all) of SSEA1, SSEA4, TRA-1-81, and TRA-1-60.

[0128] In certain embodiments, the one or more epiblast markers are selected from NANOG, TDGF1, ETV4, GDF3, NODAL, PRDM14, ETV5, CACHD1, and any combination thereof.

[0129] In certain embodiments, the pluripotent stem cells express one or more (eg, at least 1, at least 2, at least 3, at least 4, or all) of NANOG, TDGF1, ETV4, GDF3, and NODAL.

[0130] In certain embodiments, the pluripotent stem cells express one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all) of NANOG, TDGF1, ETV4, GDF3, NODAL, PRDM14, ETV5, and CACHD1.

[0131] In certain embodiments, the pluripotent stem cells of the present invention do not express or underexpress at least one hypoblast marker; alternatively, the expression level of at least one hypoblast marker in the pluripotent stem cells is reduced compared to the expression level of the marker in the hypoblast cells of pig embryos at E8 to E10 (e.g., E8, E9 or E10).

[0132] In certain embodiments, the hypoblast marker is selected from IGF1, SRC, HNF4A, BMP2, SOX17, PDGFRA, NID2, RSPO3, GATA4, LAMA1, or any combination thereof.

[0133] In certain embodiments, the pluripotent stem cells do not express or underexpress one or more (eg, at least one, at least two, or all) of HNF4A, SOX17, and GATA4.

[0134] In certain embodiments, the pluripotent stem cells have reduced expression levels of at least one (e.g., at least two or all) genes selected from the following: HNF4A, SOX17, and GATA4, as compared to the expression levels of these genes in E8 to E10 (e.g., E8, E9, or E10) pig embryonic hypoblast cells.

[0135] In certain embodiments, the pluripotent stem cells of the present invention do not express or lowly express at least one gastrulation marker; alternatively, the expression level of at least one gastrulation marker in the pluripotent stem cells is reduced compared to the expression level of the marker in E11 to E14 (e.g., E11, E12, E13 or E14) pig embryonic ectoderm cells.

[0136] In certain embodiments, the gastrulation marker is selected from EOMES, WNT5A, BMP4, LEF1, HAND1, and any combination thereof.

[0137] In certain embodiments, the pluripotent stem cells have reduced expression levels of at least one (e.g., at least two, at least three, at least four, or all) genes selected from the following: EOMES, WNT5A, BMP4, LEF1, and HAND1, as compared to the expression levels of these genes in pig embryonic ectoderm cells from E11 to E14 (e.g., E11, E12, E13, or E14).

[0138] In certain embodiments, the pluripotent stem cells exhibit at least about a 2-fold increase in the expression levels of at least one (e.g., at least two, at least five, at least 10, at least 15, at least 20, or all) genes selected from the group consisting of ADPRM, FRG1, GAS2, HK3, NCAN, POU5F1B, ZFP2, CLDND2, CRK, DMP1, GATD3B, H3F3A, IRF8, ITGA4, KRT14, MPC1, MSH4, NDE1, PBX2, PRKY, RGL2, SOX10, VHLL, as compared to the expression levels of these genes in human embryonic stem cells.

[0139] In certain embodiments, the pluripotent stem cells exhibit at least about a 2-fold decrease in the expression levels of at least one (e.g., at least two, at least five, at least ten, at least fifteen, or all) genes selected from ABCC4, ADCY2, AK2, AKT1, BMP2, CD46, CDH3, DNM1, DPPA4, ETS1, GAB2, ID2, KDR, MMP24, TGFB1, VGLL3, ZNF195, ZNF519, as compared to the expression levels of these genes in human embryonic stem cells.

[0140] In certain embodiments, the human embryonic stem cells compared with the pluripotent stem cells of the present invention refer to conventional human embryonic stem cells (hESCs) or primed human embryonic stem cells.

[0141] As used herein, the terms "conventional human embryonic stem cells (hESCs)" and "primed human embryonic stem cells" have the same meaning. For a definition of primed pluripotency, reference may be made, for example, to Weinberger, L., Ayyash, M., Novershtern, N. & Hanna, J. H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155-169, doi: 10.1038 / nrm.2015.28 (2016).

[0142] In certain embodiments, the pluripotent stem cells comprise genes with co-variation between expression and regulatory potential score (RPS) compared to porcine embryonic fibroblasts (pEF), wherein the genes are referred to as co-variation genes. The co-variation genes are selected from at least one (e.g., at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or all) of the genes listed in Table 1. In certain embodiments, the co-variant genes are selected from at least one (e.g., at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or all) of the following: METTL3, FGFR1, CYC1, ETV5, SOD1, KIF21B, DNMT3A, NOD2, SOX11, MCM7, ITGA4, MYB, UPP1, GSC, ZSCAN21, TFAP2C, ZIC2, LIN28B, ZIC5, HNF4G, MYCN, SALL4, CDH1, DNMT3B, ZFP42, SOX2, UTF1, PRDM14, LEFTY2, OTX2, LIN28A. In certain embodiments, genes with covariation between RPS and gene expression (Genes with Co-variation Between Expression and RPS) refer to genes with higher RPS values ​​that are generally upregulated in pgEpiSCs compared to pEFs (log2 fold change [FC]>1, FDR<0.05). In certain embodiments, the above genes are identified using ultra-deep in situ high-throughput chromatin conformation capture (Hi-C) sequencing technology.

[0143] In certain embodiments, representative covariant genes are as follows:

[0144] In some embodiments, the pluripotent stem cells express increased levels of at least one (such as at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or all) gene selected from Table 1 as compared to the expression levels of these genes in porcine embryonic fibroblasts. In some embodiments, the pluripotent stem cells have increased expression levels of at least one (e.g., at least two, at least five, at least 10, at least 15, at least 20, or all) genes selected from the group consisting of ZSCAN21, LIN28B, MYCN, SALL4, CDH1, DNMT3B, ZFP42, SOX2, UTF1, PRDM14, LEFTY2, OTX2, LIN28A, ACVR2B, HESX1, FZD5, PPP1R1A, VMO1, NANOG, KRT8, KRT18, EPCAM, as compared to the expression levels of these genes in porcine embryonic fibroblasts.

[0145] In certain embodiments, compared to porcine embryonic fibroblasts, in the genome of the pluripotent stem cells, at least one (e.g., at least two, at least three, at least four, at least five, or all) transcription factors selected from the group consisting of OTX2, LIN28A, NANOG, PRDM14, SALL4, UTF1, ZFP42, CDH1, DNMT3B, and LEFTY2 specifically interacts with enhancers. In certain embodiments, the specific interaction with enhancers means that the transcription factors interact with enhancers, as determined by ultra-deep in situ high-throughput chromatin conformation capture (Hi-C) sequencing technology, and the interaction is absent or relatively low in porcine embryonic fibroblasts.

[0146] In certain embodiments, the pluripotent stem cells have the ability to differentiate into cells of any of the endoderm, ectoderm, and mesoderm.

[0147] In certain embodiments, the pluripotent stem cells are capable of forming a dome-shaped colony morphology.

[0148] In certain embodiments, the pluripotent stem cells are capable of stably passaged at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, or more.

[0149] In certain embodiments, the pluripotent stem cells are derived from the epiblast of a pig embryo before gastrulation. In certain embodiments, the pluripotent stem cells are derived from the epiblast of a pig embryo at E8 to E10 (e.g., E8, E9, or E10). In certain embodiments, the pluripotent stem cells are derived from the epiblast of a pig embryo at E10.

[0150] In certain embodiments, the pluripotent stem cell is a cell line.In certain embodiments, the pluripotent stem cell is an epiblast stem cell.

[0151] In certain embodiments, the porcine pluripotent stem cells are prepared according to the following method:

[0152] 1) Providing porcine embryonic epiblast or its inner cell mass;

[0153] 2) Cultivating the porcine embryonic epiblast or its inner cell mass in a culture medium to obtain the porcine pluripotent stem cells.

[0154] In some embodiments, the culture medium comprises:

[0155] A first component, wherein the first component is IWR-1-endo;

[0156] A second component, wherein the second component is selected from WH-4-023 and A419259;

[0157] The third component is selected from fibroblast growth factors.

[0158] In some embodiments, the culture medium further comprises:

[0159] a fourth component selected from CHIR99021 and WNT3a;

[0160] a fifth component selected from members of the TGF-β superfamily;

[0161] The sixth component is LIF.

[0162] In some embodiments, the second component is WH-4-023.

[0163] In some embodiments, the third component is selected from FGF2, FGF1. In some embodiments, the third component is FGF2. In some embodiments, the third component is recombinant human FGF2.

[0164] In some embodiments, the fourth component is CHIR99021.

[0165] In some embodiments, the fifth component is selected from Activin A and Nodal. In some embodiments, the fifth component is Activin A. In some embodiments, the fifth component is recombinant human Activin A.

[0166] In some embodiments, the sixth component is selected from recombinant human LIF, recombinant mouse LIF. In some embodiments, the sixth component is recombinant human LIF.

[0167] In some embodiments, the concentration of the first component is 0.1-10 μM. In some embodiments, the concentration of the first component is 0.9-3 μM. In some embodiments, the concentration of the first component is 1-3 μM. In some embodiments, the concentration of the first component is 2.5 μM.

[0168] In some embodiments, the concentration of the second component is 3 nM-30 μM. In some embodiments, the concentration of the second component is 0.01-5 μM. In some embodiments, the concentration of the second component is 1 μM.

[0169] In some embodiments, the concentration of the third component is 0.01-100 ng / mL. In some embodiments, the concentration of the third component is 1-100 ng / mL. In some embodiments, the concentration of the third component is 10 ng / mL.

[0170] In some embodiments, the concentration of the fourth component is 0.0025 nM-3 μM. In some embodiments, the concentration of the fourth component is 0.01-3 μM. In some embodiments, the concentration of the fourth component is 1 μM.

[0171] In some embodiments, the concentration of the fifth component is 0.01-100 ng / mL. In some embodiments, the concentration of the fifth component is 25 ng / mL.

[0172] In some embodiments, the concentration of the sixth component is 0.01-100 ng / mL. In some embodiments, the concentration of the sixth component is 1-100 ng / mL. In some embodiments, the concentration of the sixth component is 10 ng / mL.

[0173] In some embodiments, the concentration ratio of the fourth component to the first component is 25:1 to 1:25. In some embodiments, the concentration ratio of the fourth component to the first component is 2:3 to 1:3. In some embodiments, the concentration ratio of the fourth component to the first component is 1:2 to 1:3.

[0174] In some embodiments, the culture medium comprises:

[0175] In some embodiments, the culture medium further comprises a seventh component, which is a ROCK inhibitor. Adding a ROCK inhibitor, such as Y-27632, can promote the proliferation of pluripotent stem cells. In some embodiments, the seventh component is Y-27632.

[0176] In some embodiments, the concentration of the seventh component is 0.01-50 μM.

[0177] In some embodiments, the culture medium further comprises: an eighth component, wherein the eighth component is a basal culture medium. In some embodiments, the basal culture medium is a basal culture medium for culturing mammalian (preferably porcine) pluripotent stem cells.

[0178] In some embodiments, the basal medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, β-mercaptoethanol, knockout serum replacement, and any one selected from GlutaMAX and glutamine.

[0179] In some embodiments, the base medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, β-mercaptoethanol, knockout serum replacement, and GlutaMAX.

[0180] In some embodiments, the base medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, β-mercaptoethanol, knockout serum replacement, ascorbic acid, GlutaMAX, and penicillin-streptomycin.

[0181] In some embodiments, the basic culture medium is selected from DMEM / F12, Neurobasal, DMEM, KO-DMEM, RPMI1640, MEM, mTeSR1, or any combination thereof.

[0182] In some embodiments, the basic culture medium is selected from DMEM / F12, Neurobasal or a combination thereof. In some embodiments, the basic culture medium is DMEM / F12 and Neurobasal.

[0183] In some embodiments, the volume fraction of the N2 supplement is 0.002%-10%. In some embodiments, the volume fraction of the N2 supplement is 0.5%.

[0184] In some embodiments, the volume fraction of the B27 supplement is 0.002%-20%. In some embodiments, the volume fraction of the B27 supplement is 1%.

[0185] In some embodiments, the volume fraction of the non-essential amino acids is 0.01%-10%. In some embodiments, the volume fraction of the non-essential amino acids is 1%.

[0186] In some embodiments, the concentration of the β-mercaptoethanol is 0.01 mM to 1 mM. In some embodiments, the concentration of the β-mercaptoethanol is 0.1 mM.

[0187] In some embodiments, the volume fraction of the knockout serum replacement is 0.01%-50%. In some embodiments, the volume fraction of the knockout serum replacement is 5%.

[0188] In some embodiments, the concentration of ascorbic acid is 1 μg / mL to 5000 μg / mL. In some embodiments, the concentration of ascorbic acid is 50 μg / mL.

[0189] In some embodiments, the volume fraction of GlutaMAX or glutamine (preferably GlutaMAX) is 0.01%-10%. In some embodiments, the volume fraction of GlutaMAX or glutamine (preferably GlutaMAX) is 0.5%.

[0190] In some embodiments, the volume fraction of the penicillin-streptomycin is 0.01%-20%. In some embodiments, the volume fraction of the penicillin-streptomycin is 1%.

[0191] In some embodiments, the volume ratio of the DMEM / F12 to the Neurobasal is 5:1-1:5. In some embodiments, the volume ratio of the DMEM / F12 to the Neurobasal is 1:1.

[0192] It should be noted that the concentration of each specific component in the eighth component refers to the final concentration of each specific component in the culture medium. In addition, the volume fraction of each specific component in the eighth component refers to the volume of the specific component / total volume of the culture medium.

[0193] In some embodiments, each 500 mL of culture medium comprises:

[0194] It should also be noted that the components of the above culture medium or the specific ingredients in each component are reagents commonly used by those skilled in the art and can be purchased commercially.

[0195] In certain embodiments, the porcine pluripotent stem cells are described in patent application PCT / CN2022 / 117588.

[0196] In certain embodiments, the porcine pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0197] In certain embodiments, the iPSCs express OCT4, SOX2, and NANOG. Preferably, the iPSCs further express KLF4, C-MYC, REX1, LIN28A, SALL4, and OTX2.

[0198] In certain embodiments, the iPSCs have downregulated, for example, at least 4-fold reduction, in the expression level of at least one (e.g., at least two, at least five, or all ten) gene selected from the group consisting of NR4A1, NR4A2, NR4A3, FOSB, CCN2, CCN1, THBS1, RPL26, EGR4, and DUSP2, compared to porcine pre-gastrulation epiblast pluripotent stem cells (pgEpiSCs).

[0199] In certain embodiments, the iPSCs have an upregulated expression level of at least one (e.g., at least two, at least five, or all six) gene selected from the group consisting of FABP3, FN3KRP, DNPH1, ANXA1, RUSC1, and SNX1, compared to porcine pre-gastrulation epiblast pluripotent stem cells (pgEpiSCs), for example, at least a 6-fold increase.

[0200] In certain embodiments, the iPSCs have the ability to differentiate into cells of any of the endoderm, ectoderm, and mesoderm.

[0201] In certain embodiments, the iPSCs are capable of forming domed-shaped clones.

[0202] In certain embodiments, the iPSCs are capable of stable passage for at least 50 times or more. Preferably, the iPSCs are capable of stable passage for at least 100 times or more.

[0203] In certain embodiments, the iPSCs are exogenous gene-independent porcine iPSCs.

[0204] In certain embodiments, the iPSCs are prepared according to the following method:

[0205] (i) Reprogramming porcine somatic cells;

[0206] (ii) culturing the cells of step (i) in a medium comprising a WNT signaling pathway inhibitor, CHIR99021, a Src inhibitor, LIF, a TGF-β superfamily member, and a fibroblast growth factor to generate iPSCs.

[0207] In certain embodiments, the WNT signaling pathway inhibitor is IWR-1.

[0208] In certain embodiments, the Src inhibitor is WH-4-023.

[0209] In certain embodiments, the TGF-β superfamily member is Activin A, such as human Activin A.

[0210] In certain embodiments, the fibroblast growth factor is FGF2, such as human FGF2.

[0211] In certain embodiments, the LIF is human LIF.

[0212] In certain embodiments, the WNT signaling pathway inhibitor is IWR-1, and the content ratio of IWR-1 to CHIR99021 is 2:3-1:3, such as 1:2-1:3.

[0213] In certain embodiments, the content ratio of the WNT signaling pathway inhibitor to CHIR99021 is 25:1-1:25, for example, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20 or 1:25. In certain embodiments, the content ratio of the WNT signaling pathway inhibitor to CHIR99021 is 2:3-1:3, for example, 1:2-1:3.

[0214] In certain embodiments, the concentration of the WNT signaling pathway inhibitor is 1-5 μM, such as 1 μM, 2.5 μM, 3 μM, or 5 μM. In certain embodiments, the concentration of the WNT signaling pathway inhibitor is 2.5 μM.

[0215] In certain embodiments, the concentration of CHIR99021 is 0.01-3 μM, such as 0.01 μM, 0.1 μM, 1 μM, 2 μM, 3 μM. In certain embodiments, the concentration of CHIR99021 is 1 μM.

[0216] In certain embodiments, the concentration of the Src inhibitor is 0.01-5 μM, such as 0.01 μM, 0.1 μM, 1 μM, 3 μM, 5 μM. In certain embodiments, the concentration of the Src inhibitor is 1 μM.

[0217] In certain embodiments, the concentration of LIF is 1-100 ng / mL, such as 1 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL. In certain embodiments, the concentration of LIF is 10 ng / mL.

[0218] In certain embodiments, the concentration of the TGF-β superfamily member is 1-100 ng / mL, such as 1 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL. In certain embodiments, the concentration of the TGF-β superfamily member is 25 ng / mL.

[0219] In certain embodiments, the concentration of the fibroblast growth factor is 1-100 ng / mL, such as 1 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL. In certain embodiments, the concentration of the fibroblast growth factor is 10 ng / mL.

[0220] In certain preferred embodiments, the culture medium comprises: 1-5 μM WNT signaling pathway inhibitor (e.g., IWR-1), 0.01-3 μM CHIR99021, 0.01-5 μM Src inhibitor (e.g., WH-4-023), 1-100 ng / mL TGF-β superfamily member (e.g., Activin A, such as human Activin A), 1-100 ng / mL fibroblast growth factor (e.g., FGF2, human FGF2), and 1-100 ng / mL LIF (e.g., human LIF).

[0221] In certain exemplary embodiments, the culture medium comprises: 2.5 μM WNT signaling pathway inhibitor (e.g., IWR-1), 1 μM CHIR99021, 1 μM Src inhibitor (e.g., WH-4-023), 25 ng / mL TGF-β superfamily member (e.g., Activin A, such as human Activin A), 10 ng / mL fibroblast growth factor (e.g., FGF2, human FGF2), and 10 ng / mL LIF (e.g., human LIF).

[0222] In certain exemplary embodiments, the culture medium comprises: 2.5 μM IWR-1, 1 μM CHIR99021, 1 μM WH-4-023, 25 ng / mL Activin A (eg, human Activin A), 10 ng / mL FGF2 (eg, human FGF2), and 10 ng / mL LIF (eg, human LIF).

[0223] It should be noted that the concentrations of the above components refer to the final concentrations of the components in the culture medium.

[0224] In certain embodiments, the culture medium of step (ii) further comprises a basal culture medium.

[0225] In certain embodiments, the basal medium is a basal medium for culturing mammalian (preferably porcine) pluripotent stem cells.

[0226] In certain embodiments, the base medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, β-mercaptoethanol, serum replacement (eg, knockout serum replacement), and glutamine or a derivative thereof (eg, GlutaMAX).

[0227] In certain embodiments, the base medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, β-mercaptoethanol, serum replacement (eg, knockout serum replacement), and GlutaMAX.

[0228] In certain embodiments, the basal medium further comprises ascorbic acid.

[0229] In certain embodiments, the basal medium further comprises penicillin-streptomycin.

[0230] In certain embodiments, the basic culture medium is selected from DMEM / F12, Neurobasal, DMEM, KO-DMEM, RPMI1640, MEM, mTeSR1, or any combination thereof.

[0231] In certain embodiments, the base medium is selected from DMEM / F12, Neurobasal or a combination thereof. In certain embodiments, the base medium is DMEM / F12 and Neurobasal.

[0232] In certain embodiments, the volume fraction of the N2 supplement is 0.002%-10%, for example 0.1%-10%, such as 0.1%-5%, such as 0.1%-2%, such as 0.1%-1%, such as 0.5%-2%, such as 0.5%-1%, such as 0.2%-2%, such as 0.2%-1%, preferably 0.5%.

[0233] In certain embodiments, the volume fraction of the B27 supplement is 0.002%-20%, such as 0.1%-20%, such as 0.1%-10%, such as 0.1%-5%, such as 0.1%-2%, such as 0.5%-5%, such as 0.5%-2%, such as 1%-5%, such as 1%-2%, preferably 1%.

[0234] In certain embodiments, the volume fraction of the non-essential amino acids is 0.01%-10%, for example 0.1%-20%, such as 0.1%-10%, such as 0.1%-5%, such as 0.1%-2%, such as 0.5%-5%, such as 0.5%-2%, such as 1%-5%, such as 1%-2%, and preferably 1%.

[0235] In certain embodiments, the concentration of β-mercaptoethanol is 0.01%-1%, such as 0.05%-1%, such as 0.08%-1%, such as 0.1%-1%, such as 0.05%-0.5%, such as 0.08%-0.5%, such as 0.1%-0.5%, preferably 0.1%.

[0236] In certain embodiments, the volume fraction of the serum substitute (eg, knockout serum replacement) is 0.01%-50%, such as 1-50%, 1-30%, 1-20%, 2-30%, 2-20%, 5-20%, preferably 5%.

[0237] In certain embodiments, the concentration of ascorbic acid is 1 μg / mL-5000 μg / mL, for example, 1 μg / mL-100 μg / mL, 10 μg / mL-100 μg / mL, 20 μg / mL-100 μg / mL, 20 μg / mL-80 μg / mL, 30 μg / mL-80 μg / mL, 30 μg / mL-60 μg / mL, 40 μg / mL-60 μg / mL, and preferably 50 μg / mL.

[0238] In certain embodiments, the volume fraction of glutamine or its derivative (e.g., GlutaMAX) is 0.01%-10%, for example, 0.1%-10%, such as 0.1%-5%, such as 0.1%-2%, such as 0.1%-1%, such as 0.5%-2%, such as 0.5%-1%, such as 0.2%-2%, such as 0.2%-1%, and preferably 0.5%.

[0239] In certain embodiments, the volume fraction of penicillin-streptomycin is 0.01%-20%, for example 0.1%-20%, such as 0.1%-10%, such as 0.1%-5%, such as 0.1%-2%, such as 0.5%-5%, such as 0.5%-2%, such as 1%-5%, such as 1%-2%, and preferably 1%.

[0240] In certain embodiments, the volume ratio of the DMEM / F12 to the Neurobasal is 5:1-1:5, such as 2:1-1:2, preferably 1:1.

[0241] In certain embodiments, the volume fraction of the basic culture medium is 1%-99%, such as 50%-99%, 60%-99%, 50%-95%, 60%-95%, 80%-95%, 85%-95%, 90%-95%, and preferably 91%.

[0242] In certain embodiments, the volume fraction of DMEM / F12 is 1%-99%, such as 40%-60%, 40%-50%, and preferably 45%-50% (such as 45.5%).

[0243] In certain embodiments, the volume fraction of Neurobasal is 1%-99%, such as 40%-60%, 40%-50%, and preferably 45%-50% (such as 45.5%).

[0244] In certain exemplary embodiments, the base medium is a minimal medium (e.g., DMEM / F12 / Neurobasal in a volume ratio of 2:1-1:2, preferably 1:1) supplemented with the following components: 0.1%-5% (e.g., 0.1%-2%, 0.1%-1%, 0.5%-2%, 0.5%-1%, 0.2%-2%, 0.2%-1%) N2 supplement, 0.1%-5% (e.g., 0.1%-2%, 0.5%-5%, 0.5%-2%, 1%-5%, 1%-2%) B27 supplement. supplement, 0.1%-5% (e.g., 0.1%-2%, 0.5%-5%, 0.5%-2%, 1%-5%, 1%-2%) non-essential amino acids, 0.1%-1% (0.05%-0.5%, such as 0.08%-0.5%, 0.1%-0.5%) beta-mercaptoethanol, 1-30% (e.g., 1-20%, 2-30%, 2-20%, 5-20%) serum replacement, and 0.1%-5% (e.g., 0.1%-2%, 0.1%-1%, 0.5%-2%, 0.5%-1%, 0.2%-2%, 0.2%-1%) glutamine or its derivatives. Preferably, the invention further comprises 20 μg / mL-80 μg / mL (e.g., 30 μg / mL-80 μg / mL, 30 μg / mL-60 μg / mL, 40 μg / mL-60 μg / mL) of ascorbic acid. Preferably, the invention further comprises 0.1%-5% (e.g., 0.1%-2%, 0.5%-5%, 0.5%-2%, 1%-5%, 1%-2%) of penicillin-streptomycin.

[0245] In certain exemplary embodiments, the basal medium is a minimal medium supplemented with the following components (e.g., DMEM / F12 / Neurobasal at a volume ratio of 2:1-1:2, preferably 1:1): 0.5% N2 supplement, 1% B27 supplement, 1% non-essential amino acids, 0.1% β-mercaptoethanol, 5% serum replacement, 0.5% glutamine or its derivatives, and 50 μg / mL ascorbic acid.

[0246] It should be noted that the concentration of each specific component in the above-mentioned basal culture medium refers to the final concentration of each specific component in the culture medium, and the volume fraction of each specific component refers to the volume of the specific component / total volume of the culture medium.

[0247] In certain embodiments, the somatic cells are cultured after the reprogramming factors are introduced into the somatic cells in the presence of a feeder layer. In certain embodiments, the feeder layer is mouse embryonic fibroblasts.

[0248] In certain embodiments, the method further comprises a passaging step.

[0249] In certain embodiments, the culture medium used in the passaging step is the culture medium described in step (ii).

[0250] In certain embodiments, the culture medium used in the subculturing step is the culture medium described in step (ii) supplemented with a ROCK inhibitor. In certain embodiments, the ROCK inhibitor is Y-27632. In certain embodiments, the ROCK inhibitor is present in an amount of 0.01-50 μM, e.g., 0.01-20 μM, 0.1-20 μM, 0.1-10 μM, 0.1-5 μM, 0.5-5 μM, 1-5 μM, e.g., 2 μM.

[0251] Reprogramming of somatic cells can be performed using any method known in the art. Typically, somatic cell pluripotency reprogramming technology can convert differentiated somatic cells into induced pluripotent stem cells (iPSCs) by using reprogramming transcription factors (such as OCT4, SOX2, KLF4, C-MYC).

[0252] In certain embodiments, the reprogramming comprises introducing reprogramming factors into pig somatic cells. In certain embodiments, the somatic cells are fibroblasts (e.g., embryonic fibroblasts or adult fibroblasts), mesenchymal cells, perivascular cells, renal epithelial cells in urine, or peripheral blood mononuclear cells.

[0253] In certain embodiments, the reprogramming factors include OCT4, SOX2, KLF4 and C-MYC. Preferably, the reprogramming factors include hOCT4, hSOX2, hKLF4 and hC-MYC.

[0254] In certain embodiments, the reprogramming factors further include BCL2L1. Preferably, the reprogramming factors include hBCL2L1.

[0255] In certain embodiments, the reprogramming factors include hOCT4, hSOX2, hKLF4, hC-MYC, and hBCL2L1.

[0256] In certain embodiments, for more differentiated somatic cells (e.g., adult fibroblasts), the reprogramming factors may further include LIN28A and NANOG. In certain embodiments, preferably, the LIN28A and NANOG are derived from pigs, and thus the reprogramming factors further include pLIN28A and pNANOG.

[0257] In certain embodiments, the reprogramming factors include hOCT4, hSOX2, hKLF4, hC-MYC, hBCL2L1, pLIN28A, and pNANOG.

[0258] In certain embodiments, the reprogramming factors are encoded by one or more exogenous expression cassettes.

[0259] In certain embodiments, the nucleotide sequences encoding each of the reprogramming factors are optionally located in the same or different expression cassettes. For example, nucleic acid molecules encoding OCT4 and hSOX2 can be located in the same expression cassette, while nucleic acid molecules encoding other factors can be located in different expression cassettes.

[0260] In certain embodiments, the introduction of the reprogramming factors is achieved in a non-integrating form.

[0261] In certain embodiments, the one or more exogenous expression cassettes are contained in a non-integrating vector.

[0262] In certain embodiments, the non-integrating vector is introduced into the cell by electrofection.

[0263] In certain embodiments, the non-integrating vector is an episomal vector. Typically, prolonged expression of reprogramming factors can be achieved using episomal vectors based on oriP / EBNA1. These plasmids contain the oriP / EBNA1 viral element, derived from the Epstein-Barr virus. The oriP / EBNA1 element promotes the replication of episomal plasmid DNA in dividing cells, thereby allowing the expression of reprogramming factors long enough to initiate the reprogramming process. The plasmid is eventually lost from the proliferating cells, leaving no trace of the plasmid transfection.

[0264] In certain embodiments, the non-integrating vector is a pEV vector comprising the spleen focus forming virus promoter (SFFV), WPRE, OriP, and EBNA1 elements.

[0265] In certain embodiments, the method further comprises: (iii) detecting the residual state of the exogenous gene at the endogenous locus to screen cells without the exogenous gene remaining at the endogenous locus, thereby obtaining porcine iPSCs without exogenous gene modification.

[0266] In certain embodiments, the introduction of the reprogramming factors is achieved in an integrated form.

[0267] In certain embodiments, the one or more exogenous expression cassettes are contained in an integrative vector.

[0268] In certain embodiments, the integrating vector is introduced into the cell by viral transfection.

[0269] In certain embodiments, the integrating vector is a retroviral, lentiviral, or transposase vector.

[0270] In certain embodiments, the method further comprises: (iii) detecting the expression of the exogenous gene (e.g., by quantitative PCR or semi-quantitative PCR) to screen for cells in which the exogenous gene is silenced, thereby obtaining exogenous gene-silenced porcine iPSCs.

[0271] In certain embodiments, the reprogramming step is performed in a culture medium, which is any culture medium suitable for somatic cells. For example, for fibroblasts, the culture medium can be a minimal medium (e.g., DMEM) supplemented with serum (e.g., FBS); preferably, the culture medium is also supplemented with non-essential amino acids (e.g., NEAA); preferably, the culture medium comprises 5-20% serum (e.g., 5-15%, 10-20%, 10-15%; e.g., 5%, 8%, 10%, 12% or 15%) and 0.01%-10% non-essential amino acids (e.g., 1-10%, 1-5%, 1-2%, 1%); preferably, the culture medium also comprises penicillin-streptomycin; e.g., a volume fraction of 0.01%-20%, preferably 1%.

[0272] In certain embodiments, the method comprises: introducing the reprogramming factors into somatic cells, pre-culturing the cells in a culture medium suitable for the somatic cells, and then gradually replacing the pre-culture medium with the culture medium described in step (ii).

[0273] In certain embodiments, the iPSCs are described in patent application CN2023105046956.

[0274] In another aspect, the present application provides a method for preparing cell-cultured meat, comprising:

[0275] (a) providing: a scaffold, and myoblasts obtained according to the method described above;

[0276] (b) seeding the myoblasts on the scaffold and culturing them in a fifth stage differentiation medium, wherein the fifth stage differentiation medium is as defined above;

[0277] (c) culturing the cells obtained in step (b) using a sixth stage differentiation medium, wherein the sixth stage differentiation medium is as defined above;

[0278] Thus, cell cultured meat is obtained.

[0279] In certain embodiments, the method comprises one or more of the following features:

[0280] (i) in step (b), the cell culture time is 1-15 days (e.g., 1-8 days, 1-10 days, 1-12 days, 3-8 days, 3-10 days, 3-12 days, 3-15 days, 5-8 days, 5-10 days, 5-12 days, 5-15 days, 8-10 days, 8-12 days, 8-15 days, 8 days);

[0281] (ii) in step (c), the cell culture time is 1-15 days (e.g., 1-7 days, 1-10 days, 1-12 days, 3-7 days, 3-10 days, 3-12 days, 3-15 days, 5-7 days, 5-10 days, 5-12 days, 5-15 days, 7-10 days, 7-12 days, 7-15 days, 7 days);

[0282] (iii) In step (c), the culture medium of the cells obtained in step (b) is replaced with the sixth stage differentiation medium, and the cells are cultured.

[0283] In certain embodiments, the scaffold is a three-dimensional edible scaffold.

[0284] In certain embodiments, the scaffold has a porous layered structure.

[0285] In another aspect, the present application provides cell-cultured meat prepared by the method described above.

[0286] Advantageous Effects of the Invention

[0287] The present application provides a method for inducing myogenic differentiation of porcine pluripotent stem cells, which does not involve transgenics and is serum-free throughout the process. The method further realizes the preparation of CM derived from PSCs based on the myogenic differentiation induction method, providing a new seed cell and serum-free and transgenic-free induced differentiation technology system for the research and development of CM. At the same time, it also overcomes the problems faced by the existing technology of preparing CM based on muscle stem cell induced differentiation, such as serum dependence and inability to maintain long-term stable passage in vitro.

[0288] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0289] Figure 1: Schematic diagram of pgEpiSCs myogenic differentiation induction technology

[0290] Among them, Stage IV corresponds to MDM IV in the technical approach.

[0291] Figure 2: Optimization of pgEpiSCs differentiation basal medium

[0292] (A) Morphological observation of cell adhesion under different culture medium formulations without the addition of any small molecules or growth factors. Scale bar, 200 μm.

[0293] (B) Expression of pluripotency genes OCT4, SOX2, NANOG and three-germ layer differentiation-related genes PAX6, T and EOMES in different differentiation-based culture systems.

[0294] ① Insulin-Transferrin-Selenium (ITS) group: DMEM / F12 supplemented with 1% ITS, 0.1 mM β-mercaptoethanol, 1% NEAA, 1% penicillin-streptomycin, and 200 μM ascorbic acid;

[0295] ②YH BM group: DMEM / F12 and Neurobasal (1:1) supplemented with 0.5% N2, 1% B27, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR, and 200 μM ascorbic acid;

[0296] ③YH BM-Neur group: DMEM / F12 supplemented with 0.5% N2, 1% B27, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR and 200 μM ascorbic acid;

[0297] ④F12+N2 group: DMEM / F12 supplemented with 0.5% N2, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR and 200 μM ascorbic acid;

[0298] ⑤F12+B27 group: DMEM / F12 supplemented with 1% B27, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR and 200 μM ascorbic acid;

[0299] Error bars represent mean ± SD, and different letters indicate differences between groups.

[0300] Figure 3 Small molecule screening of pgEpiSCs in the early stages of myogenic differentiation

[0301] (A) Morphological observation of pgEpiSCs attachment and differentiation in the presence of different small molecule combinations in myogenic differentiation medium MDM I. Scale bar, 50 μm.

[0302] (BD) Expression of genes related to pluripotency (OCT4, SOX2, NANOG) and paraxial mesoderm differentiation (T, PDGFRα, and MGGN1) in different systems; Figure B shows the culture medium: BM culture medium supplemented with 1% B27, 3 μM CHIR99021, and 0.5 μM LDN193189; Figure C shows the culture medium: BM culture medium supplemented with 1% B27, 3 μM CHIR99021, and 2 μM SB431542; Figure D shows the culture medium: BM culture medium supplemented with 1% B27, 3 μM CHIR99021, 0.5 μM LDN193189, and 2 μM SB431542; Figure BM culture medium (basal medium) is DMEM / F12 supplemented with 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR, and 200 μM ascorbic acid. acid.

[0303] (E) Expression of differentiation-related genes T (differentiation day 2), PDGFRα (differentiation day 3), MGGN 1 (differentiation day 3), and BMP4 (differentiation day 3) under different systems.

[0304] (F) Alkaline phosphatase staining of pgEpiSCs after differentiation under Wnt activation and TGF-β inhibition conditions. Scale bar, 50 μm.

[0305] (G) Immunostaining of T, NANOG, and SOX2 markers in the presence of Wnt activation and TGF-β inhibition, with DAPI used for nuclear staining. Scale bar, 10 μm.

[0306] For (A), CHIR: CHIR99021; LDN: LDN193189; SB: SB431542. For (B, D, and F), Mes-Dif represents paraxial mesoderm differentiation. Error bars are shown as mean ± SD based on three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For E, differences between groups are indicated by different letters.

[0307] Figure 4 Cell characteristics analysis of terminally differentiated myogenic pgEpiSCs

[0308] (A) Karyotype analysis of pgEpiSCs myogenic terminally differentiated cells.

[0309] (B) Gene expression analysis of pgEpiSCs myogenic terminally differentiated cells.

[0310] Myo-Dif represents the terminal myogenic differentiation of pgEpiSCs. Error bars represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0311] Figure 5 Cluster analysis of different gene expression patterns during in vitro myogenic differentiation of pgEpiSCs

[0312] (A) Principal component analysis of three populations (pgEpiSCs, pgEpiSCs-MPCs, and pgEpiSCs-MCs) during in vitro myogenic differentiation of pgEpiSCs. Colors represent different cell populations.

[0313] (B) Ternary plot of the three populations (pgEpiSCs, pgEpiSCs-MPCs, and pgEpiSCs-MCs) during in vitro myogenic differentiation of pgEpiSCs. Key markers of different cell populations are represented by different colors.

[0314] (C) PC1 gene loading fraction during pgEpiSCs myogenesis in vitro.

[0315] (D) Heatmap of clusters representing each cell population during in vitro myogenic differentiation of pgEpiSCs showing similar expression patterns of genes in the same cluster.

[0316] (E) Analysis of enriched gene ontology (GO) terms in representative clusters with high q-values ​​in different cell populations during myogenic differentiation of pgEpiSCs.

[0317] (F and G) Expression of genes related to myofiber maturation and collagen formation during myogenic differentiation of pgEpiSCs assessed by qPCR, n = 3, WT: undifferentiated pgEpiSCs, Myo-Dif: terminally differentiated cells after N2 treatment.

[0318] For (A, B, and D), pgEpiSCs-MPCs: pgEpiSC-derived myogenic progenitor cells; pgEpiSCs-MCs: ​​mature muscle fiber cells after N2 treatment. For (F and G), error bars represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0319] Figure 6 pgEpiSCs terminally differentiated myogenic cells have mature muscle fiber morphology

[0320] (A) Cell morphology of mature myofibroblasts derived from pgEpiSCs after treatment with 2% horse serum (HS) or N2. Scale bar, 500 μm (left) or 100 μm (right).

[0321] (B) Immunofluorescence staining of MF20 (red), myosin (green), and F-actin (red) in myotubes derived from terminally differentiated pgEpiSCs. DAPI was used for nuclear staining. Scale bars, 100 μm (left) or 20 μm (right).

[0322] Figure 7 Characterization of myoblasts derived from pgEpiSCs after long-term differentiation

[0323] (A) Appearance and morphology of pgEpiSCs-derived myoblasts at different days. Scale bar, 500 μm.

[0324] (B) Survival of pgEpiSCs-derived myoblasts during long-term differentiation. Calcein-AM (green) indicates live cells, and PI (red) indicates dead cells. Scale bar, 100 μm.

[0325] (C) Flow cytometric analysis of cell viability.

[0326] (D) Immunofluorescence staining of pgEpiSCs-MCs subcultures at days 30, 60, 90, and 120. Multinucleated myofibers derived from pgEpiSCs redifferentiated in N2 medium, with nuclei stained with DAPI (blue), and abundant skeletal myofibers demonstrated by myosin expression. Scale bar, 20 μm.

[0327] Figure 8 Differentiation and CM formation assay of pgEpiSCs-derived myoblasts on 2-CS-SA-Col1-Gel1 scaffolds

[0328] (A) Confocal microscopy observation of differentiation status on 3D edible scaffolds. Representative fluorescence images of the cytoskeletal protein F-actin on day 15. Red, Factin; blue, DAPI. Scale bar, 100 μm.

[0329] (B) Appearance of pgEpiSC-derived CMs cultured for 15 days after staining and cooking.

[0330] (C) Karyotype analysis of pgEpiSCs-MCs after differentiation on 2-CS-SA-Col1-Gel1 scaffolds.

[0331] (D) Textural analysis of pgEpiSC-derived CM and fresh pork after 15 days of culture. Error bars represent mean ± SD, and different letters indicate differences between groups.

[0332] Figure 9 Microbial detection of pgEpiSCs-derived CM

[0333] (A) Colony formation of Escherichia coli (E. coli) after plating.

[0334] (B) Colony formation of Staphylococcus aureus (S. aureus) after plating.

[0335] (C) Colony formation of Salmonella enterica (S. enterica) after plating.

[0336] (D) shows the colony formation after plating the culture medium of MDM V cells.

[0337] (E) is a picture of colony formation after plating the culture medium after terminal differentiation of N2.

[0338] (F) Colony formation images of the pgEpiSCs-derived CM extract after plating.

[0339] Figure 10 shows the cell morphology of each group after Stage I culture using MDM I culture medium containing different concentrations of CHIR99021 to induce differentiation.

[0340] Figure 11 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM I culture medium containing different concentrations of CHIR99021 and undergoing Stage III culture.

[0341] Figure 12 shows the cell morphology of each group after Stage I culture using MDM I culture medium containing different concentrations of SB431542 to induce differentiation.

[0342] FIG13 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM I culture medium containing different concentrations of SB431542 and undergoing Stage III culture.

[0343] FIG14 shows the cell morphology of each group after inducing differentiation using MDM II culture medium containing different concentrations of CHIR99021 and undergoing Stage II culture.

[0344] FIG15 shows the results of immunofluorescence staining of the marker protein PAX7 of each group of cells after inducing differentiation using MDM II culture medium containing different concentrations of CHIR99021 and undergoing Stage III culture.

[0345] FIG16 shows the cell morphology of each group after inducing differentiation using MDM II culture medium containing different concentrations of LDN193189 and undergoing Stage II culture.

[0346] FIG17 shows the results of immunofluorescence staining of PAX7, a marker protein of cells in each group after inducing differentiation using MDM II culture medium containing different concentrations of LDN193189 and undergoing Stage III culture.

[0347] FIG18 shows the cell morphology of each group after inducing differentiation using MDM II culture medium containing different concentrations of FGF2 and undergoing Stage II culture.

[0348] FIG19 shows the results of immunofluorescence staining of the marker protein PAX7 of each group of cells after inducing differentiation using MDM II culture medium containing different concentrations of FGF2 and undergoing Stage III culture.

[0349] FIG20 shows the cell morphology of each group after inducing differentiation using MDM III culture medium containing different concentrations of FGF2 and undergoing Stage III culture.

[0350] Figure 21 shows the results of immunofluorescence staining of PAX7, a marker protein of cells in each group after inducing differentiation using MDM III culture medium containing different concentrations of FGF2 and undergoing Stage III culture.

[0351] Figure 22 shows the cell morphology of each group after inducing differentiation using MDM III culture medium containing different concentrations of IGF-1 and undergoing Stage III culture.

[0352] FIG23 shows the results of immunofluorescence staining of PAX7, a marker protein of cells in each group after inducing differentiation using MDM III culture medium containing different concentrations of IGF-1 and undergoing Stage III culture.

[0353] FIG24 shows the cell morphology of each group after inducing differentiation using MDM III culture medium containing different concentrations of DN193189 and undergoing Stage III culture.

[0354] FIG25 shows the results of immunofluorescence staining of PAX7, a marker protein of cells in each group after inducing differentiation using MDM III culture medium containing different concentrations of DN193189 and undergoing Stage III culture.

[0355] FIG26 shows the cell morphology of each group after inducing differentiation using MDM III culture medium containing different concentrations of HGF and undergoing Stage III culture; the image scale is 50 μm.

[0356] FIG27 shows the results of immunofluorescence staining of PAX7, a marker protein of cells in each group after inducing differentiation using MDM III culture medium containing different concentrations of HGF and undergoing Stage III culture.

[0357] FIG28 shows the cell morphology of each group after inducing differentiation using MDM IV culture medium containing different concentrations of IGF-1 and undergoing Stage IV culture; the image scale is 50 μm.

[0358] Figure 29 shows the cell morphology of each group after inducing differentiation using MDM V culture medium containing different concentrations of IGF-1 and undergoing Stage V culture.

[0359] Figure 30 shows the cell morphology of each group after inducing differentiation using MDM V culture medium containing different concentrations of HGF and undergoing Stage V culture. DETAILED DESCRIPTION

[0360] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0361] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0362] Experimental cells:

[0363] Porcine pgEpiSCs, i.e. porcine Pre-gastrulation epiblast stem cells (pre-gastrulation epiblast stem cells) that can be stably passaged, are also referred to herein as porcine stable epiblast stem cells.

[0364] Example 1: pgEpiSCs serum-free myogenic differentiation technology system and its application in the preparation of cell-cultured meat:

[0365] (1) pgEpiSCs serum-free myogenic differentiation technology system

[0366] pgEpiSCs were differentiated into mature myocytes using a feeder-free method. By changing the culture system at different stages as shown in Figure 1 , pgEpiSCs were directed to differentiate into mature myocytes. The BM culture medium consisted of DMEM / F12 supplemented with NEAA, β-mercaptoethanol, penicillin-streptomycin, KOSR, and ascorbic acid.

[0367] The specific process of directed differentiation shown in Figure 1 is as follows:

[0368] ① (Stage I) Resuspend single cells of pgEpiSCs in MDM I myogenic differentiation medium for 3 days. The MDM I medium is the above-mentioned BM medium supplemented with B27, CHIR99021, and SB431542.

[0369] ② (Stage II) MDM I stage cells were dissociated into single cells using TryPLE, harvested by centrifugation at 1000 rpm for 5 min, and the cell pellet was resuspended in MDM II myogenic differentiation medium and then inoculated into cell culture plates containing MDM II medium for 3 days. The MDM II medium is the above-mentioned BM medium supplemented with CHIR99021, LDN193189, and FGF2.

[0370] ③ (Stage III) Change to MDM stage III (pgEpiSCs-MPCs) medium (i.e., the above BM medium supplemented with HGF, IGF-1, FGF2, and LDN193189) for 2 days.

[0371] ④ (Stage IV) After 2 days of MDM III induction, the differentiation medium was changed to MDM IV (ie, the above-mentioned BM medium supplemented with IGF-1) and maintained for 4 days.

[0372] ⑤ (Stage V) The cells were cultured in MDM V myogenic differentiation medium (i.e., the above-mentioned BM medium supplemented with IGF-1 and HGF) for 20-25 days, and the generation of fibroblasts was clearly observed.

[0373] ⑥(N2) Replace the myogenic differentiation medium at MDM stage V with N2 differentiation medium (i.e., the above BM medium supplemented with N2), with an induction period of 5-7 days, and use the HS group (DMEM / F12 supplemented with KOSR, HS, penicillin-streptomycin and NEAA) as a positive control.

[0374] During the above-mentioned pgEpiSCs myogenic differentiation process, the cells at the intermediate stage can be frozen according to experimental requirements, and the pgEpiSCs myogenically differentiated cells can be frozen using freezing medium (DMSO+FBS).

[0375] The formulas of the above-mentioned culture media are shown in Table 2:

[0376] Table 2: Formula of culture medium for each differentiation induction stage

[0377] (2) Preparation of pgEpiSCs-derived CM (Cultured Meat)

[0378] Resuspend pgEpiSCs-MCs (MDM V, i.e., Stage V) in culture medium at a concentration of 1.0×10 6 Cells were uniformly seeded onto scaffolds (for an exemplary preparation method of the scaffold, see Li L, et al. Chitosan-sodium alginate-collagen / gelatin three-dimensional edible scaffolds for building a structured model for cell cultured meat. Int J Biol Macromol. 2022 Jun 1; 209(Pt A): 668-679. doi: 10.1016 / j.ijbiomac.2022.04.052.) at a density of 10 cells / mL. Adherence was followed by supplementation with adapted culture medium after 4 hours of adhesion. pgEpiSCs-MCs were maintained in MDM V myogenic differentiation medium for 8 days and then in differentiation medium containing N2 for 7 days.

[0379] ① All three-dimensional cell cultures were performed in an incubator at 37°C and 5% CO2, and the medium was changed individually.

[0380] ②The three-dimensional differentiation of pgEpiSCs-MCs was observed using confocal microscopy.

[0381] After seeding, culture, and differentiation, pgEpiSCs-MCs were washed once with DPBS and then fixed with 4% PFA at room temperature. Three-dimensional staining was performed using the same method as immunofluorescence staining, using Actin-Tracker Red-594 to observe F-actin expression. Cell nuclei were stained with DAPI and washed with DPBS. Images were captured using a laser scanning confocal microscope.

[0382] ③ Detection of texture characteristics of pgEpiSCs-derived CM

[0383] The texture profile analysis (TPA) of pgEpiSCs-derived CM was measured using a texture analyzer. pgEpiSCs-MCs were cultured and differentiated on a three-dimensional edible scaffold. The surface culture medium was gently aspirated using filter paper. The scaffold was then subjected to a double compression cycle test, compressed to 50% of its original height at a strain rate of 5 mm s⁻¹. Using an unseeded three-dimensional edible scaffold as a negative control and commercially available pork tenderloin as a positive control, the physical properties of the pgEpiSCs-derived CM, including springiness, cohesiveness, gumminess, chewiness, resilience, and hardness, were measured to determine their similarity to authentic pork products.

[0384] ④ Food coloring of pgEpiSCs-derived CMs

[0385] The pgEpiSC-derived CMs were stained with food pigments (lycopene and betalain) at room temperature for 10 minutes. The stained biomimetic tissues were then placed in a pan and fried with a small amount of cooking oil. Their deformation state was observed, and photos were taken to record the changes in their appearance.

[0386] Experimental results:

[0387] From the perspective of cell appearance, pgEpiSCs changed from a clone morphology with smooth and clear boundaries to a myofiber morphology visible to the naked eye, as shown in the technical flow chart 1.

[0388] We observed that the differentiated cells in the B27 group not only adhered better but also exhibited greater random differentiation potential, with downregulation of pluripotency-related genes OCT4, SOX2, and NANOG, and upregulation of genes associated with three germ layer differentiation: PAX6 (ectoderm), T (mesoderm), and EOMES (endoderm). Therefore, we selected this medium for subsequent differentiation (as shown in Figure 2).

[0389] Muscle development originates from the paraxial mesoderm (PM). Early myogenic differentiation of PSCs toward the PM and muscle can be achieved by regulating key signaling pathways, such as WNT, BMP, and TGF-β (Wu et al., 2018). However, the efficiency and consistency of directed differentiation protocols often vary greatly (Kim et al., 2017), primarily due to differences in culture medium composition between reports. Therefore, based on the previously screened differentiation basal culture system, different small molecules were added to promote the differentiation of pgEpiSCs toward the PM stage.

[0390] Based on existing literature reports, this application optimized the early PM differentiation system, namely WNT activation and BMP inhibition (CHIR+LDN), WNT activation and TGF-β inhibition (CHIR+SB), and WNT activation with BMP inhibition and TGF-β inhibition (CHIR+LDN+SB), and differentiated pgEpiSCs into PMs under feeder-free conditions. As differentiation progressed, the cell morphology under the three differentiation systems changed from clonal to flat, and there was no significant difference in the three cell appearance morphologies (Figure 3A). In addition, all three differentiation systems could downregulate the expression of pluripotency genes OCT4, SOX2, and NANOG, and upregulate the expression of PM differentiation-related genes PDGFRα, T, and MSGN1 (Figure 3B, C, D). Compared with undifferentiated pgEpiSCs, the expression of pluripotency and PM differentiation-related genes in differentiated cells was significantly different. Among them, WNT activation and TGF-β inhibition (CHIR+SB) showed the highest expression of PM differentiation-related genes compared with the other two groups (Figure 3E). After differentiation, cells in this system showed decreased AP positivity (Figure 3F) and expressed protein T, a protein associated with primitive streak development (Figure 3G), indicating that pgEpiSCs gradually exited pluripotency and progressed toward PM differentiation. Therefore, this system was selected for subsequent differentiation.

[0391] RT-PCR analysis revealed that mature muscle cells no longer expressed pluripotency-related markers (OCT4, NANOG), but instead expressed markers associated with mature muscle cells (MYOG, MYMK, MYH2, etc.). Furthermore, extracellular matrix formation, another prominent characteristic of mature muscle cells, was detected, and detection of extracellular matrix-related genes revealed high expression of COL3A1, COL5A2, COL6A3, COL11A1, FBN1, LAMA4, and FLN. The karyotype of the differentiated cells remained unchanged. Transcriptomic analysis revealed that the differentiated myoblasts possessed typical muscle-related features, enriched for functions related to muscle development, and gene expression was consistent with the RT-PCR results (Figures 4 and 5).

[0392] Immunofluorescence staining experiments showed ( FIG6 ) that, compared with the results induced by 2% HS, the terminally differentiated cells in the absence of serum (N2) expressed myofiber-related proteins Myosin and F-actin and could maintain long-term myogenic differentiation ( FIG7 ).

[0393] After seeding, the scaffolds were harvested and analyzed. Immunostaining for F-actin was performed to assess the maturity of myotubes derived from pgEpiSCs-derived myoblasts in 3D differentiation. Confocal microscopy (Figure 8) revealed that pgEpiSCs-derived myoblasts were able to achieve 3D differentiation within the edible scaffolds, exhibiting a highly connected, extended cytoskeleton morphology with a striped pattern of F-actin. This further demonstrates that pgEpiSCs-derived myoblasts possessed excellent adhesion, survival, elongation, proliferation, and differentiation abilities on the edible scaffolds, ultimately forming myotubes. Furthermore, the karyotype of the pgEpiSCs-derived myoblasts remained unchanged, maintaining a normal number of 38 chromosomes. This indicates that 3D differentiation of the cells on the edible scaffolds did not result in chromosome loss, confirming the safety of both the cells and the scaffold. Finally, the edible properties of the pgEpiSCs-derived CM were evaluated. After 15 days of culture, the simulated muscle tissue was colored with food coloring and deep-fried, resulting in a round cake approximately 15 mm in diameter with a color similar to that of real meat. Textural analysis revealed that pgEpiSCs-derived CM exhibited no significant differences from real meat products in terms of elasticity and cohesion, while other parameters showed significant differences. However, cell-inoculated CM exhibited significantly enhanced textural properties compared to uninoculated scaffolds. While pgEpiSCs-derived CM still exhibited some textural differences from fresh pork, its large, blocky structure was visually striking.

[0394] One of the advantages of pgEpiSCs-derived CM over traditional cultured meat is its sterility due to the control of the culture environment. In addition, foodborne pathogens are an important source of food safety issues. Therefore, common foodborne pathogens (such as Escherichia coli, Staphylococcus aureus, and Salmonella) were tested (Figure 9) to evaluate the microbial contamination of pgEpiSCs-derived CM to confirm its safety as a new meat product. Specifically, the diluted bacterial solutions of Escherichia coli, Staphylococcus aureus, and Salmonella, the pgEpiSCs myogenic differentiation medium after cell culture (MDM V, N2 terminal differentiation), and the homogenate of pgEpiSCs-derived CM were spread on LB solid culture medium. The results of the viable bacteria test showed that Escherichia coli (5.82×10 9 CFU), Staphylococcus aureus (6.14×10 9 CFU) and Salmonella (5.52×10 9CFU), while no colonies grew in the pgEpiSCs myogenic differentiation medium and homogenate after cell culture, which clearly showed that the pgEpiSCs-derived meat-like tissue was cleaner in terms of microbial contamination.

[0395] Taken together, these results indicate that the muscle cells obtained by serum-free induction differentiation of pgEpiSCs possess typical muscle characteristics and can be used to prepare CM, which is expected to be used in the future research and development of cell-cultured meat.

[0396] Example 2: Optimization of the serum-free myogenic differentiation system of pgEpiSCs

[0397] 1. Optimization of CHIR99021 concentration in MDM I medium (corresponding to Stage I culture stage)

[0398] The CHIR99021 concentrations in the MDM I medium shown in Table 2 were adjusted to 0.1 μM, 3 μM, and 10 μM, respectively, while the other components and their concentrations remained unchanged; except for the MDM I medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0399] The cell morphology after Stage I culture in MDM I medium containing different concentrations of CHIR99021 is shown in Figure 10. The results show that there is little difference in the cell morphology of each group, indicating that CHIR99021 has little effect on the morphology of cells undergoing Stage I differentiation culture within the concentration range of 0.1 μM-10 μM.

[0400] Furthermore, each of the above groups of cells was further subjected to differentiation culture in Stage II and Stage III (the differentiation culture conditions for Stage II and Stage III were the same as in Example 1), and immunofluorescence staining of the marker protein PAX7 of each group of cells was performed. The staining results are shown in Figure 11, which show that each group of cells can differentiate into muscle progenitor cells (MPCs) stage, among which, the effect is better when the concentration of CHIR99021 in MDM I medium is 10 μM, and the differentiation efficiency is higher.

[0401] 2. Optimization of SB431542 concentration in MDM I medium (corresponding to Stage I culture stage)

[0402] The SB431542 concentrations in the MDM I medium shown in Table 2 were adjusted to 0.1 μM, 2 μM, and 10 μM, respectively, while the other components and their concentrations remained unchanged; except for the MDM I medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0403] The cell morphology after Stage I culture in MDM I medium containing different concentrations of SB431542 is shown in Figure 12. The results show that there is little difference in the cell morphology of each group, indicating that SB431542 has little effect on the morphology of cells undergoing Stage I differentiation culture within the concentration range of 0.1 μM-10 μM.

[0404] Furthermore, each of the above groups of cells was further subjected to differentiation culture in Stage II and Stage III (the differentiation culture conditions for Stage II and Stage III were the same as in Example 1), and immunofluorescence staining of the marker protein PAX7 in each group of cells was performed. The staining results are shown in Figure 13, which show that each group of cells can differentiate into muscle progenitor cells (MPCs). Among them, the SB431542 concentration in the MDM I medium was better at 10 μM, and the differentiation efficiency was higher.

[0405] 3. Optimization of CHIR99021 concentration in MDM II medium (corresponding to Stage II culture stage)

[0406] The CHIR99021 concentrations in the MDM II medium shown in Table 2 were adjusted to 0.1 μM, 3 μM, and 10 μM, respectively, while the other components and their concentrations remained unchanged; except for the MDM II medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0407] Referring to Example 1, the cells were cultured in MDM I medium for Stage I and then further cultured in MDM II medium containing different concentrations of CHIR99021 for Stage II. The cell morphology after Stage II culture is shown in Figure 14. The results show that there is little difference in the cell morphology of each group, indicating that CHIR99021 has little effect on the morphology of cells undergoing Stage II differentiation culture within the concentration range of 0.1 μM-10 μM.

[0408] Furthermore, each of the above groups of cells was further subjected to Stage III differentiation culture (Stage III differentiation culture conditions were the same as in Example 1), and immunofluorescence staining of the marker protein PAX7 in each group of cells was performed. The staining results are shown in Figure 15, and the results show that each group of cells can differentiate into muscle progenitor cells (MPCs) stage, among which, the effect is better when the concentration of CHIR99021 in MDM II medium is 10 μM, and the differentiation efficiency is higher.

[0409] 4. Optimization of LDN193189 concentration in MDM II medium (corresponding to Stage II culture stage)

[0410] The concentrations of LDN193189 in the MDM II medium shown in Table 2 were adjusted to 0.1 μM and 0.5 μM, respectively, while the other components and their concentrations remained unchanged; except for the MDM II medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0411] Referring to Example 1, the cells were cultured in MDM I medium for Stage I and then further cultured in MDM II medium containing different concentrations of LDN193189 for Stage II. The cell morphology after Stage II culture is shown in Figure 16. The results show that there is little difference in the cell morphology of each group, indicating that LDN193189 has little effect on the morphology of cells undergoing Stage II differentiation culture within the concentration range of 0.1 μM-0.5 μM.

[0412] Furthermore, each of the above groups of cells was further subjected to Stage III differentiation culture (Stage III differentiation culture conditions were the same as in Example 1), and immunofluorescence staining of the marker protein PAX7 was performed on each group of cells. The staining results are shown in Figure 17, which show that each group of cells can differentiate into muscle progenitor cells (MPCs). Among them, the effect is better when the concentration of LDN193189 in MDM II medium is 0.1 μM, and the differentiation efficiency is higher.

[0413] 5. Optimization of FGF2 concentration in MDM II medium (corresponding to Stage II culture stage)

[0414] The FGF2 concentrations in the MDM II medium shown in Table 2 were adjusted to 1 ng / mL, 20 ng / mL, and 100 ng / mL, respectively, while the other components and their concentrations remained unchanged; except for the MDM II medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0415] Referring to Example 1, the cells were cultured in MDM I medium for Stage I and then further cultured in MDM II medium containing different concentrations of FGF2 for Stage II. The cell morphology after Stage II culture is shown in Figure 18. The results show that there is little difference in the cell morphology of each group, indicating that FGF2 has little effect on the morphology of cells undergoing Stage II differentiation culture within the concentration range of 1 ng / mL to 100 ng / mL.

[0416] Furthermore, each of the above groups of cells was further subjected to Stage III differentiation culture (Stage III differentiation culture conditions were the same as in Example 1), and immunofluorescence staining of the marker protein PAX7 in each group of cells was performed. The staining results are shown in Figure 19, which show that each group of cells can differentiate into muscle progenitor cells (MPCs). Among them, the effect is better when the FGF2 concentration in the MDM II culture medium is 100 ng / mL, and the differentiation efficiency is higher.

[0417] 6. Optimization of FGF2 concentration in MDM III medium (corresponding to Stage III culture stage)

[0418] The FGF2 concentrations in the MDM III medium shown in Table 2 were adjusted to 1 ng / mL, 20 ng / mL, and 100 ng / mL, respectively, while the other components and their concentrations remained unchanged; except for the MDM III medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0419] With reference to Example 1, after the cells were cultured in the Stage I and Stage II stages, they were further placed in MDM III culture medium containing different concentrations of FGF2 to culture in the Stage III stage. The cell morphology after the Stage III stage culture was shown in Figure 20, and the results showed that the morphological differences of the cells in each group were not large, indicating that FGF2 had little effect on the cell morphology of the cells undergoing the Stage III stage differentiation culture within a concentration range of 1 ng / mL to 100 ng / mL. Further, immunofluorescence staining was performed on the marker protein PAX7 of each group of cells. The staining results are shown in Figure 21, and the results show that each group of cells can differentiate into the muscle progenitor cell (MPCs) stage, wherein the FGF2 concentration in the MDM III culture medium is better at 100 ng / mL, and the differentiation efficiency is higher.

[0420] 7. Optimization of IGF-1 concentration in MDM III medium (corresponding to Stage III culture stage)

[0421] The IGF-1 concentrations in the MDM III medium shown in Table 2 were adjusted to 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively, while the other components and their concentrations remained unchanged; except for the MDM III medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0422] With reference to Example 1, after the cells were cultured in the Stage I and Stage II stages, they were further placed in MDM III culture medium containing different concentrations of IGF-1 to culture in the Stage III stage. The cell morphology after the Stage III stage culture is shown in Figure 22, and the results show that the morphology of each group of cells is not much different, indicating that IGF-1 has little effect on the morphology of cells undergoing Stage III stage differentiation culture within a concentration range of 1 ng / mL to 100 ng / mL. Further, immunofluorescence staining was performed on the marker protein PAX7 of each group of cells. The staining results are shown in Figure 23, which shows that each group of cells can differentiate into muscle progenitor cells (MPCs) stage, wherein the IGF-1 concentration in the MDM III culture medium is better at 100 ng / mL, and the differentiation efficiency is higher.

[0423] 8. Optimization of LDN193189 concentration in MDM III medium (corresponding to Stage III culture stage)

[0424] The concentrations of LDN193189 in the MDM III medium shown in Table 2 were adjusted to 0.1 μM and 0.5 μM, respectively, while the other components and their concentrations remained unchanged; except for the MDM III medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0425] With reference to Example 1, after the cells were cultured in the Stage I and Stage II stages, they were further placed in MDM III culture medium containing different concentrations of LDN193189 to culture in the Stage III stage. The cell morphology after culture in the Stage III stage is shown in Figure 24, and the results show that the morphological differences of each group of cells are not large, indicating that LDN193189 has little effect on the cell morphology of the differentiation cultured in the Stage III stage within the concentration range of 0.1 μM to 0.5 μM. Further, immunofluorescence staining was performed on the marker protein PAX7 of each group of cells. The staining results are shown in Figure 25, and the results show that each group of cells can be differentiated to the muscle progenitor cell (MPCs) stage, wherein the concentration of LDN193189 in the MDM III culture medium is better at 0.1 μM, and the differentiation efficiency is higher.

[0426] 9. Optimization of HGF concentration in MDM III medium (corresponding to Stage III culture stage)

[0427] The HGF concentrations in the MDM III medium shown in Table 2 were adjusted to 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively, while the other components and their concentrations remained unchanged; except for the MDM III medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0428] With reference to Example 1, after the cells were cultured in Stage I and Stage II, they were further placed in MDM III culture medium containing different concentrations of HGF to culture in the Stage III stage. The cell morphology after the Stage III stage culture is shown in Figure 26. The results show that the morphology of each group of cells is not much different, indicating that HGF has little effect on the morphology of cells undergoing Stage III stage differentiation culture within a concentration range of 1 ng / mL to 100 ng / mL. Further, immunofluorescence staining was performed on the marker protein PAX7 of each group of cells. The staining results are shown in Figure 27, which shows that each group of cells can differentiate into muscle progenitor cells (MPCs) stage, wherein the HGF concentration in the MDM III culture medium is better at 100 ng / mL, and the differentiation efficiency is higher.

[0429] 10. Optimization of IGF-1 concentration in MDM IV medium (corresponding to Stage IV culture stage)

[0430] The IGF-1 concentrations in the MDM IV medium shown in Table 2 were adjusted to 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively, while the remaining components and their concentrations remained unchanged; except for the MDM IV medium, the culture media corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0431] Referring to Example 1, after the cells underwent culture from Stage I to Stage III, they were further placed in MDM IV culture medium containing different concentrations of IGF-1 for Stage IV culture. The cell morphology after Stage IV culture is shown in Figure 28. The results showed that there was little difference in the cell morphology of each group, indicating that IGF-1 had little effect on the morphology of cells undergoing Stage IV differentiation culture within the concentration range of 1 ng / mL to 100 ng / mL.

[0432] 11. Optimization of IGF-1 concentration in MDM V medium (corresponding to Stage V culture stage)

[0433] The IGF-1 concentrations in the MDM V medium shown in Table 2 were adjusted to 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively, while the other components and their concentrations remained unchanged; except for the MDM V medium, the culture media corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0434] Referring to Example 1, after the cells underwent culture from Stage I to Stage IV, they were further placed in MDM V culture medium containing different concentrations of IGF-1 for Stage V culture. The cell morphology after Stage V culture is shown in Figure 29. The results showed that there was little difference in the cell morphology of each group, indicating that IGF-1 had little effect on the morphology of cells undergoing Stage V differentiation culture within the concentration range of 1 ng / mL to 100 ng / mL.

[0435] 12. Optimization of HGF concentration in MDM V medium (corresponding to Stage V culture stage)

[0436] The HGF concentrations in the MDM V medium shown in Table 2 were adjusted to 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively, while the other components and their concentrations remained unchanged; except for the MDM V medium, the culture medium corresponding to each differentiation stage remained consistent with Table 2; and serum-free myogenic differentiation of pgEpiSCs was performed with reference to Example 1.

[0437] Referring to Example 1, after the cells underwent culture from Stage I to Stage IV, they were further placed in MDM V culture medium containing different concentrations of HGF for Stage V culture. The cell morphology after Stage V culture is shown in Figure 30. The results showed that there was little difference in the cell morphology of each group, indicating that HGF had little effect on the morphology of cells undergoing Stage V differentiation culture within the concentration range of 1 ng / mL to 100 ng / mL.

[0438] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for inducing myogenic differentiation of porcine pluripotent stem cells, comprising: (1) Providing porcine pluripotent stem cells; (2) culturing the pluripotent stem cells using a first stage differentiation medium, wherein the first stage differentiation medium contains B27 supplement, CHIR99021 and SB431542; (3) culturing the cells obtained in step (2) using a second-stage differentiation medium, wherein the second-stage differentiation medium contains CHIR99021, LDN193189 and FGF2; (4) culturing the cells obtained in step (3) using a third-stage differentiation medium, wherein the third-stage differentiation medium contains HGF, IGF-1, FGF2, and LDN193189; (5) culturing the cells obtained in step (4) using a fourth stage differentiation medium, wherein the fourth stage differentiation medium contains IGF-1; and (6) culturing the cells obtained in step (5) using a fifth stage differentiation medium, wherein the fifth stage differentiation medium contains IGF-1 and HGF; Thus, myoblasts are obtained.

2. The method of claim 1, further comprising step (7): culturing the cells obtained in step (6) using a sixth stage differentiation medium, wherein the sixth stage differentiation medium contains N2 supplement; thereby obtaining muscle cells with mature skeletal muscle fibers.

3. The method of claim 1 or 2, wherein The first stage differentiation medium is a basic medium containing B27 supplement, CHIR99021 and SB431542; Preferably, the basal culture medium is selected from DMEM / F12, IMDM; Preferably, the first stage differentiation medium has one or more selected from the following characteristics: (i) the first stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement) and ascorbic acid; (ii) the first stage differentiation medium does not contain serum; (iii) in the first stage differentiation medium, the volume fraction of B27 supplement is 0.5%-5%; (iv) in the first stage differentiation medium, the concentration of CHIR99021 is 0.05-20 μM (e.g., 0.05-15 μM, 0.05-10 μM, 0.1-20 μM, 0.1-15 μM, 0.1-10 μM, 1-20 μM, 1-15 μM, 1-10 μM, 3.5-20 μM, 3.5-15 μM, 3.5-10 μM, 5-20 μM, 5-15 μM, 5-10 μM, 10 μM); (v) In the first stage differentiation medium, the concentration of SB431542 is 0.05-20 μM (e.g., 1-5 μM, 0.05-15μM, 0.05-10μM, 0.1-20μM, 0.1-15μM, 0.1-10μM, 1-20μM, 1-15μM, 1-10μM, 2.5-20μM, 2.5-15μM, 2.5-10μM, 5-20μM, 5-15μM, 5-10μM, 10μM).

4. The method of any one of claims 1 to 3, wherein The second stage differentiation medium is a basic medium containing CHIR99021, LDN193189 and FGF2; Preferably, the basal culture medium is selected from DMEM / F12, IMDM; Preferably, the second stage differentiation medium has one or more selected from the following characteristics: (i) the second stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement) and ascorbic acid; (ii) the second stage differentiation medium does not contain serum; (iii) in the second stage differentiation medium, the concentration of CHIR99021 is 0.05-20 μM (e.g., 0.05-15 μM, 0.05-10 μM, 0.1-20 μM, 0.1-15 μM, 0.1-10 μM, 1-20 μM, 1-15 μM, 1-10 μM, 3.5-20 μM, 3.5-15 μM, 3.5-10 μM, 5-20 μM, 5-15 μM, 5-10 μM, 10 μM); (iv) in the second stage differentiation medium, the concentration of LDN193189 is 0.01-3 μM (e.g., 0.1-3 μM, 0.01-1 μM, 0.01-0.5 μM, 0.01-0.4 μM, 0.05-3 μM, 0.05-1 μM, 0.05-0.5 μM, 0.05-0.4 μM, 0.1-3 μM, 0.1-1 μM, 0.1-0.5 μM, 0.1-0.4 μM, 0.1 μM); (v) in the second stage differentiation medium, the concentration of FGF2 is 0.5-200 ng / mL (e.g., 5-50 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 30-200 ng / mL, 30-150 ng / mL, 30-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (vi) The FGF2 is human FGF2 (eg, recombinant human FGF2).

5. The method of any one of claims 1 to 4, wherein The third stage differentiation medium is a basic medium containing HGF, IGF-1, FGF2 and LDN193189; Preferably, the basal culture medium is selected from DMEM / F12, IMDM; Preferably, the third stage differentiation medium has one or more selected from the following characteristics: (i) the third stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement) and ascorbic acid; (ii) the third stage differentiation medium does not contain serum; (iii) in the third stage differentiation medium, the concentration of HGF is 0.5-200 ng / mL (e.g., 2-15 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (iv) in the third stage differentiation medium, the concentration of IGF-1 is 0.5-200 ng / mL (e.g., 1-20 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (v) in the third stage differentiation medium, the concentration of FGF2 is 0.5-200 ng / mL (e.g., 5-50 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 30-200 ng / mL, 30-150 ng / mL, 30-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (vi) in the third stage differentiation medium, the concentration of LDN193189 is 0.01-3 μM (e.g., 0.1-3 μM, 0.01-1 μM, 0.01-0.5 μM, 0.01-0.4 μM, 0.05-3 μM, 0.05-1 μM, 0.05-0.5 μM, 0.05-0.4 μM, 0.1-3 μM, 0.1-1 μM, 0.1-0.5 μM, 0.1-0.4 μM, 0.1 μM); (vii) the HGF is human HGF (e.g., recombinant human HGF); (viii) the IGF-1 is human IGF-1 (e.g., recombinant human IGF-1); (ix) The FGF2 is human FGF2 (eg, recombinant human FGF2).

6. The method of any one of claims 1 to 5, wherein The fourth stage differentiation medium is a basic medium containing IGF-1; Preferably, the basal culture medium is selected from DMEM / F12, IMDM; Preferably, the fourth stage differentiation medium has one or more selected from the following characteristics: (i) the fourth stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement) and ascorbic acid; (ii) the fourth stage differentiation medium does not contain serum; (iii) in the fourth stage differentiation medium, the concentration of IGF-1 is 0.5-200 ng / mL (e.g., 1-20 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (iv) The IGF-1 is human IGF-1 (eg, recombinant human IGF-1).

7. The method of any one of claims 1 to 6, wherein The fifth stage differentiation medium is a basic medium containing IGF-1 and HGF; Preferably, the basal culture medium is selected from DMEM / F12, IMDM; Preferably, the fifth stage differentiation medium has one or more selected from the following characteristics: (i) the fifth stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement) and ascorbic acid; (ii) the fifth stage differentiation medium does not contain serum; (iii) in the fifth stage differentiation medium, the concentration of HGF is 0.5-200 ng / mL (e.g., 2-15 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (iv) in the fifth stage differentiation medium, the concentration of IGF-1 is 0.5-200 ng / mL (e.g., 1-20 ng / mL, 0.5-150 ng / mL, 0.5-100 ng / mL, 1-200 ng / mL, 1-150 ng / mL, 1-100 ng / mL, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 20-200 ng / mL, 20-150 ng / mL, 20-100 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 100 ng / mL); (v) the HGF is human HGF (e.g., recombinant human HGF); (vi) The IGF-1 is human IGF-1 (eg, recombinant human IGF-1).

8. The method of any one of claims 2 to 7, wherein The sixth stage differentiation medium is a basic medium containing N2 supplement; Preferably, the basal culture medium is selected from DMEM / F12, IMDM; Preferably, the sixth stage differentiation medium has one or more selected from the following characteristics: (i) the sixth stage differentiation medium further comprises one or more selected from the group consisting of non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (KnockOut Serum Replacement) and ascorbic acid; (ii) the sixth stage differentiation medium does not contain serum; (iii) In the sixth stage differentiation medium, the volume fraction of N2 supplement is 0.5%-5%.

9. The method of any one of claims 1 to 8, comprising one or more of the following features: (i) in step (2), the cell culture time is 1-5 days (e.g., 1-3 days, 1-3.5 days, 1-4 days, 2-3 days, 2-3.5 days, 2-4 days, 2-5 days, 2.5-3 days, 2.5-3.5 days, 2.5-4 days, 2.5-5 days, 3-3.5 days, 3-4 days, 3-5 days, 3 days); (ii) in step (3), the cell culture time is 1-5 days (e.g., 1-3 days, 1-3.5 days, 1-4 days, 2-3 days, 2-3.5 days, 2-4 days, 2-5 days, 2.5-3 days, 2.5-3.5 days, 2.5-4 days, 2.5-5 days, 3-3.5 days, 3-4 days, 3-5 days, 3 days); (iii) in step (4), the cell culture time is 1-4 days (e.g., 1-2 days, 1-2.5 days, 1-3 days, 1.5-2 days, 1.5-2.5 days, 1.5-3 days, 1.5-4 days, 2-2.5 days, 2-3 days, 2-4 days, 2 days); (iv) in step (5), the cell culture time is 1-8 days (e.g., 1-4 days, 1-5 days, 1-6 days, 1-7 days, 2-4 days, 2-5 days, 2-6 days, 2-7 days, 2-8 days, 3-4 days, 3-5 days, 3-6 days, 3-7 days, 3-8 days, 4-5 days, 4-6 days, 4-7 days, 4-8 days, 4 days); (v) In step (6), the cell culture time is 5-120 days (e.g., 5-20 days, 5-25 days, 5-30 days, 5-40 days, 5-50 days, 5-80 days, 5-100 days, 10-20 days, 10-25 days, 10-30 days, 10-40 days, 10-50 days, 10-80 days, 10-100 days, 10-120 days, 15-20 days, 15-25 days). , 15-30 days, 15-40 days, 15-50 days, 15-80 days, 15-100 days, 15-120 days, 20-25 days, 20-30 days, 20-40 days, 20-50 days, 20-80 days, 20-100 days, 10-120 days, 25-30 days, 25-40 days, 25-50 days, 25-80 days, 25-100 days, 25-120 days); (vi) in step (7), the cell culture time is 2-15 days (e.g., 2-5 days, 2-7 days, 2-10 days, 2-12 days, 5-7 days, 5-10 days, 5-12 days, 5-15 days, 7-10 days, 7-12 days, 7-15 days); (vii) in step (3), the cells obtained in step (2) are dissociated into single cells and then inoculated into the second stage differentiation medium for culture; (viii) in step (4), replacing the culture medium of the cells obtained in step (3) with the third stage differentiation medium, and performing cell culture; (ix) in step (5), the culture medium of the cells obtained in step (4) is replaced with the fourth stage differentiation medium, and the cells are cultured; (x) in step (6), the culture medium of the cells obtained in step (5) is replaced with the fifth stage differentiation medium, and the cells are cultured; (xi) In step (7), the culture medium of the cells obtained in step (6) is replaced with the sixth stage differentiation medium, and the cells are cultured.

10. A method for preparing cell cultured meat, comprising: (a) providing: a scaffold, and myoblasts obtained according to any one of claims 1, 3-7, and 9; (b) seeding the myoblasts on the scaffold and culturing them in a fifth stage differentiation medium, wherein the fifth stage differentiation medium is as defined in claim 1 or 7; (c) culturing the cells obtained in step (b) using a sixth stage differentiation medium, wherein the sixth stage differentiation medium is as defined in claim 2 or 8; Thus, cell cultured meat is obtained.

11. The method of claim 10, comprising one or more of the following features: (i) in step (b), the cell culture time is 1-15 days (e.g., 1-8 days, 1-10 days, 1-12 days, 3-8 days, 3-10 days, 3-12 days, 3-15 days, 5-8 days, 5-10 days, 5-12 days, 5-15 days, 8-10 days, 8-12 days, 8-15 days, 8 days); (ii) in step (c), the cell culture time is 1-15 days (e.g., 1-7 days, 1-10 days, 1-12 days, 3-7 days, 3-10 days, 3-12 days, 3-15 days, 5-7 days, 5-10 days, 5-12 days, 5-15 days, 7-10 days, 7-12 days, 7-15 days, 7 days); (iii) In step (c), the culture medium of the cells obtained in step (b) is replaced with the sixth stage differentiation medium, and the cells are cultured.

12. The method of claim 10 or 11, wherein The scaffold is a three-dimensional edible scaffold.

13. Cell cultured meat prepared by the method according to any one of claims 10 to 12.