WO2025011578A1 - Myogenic differentiation method for porcine pluripotent stem cell, and using same to prepare culture meat - Google Patents

Abstract

Provided is a myogenic differentiation induction method for a porcine pluripotent stem cell. The method does not involve transgenesis and does not involve serum addition in the whole process. In addition, further provided is a culture meat (CM) preparation method based on said myogenic differentiation induction method.

Description

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

This application is based on an application with CN application number 202310840978.8 and filing date July 10, 2023, and claims priority. The disclosed content of the CN application is hereby introduced as a whole into this application.

Technical Field

The present invention relates to the field of biotechnology, and in particular, to a method for inducing myogenic differentiation of porcine pluripotent stem cells, wherein the method does not involve transgenics 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

Synthetic biology is a discipline that uses engineering thinking as a guide to redesign and transform natural biological systems, design and synthesize new biological elements, components and systems, and produce target byproducts through microbial fermentation. Cultured meat (CM) is a disruptive food production technology in the future and a foreword technology for modern synthetic biology and cellular agriculture. It obtains edible meat tissue through large-scale culture of poultry or livestock cells. The acquisition of seed cells is the main bottleneck of its research and development. The purpose is to expand the replication capacity of skeletal muscle cells for industrial-scale expansion. For more than 40 years, skeletal muscle cell lines with differentiation ability have been model systems for skeletal muscle biology research. These cell lines are isolated from mice or humans and spontaneously produce corresponding muscle cells through continuous passage. However, there is a lack of large livestock species cell lines that can be used to produce meat for human consumption in cell culture. Although myoblast cell lines such as muscle satellite 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 of 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.

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 a specific tissue lineage is suppressed and they have the potential to differentiate into any somatic cell.

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, it is necessary to overcome the heavy dependence on animal-derived ingredients. At present, muscle satellite cells need to proliferate 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 the myogenic terminal differentiation of SCs can be carried out 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 early proliferation stage of SCs still relied on the addition of serum (Ding, S., et al. (2017). Characterization and isolation of highly purified porcine satellite cells. Cell Death Discov 3, 17003.), it can be seen that the current technical system for serum-free induced differentiation of muscle stem cells is still immature. In addition, muscle stem cells cannot be stably cultured 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.

So far, pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells) are used as starting cells, and the system maintained does not require the addition of serum. However, most of the research on myogenic differentiation of pluripotent stem cells is concentrated in human and mouse models, providing a theoretical basis for human myogenic-related diseases. There are few reports on myogenic differentiation of pluripotent stem cells in livestock. It is still unknown whether these technical procedures can be applied to myogenic differentiation of other species. There is still a lack of a complete technical system to obtain the corresponding differentiation lineage progenitor cells or mature cell types required for CM research and development. Therefore, it is urgent to establish a technical system for myogenic differentiation of livestock stem cells to provide a new technical approach for the research and development of CM.

Summary of the invention

At present, there is no method of using pluripotent stem cells as the starting cells for CM development to prepare CM at home and abroad.

In the present application, the inventors utilized porcine pluripotent stem cells (PSCs) that were stably passaged in vitro for a long term, and were not only able to successfully achieve 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.

Therefore, in one aspect, the present application provides 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.

In certain embodiments, the method further comprises 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 having mature skeletal muscle fibers.

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

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

In certain embodiments, the first stage differentiation medium is a basic medium containing B27 supplement, CHIR99021 and SB431542.

In some embodiments, the basal medium is a basal medium for culturing mammalian (preferably pig) pluripotent stem cells. In certain embodiments, the basal 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 non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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% (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%);

(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);

(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);

(vi) in the first stage differentiation medium, the volume fraction of non-essential amino acids (NEAA) is 0.5%-5% (for example, 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%);

(vi) in the first stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (for example, 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%);

(vii) in the first stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (for example, 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%);

(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%);

(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).

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

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

In certain embodiments, the second stage differentiation medium has one or more of the following characteristics:

(i) the second stage differentiation medium further comprises one or more selected from non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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. 5-10μM, 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);

(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);

(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);

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

(vii) in the second stage differentiation medium, the volume fraction of non-essential amino acids (NEAA) is 0.5%-5% (for example, 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%);

(viii) in the second stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (for example, 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%);

(ix) in the second stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (for example, 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%);

(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%);

(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, 150-400μM, 150-500μM, 200-250μM, 200-300μM, 200-400μM, 200-500μM).

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

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

In certain embodiments, the third stage differentiation medium has one or more of the following characteristics:

(i) the third stage differentiation medium further comprises one or more selected from non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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); ,20-100ng/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);

(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);

(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);

(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);

(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 (e.g., recombinant human FGF2);

(x) in the third stage differentiation medium, the volume fraction of non-essential amino acids (NEAA) is 0.5%-5% (for example, 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%);

(xi) in the third stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (for example, 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%);

(xii) in the third stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (for example, 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%);

(xiii) in the third stage differentiation medium, the volume fraction of KOSR (Knock Out 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%);

(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).

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

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

In certain embodiments, the fourth stage differentiation medium has one or more of the following characteristics:

(i) the fourth stage differentiation medium further comprises one or more selected from non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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). 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);

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

(v) in the fourth stage differentiation medium, the volume fraction of non-essential amino acids (NEAA) is 0.5%-5% (for example, 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%);

(vi) in the fourth stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (for example, 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%);

(vii) in the fourth stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (for example, 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%);

(viii) in the fourth stage differentiation medium, the volume fraction of KOSR (Knock Out 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%);

(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).

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

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

In certain embodiments, 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 non-essential amino acids (NEAA), 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); ,20-100ng/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);

(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);

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

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

(vii) in the fifth stage differentiation medium, the volume fraction of non-essential amino acids (NEAA) is 0.5%-5% (for example, 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%);

(viii) in the fifth stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (for example, 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%);

(ix) in the fifth stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (for example, 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%);

(x) In the fifth stage differentiation medium, the volume fraction of KOSR (Knock Out 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%);

(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).

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

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

In certain embodiments, 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 non-essential amino acids (NEAA), β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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% (for example, 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%);

(iv) in the sixth stage differentiation medium, the volume fraction of non-essential amino acids (NEAA) is 0.5%-5% (for example, 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%);

(v) in the sixth stage differentiation medium, the volume fraction of β-mercaptoethanol is 0.05%-0.5% (for example, 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%);

(vi) in the sixth stage differentiation medium, the volume fraction of penicillin-streptomycin is 0.5%-5% (for example, 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%);

(vii) in the sixth stage differentiation medium, the volume fraction of KOSR (Knock Out 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%);

(viii) In the sixth stage differentiation medium, the concentration of ascorbic acid is 100-500 μM (for example, 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).

The culture medium additives described in the present application (such as 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.

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

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.

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.

In certain embodiments, the method has 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), replacing the culture medium of the cells obtained in step (5) with the fifth stage differentiation medium, and performing cell culture;

(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.

In certain embodiments, the porcine pluripotent stem cells have no exogenous gene modification.

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

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

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.

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 stable passage.

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

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

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.

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.

In certain embodiments, the pluripotent stem cells express one or more (eg, at least 1, at least 2, at least 3, or all) of SSEA1, SSEA4, TRA-1-81, TRA-1-60.

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

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.

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.

In certain embodiments, the pluripotent stem cells of the present invention do not express or lowly express 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 E8 to E10 (e.g., E8, E9 or E10) pig embryo hypoblast cells.

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

In certain embodiments, the pluripotent stem cells do not express or underexpress one or more (eg, at least 1, at least 2, or all) of HNF4A, SOX17, and GATA4.

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.

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 pig embryonic ectoderm cells from E11 to E14 (e.g., E11, E12, E13 or E14).

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

In certain embodiments, the pluripotent stem cells have reduced expression levels of at least one (e.g., at least 2, at least 3, at least 4 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).

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 2, at least 5, at least 10, at least 15, at least 20, or all) gene 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, and VHLL, as compared to the expression levels of these genes in human embryonic stem cells.

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 2, at least 5, at least 10, at least 15, or all) gene selected from the following: 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.

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 (hESC) or primed human embryonic stem cells.

In this article, the terms "conventional hESC" or "primed hESC" have the same meaning. For the definition of primed pluripotency, please refer to, for example, 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).

In some embodiments, compared with porcine embryonic fibroblasts (pEF), the pluripotent stem cells contain genes with co-variation between regulatory potential score (RPS) and gene expression (Genes with Co-variation Between Expression and RPS), which are called co-variation genes, and the co-variation genes are selected from at least one of the genes shown in Table 1 (for example, 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). 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 co-variation between RPS and gene expression (Genes with Co-variation Between Expression and RPS) refer to genes with higher RPS values in pgEpiSCs compared to pEFs that are generally upregulated (log2 fold change [FC]>1, FDR<0.05). In certain embodiments, the above genes are determined using high-deep in situ high-throughput chromatin conformation capture (Hi-C) sequencing technology.

In certain embodiments, representative co-variant genes are as follows:

In some embodiments, the pluripotent stem cells have increased expression levels of at least 1 (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) genes selected from Table 1 as compared to the expression levels of these genes in porcine embryonic fibroblasts. In certain embodiments, the pluripotent stem cells have increased expression levels of at least one (e.g., at least 2, at least 5, at least 10, at least 15, at least 20 or all) gene 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.

In certain embodiments, compared with porcine embryonic fibroblasts, in the genome of the pluripotent stem cells, at least one (e.g., at least 2, at least 3, at least 4, at least 5 or all) transcription factors selected from the following specifically interact with enhancers: OTX2, LIN28A, NANOG, PRDM14, SALL4, UTF1, ZFP42, CDH1, DNMT3B, LEFTY2. In certain embodiments, the specific interaction with enhancers means that, as determined by ultra-deep in situ high-throughput chromatin conformation capture (Hi-C) sequencing technology, there is an interaction between the transcription factor and the enhancer, and the interaction does not exist or is relatively small in the above-mentioned porcine embryonic fibroblasts.

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

In certain embodiments, the pluripotent stem cells are capable of forming a dome-shaped clonal morphology.

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.

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.

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

In certain embodiments, the porcine pluripotent stem cells are prepared by referring to the following method:

1) Providing porcine embryo epiblast or its inner cell mass;

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

In some embodiments, the culture medium comprises:

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

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

The third component is selected from fibroblast growth factors.

In some embodiments, the culture medium further comprises:

A fourth component, wherein the fourth component is selected from CHIR99021 and WNT3a;

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

The sixth component is LIF.

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

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.

In some embodiments, the fourth component is CHIR99021.

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.

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.

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.

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.

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.

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.

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.

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.

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

In some embodiments, the culture medium comprises:

In some embodiments, the culture medium further comprises: a seventh component, the seventh component 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.

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

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

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

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

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

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

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

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%.

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%.

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%.

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

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%.

In some embodiments, the concentration of ascorbic acid is 1 μg/mL-5000 μg/mL. In some embodiments, the concentration of ascorbic acid is 50 μg/mL.

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%.

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

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.

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.

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

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

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

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

In certain embodiments, the iPSC expresses OCT4, SOX2, NANOG. Preferably, the iPSC also expresses KLF4, C-MYC, REX1, LIN28A, SALL4 and OTX2.

In certain embodiments, the iPSCs are downregulated, for example, at least 4-fold lower, in the expression level of at least 1 (e.g., at least 2, at least 5, or all 10) gene selected from the following compared to porcine pre-gastrulation epiblast pluripotent stem cells (pgEpiSCs): NR4A1, NR4A2, NR4A3, FOSB, CCN2, CCN1, THBS1, RPL26, EGR4, and DUSP2.

In certain embodiments, the iPSCs have upregulated expression levels of at least one (e.g., at least two, at least five, or all six) gene selected from the following, such as at least a 6-fold increase, compared to porcine pre-gastrulation epiblast pluripotent stem cells (pgEpiSCs): FABP3, FN3KRP, DNPH1, ANXA1, RUSC1, SNX1.

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

In some embodiments, the iPSCs are capable of forming a domed shape clone morphology.

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

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

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

(i) Reprogramming pig somatic cells;

(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.

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

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

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

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

In certain embodiments, the LIF is human LIF.

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.

In certain embodiments, the content ratio of the WNT signaling pathway inhibitor to CHIR99021 is 25: 1-1: 25, such as 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, such as 1: 2-1: 3.

In some embodiments, the concentration of the WNT signaling pathway inhibitor is 1-5 μM, such as 1 μM, 2.5 μM, 3 μM, 5 μM. In some embodiments, the concentration of the WNT signaling pathway inhibitor is 2.5 μM.

In some 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 some embodiments, the concentration of CHIR99021 is 1 μM.

In some 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 some embodiments, the concentration of the Src inhibitor is 1 μM.

In some 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 some embodiments, the concentration of LIF is 10 ng/mL.

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.

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.

In certain preferred embodiments, the culture medium contains: 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).

In certain exemplary embodiments, the culture medium contains: 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).

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 (e.g., human Activin A), 10 ng/mL FGF2 (e.g., human FGF2), and 10 ng/mL LIF (e.g., human LIF).

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

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

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

In certain embodiments, the basal medium comprises a minimal medium, an N2 supplement, a B27 supplement, non-essential amino acids, β-mercaptoethanol, a serum replacement (e.g., a knockout serum replacement), and glutamine or its derivatives (e.g., GlutaMAX).

In certain embodiments, the basal medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, β-mercaptoethanol, a serum replacement (e.g., a knockout serum replacement), and GlutaMAX.

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

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

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

In certain embodiments, the basic medium is selected from DMEM/F12, Neurobasal or a combination thereof. In certain embodiments, the basic medium is DMEM/F12 and Neurobasal.

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%, and preferably 0.5%.

In certain embodiments, the volume fraction of the B27 supplement is 0.002%-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%.

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%, preferably 1%.

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%.

In certain embodiments, the volume fraction of the serum replacement (e.g., knockout serum replacement) is 0.01%-50%, for example, 1-50%, 1-30%, 1-20%, 2-30%, 2-20%, 5-20%, and preferably 5%.

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.

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%.

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%, preferably 1%.

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.

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%, preferably 91%.

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

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%).

In certain exemplary embodiments, the basal medium is a basal medium supplemented with the following components (e.g., DMEM/F12/Neurobasal in a volume ratio of 2:1-1:2, preferably 1:1): 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 , 0.1%-5% (such as 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%) β-mercaptoethanol, 1-30% (such as 1-20%, 2-30%, 2-20%, 5-20%) serum replacement, and 0.1%-5% (such as 0.1%-2%, 0.1%-1%, 0.5%-2%, 0.5%-1%, 0.2%-2%, 0.2%-1%) glutamine or its derivatives. Preferably, it further comprises 20 μg/mL-80 μg/mL (such as 30 μg/mL-80 μg/mL, 30 μg/mL-60 μg/mL, 40 μg/mL-60 μg/mL) ascorbic acid. Preferably, it further comprises 0.1%-5% (such as 0.1%-2%, 0.5%-5%, 0.5%-2%, 1%-5%, 1%-2%) penicillin-streptomycin.

In certain exemplary embodiments, the basal culture medium is a basic culture medium supplemented with the following components (e.g., DMEM/F12/Neurobasal in 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.

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.

In certain embodiments, the culture after the reprogramming factors are introduced into the somatic cells is carried out in the presence of a feeder layer. In certain embodiments, the feeder layer is mouse embryonic fibroblasts.

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

In certain embodiments, the culture medium used in the subculturing step is the culture medium described in step (ii).

In certain embodiments, the culture medium used in the subculturing step is the culture medium in step (ii) after adding a ROCK inhibitor. In certain embodiments, the ROCK inhibitor is Y-27632. In certain embodiments, the content of the ROCK inhibitor is 0.01-50 μM, such as 0.01-20 μM, 0.1-20 μM, 0.1-10 μM, 0.1-5 μM, 0.5-5 μM, 1-5 μM, such as 2 μM.

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).

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, and peripheral blood mononuclear cells.

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

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

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

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, so the reprogramming factors also include pLIN28A and pNANOG.

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

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

In certain embodiments, the nucleotide sequence encoding each of the reprogramming factors is 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, and nucleic acid molecules encoding other factors are located in different expression cassettes.

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

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

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

In certain embodiments, the non-integrating vector is an Episomal vector. Typically, prolonged expression of reprogramming factors can be achieved by episomal vectors based on oriP/EBNA1. These plasmids contain oriP/EBNA1 viral elements derived from Epstein-Barr virus, which promote the replication of free plasmid DNA in dividing cells, thereby allowing the reprogramming factors to be expressed long enough to initiate the reprogramming process, and the plasmid will eventually be lost from the proliferating cells, leaving no trace of plasmid transfection.

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

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 residual exogenous gene at the endogenous locus, thereby obtaining pig iPSCs without exogenous gene modification.

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

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

In certain embodiments, the integrative vector is introduced into the cell by viral transfection.

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

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

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 basic culture 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 contains 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 contains penicillin-streptomycin; for example, the volume fraction is 0.01%-20%, preferably 1%.

In certain embodiments, the method comprises: introducing the reprogramming factors into somatic cells, pre-culturing 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).

In certain embodiments, the iPSCs refer to patent application CN2023105046956.

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

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

(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;

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

Thus, cell cultured meat is obtained.

In certain embodiments, the method has 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.

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

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

On the other hand, the present application provides cell-cultured meat prepared by the method described above.

Advantageous Effects of the Invention

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, while also overcoming the problems of serum dependence and inability to stably culture in vitro for a long time faced by the prior art in preparing CM based on muscle stem cell induced differentiation.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present 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

Figure 1: Schematic diagram of pgEpiSCs myogenic differentiation induction technology

Among them, Stage I-V corresponds to MDM I-V in the technical method.

Figure 2: Optimization of pgEpiSCs differentiation basal medium

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

(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.

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

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 200 μM ascorbic acid;

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

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

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;

Error bars represent mean ± S.D., and different letters are used to show the differences among groups.

Fig. 3 Small molecule screening of pgEpiSCs in the early stage of myogenic differentiation

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

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

(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.

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

(G) Immunostaining of marker proteins such as T, NANOG, and SOX2, and DAPI for nuclear staining in the presence of Wnt activation and TGF-β inhibition. Scale bar, 10 μm.

For (A), CHIR: CHIR99021; LDN: LDN193189; SB: SB431542. For (B-D and F), Mes-Dif represents paraxial mesoderm differentiation, and the error bars based on three independent experiments are represented by mean ± S.D; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For E, the differences between groups are marked with different letters.

Fig. 4 Cell characteristics analysis of terminally differentiated pgEpiSCs

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

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

Myo-Dif represents the terminal myogenic differentiation of pgEpiSCs, and the error bars are represented by mean ± S.D.; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig.5 Cluster analysis of different gene expression patterns during in vitro myogenic differentiation of pgEpiSCs

(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.

(B) Ternary plot of 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.

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

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

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

(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.

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 ± S.D; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6 pgEpiSCs terminally differentiated myogenic cells have the morphological characteristics of mature muscle fibers

(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).

(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 bar, 100 μm (left) or 20 μm (right).

Fig.7 Characterization of pgEpiSCs-derived myoblasts after long-term differentiation

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

(B) Survival rate of pgEpiSCs-derived myoblasts during long-term differentiation. Calcein-AM (green) indicates live cells, PI (red) indicates dead cells, scale bar, 100 μm.

(C) Flow cytometric analysis of cell viability.

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

Fig. 8 Differentiation and CM formation assay of pgEpiSCs-derived myoblasts on 2-CS-SA-Col1-Gel1 scaffolds

(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.

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

(C) Karyotype analysis of pgEpiSCs-MCs after differentiation on 2-CS-SA-Col ₁ -Gel ₁ scaffolds.

(D) Textural properties of pgEpiSC-derived CM and fresh pork after 15 days of culture. Error bars are expressed as mean ± S.D. Different letters indicate differences between groups.

Fig. 9 Microbial detection of pgEpiSCs-derived CM

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

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

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

(D) Colony formation image after plating the culture medium of MDM V cultured cells.

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

(F) Colony formation images of the pgEpiSCs-derived CM extracts after plating.

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.

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.

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.

Figure 13 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.

Figure 14 shows the cell morphology of each group after Stage II culture using MDM II culture medium containing different concentrations of CHIR99021 to induce differentiation.

Figure 15 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM II culture medium containing different concentrations of CHIR99021 and undergoing Stage III culture.

Figure 16 shows the cell morphology of each group after Stage II culture using MDM II culture medium containing different concentrations of LDN193189 to induce differentiation.

Figure 17 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM II culture medium containing different concentrations of LDN193189 and undergoing Stage III culture.

Figure 18 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.

Figure 19 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM II culture medium containing different concentrations of FGF2 and undergoing Stage III culture.

Figure 20 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.

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

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.

Figure 23 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM III culture medium containing different concentrations of IGF-1 during Stage III culture.

Figure 24 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.

Figure 25 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells after inducing differentiation using MDM III culture medium containing different concentrations of DN193189 during Stage III culture.

Figure 26 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 50μm.

Figure 27 shows the results of immunofluorescence staining of PAX7, a marker protein of each group of cells, after inducing differentiation using MDM III culture medium containing different concentrations of HGF and undergoing Stage III culture.

Figure 28 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 50μm.

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.

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

The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

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 F. M. Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention.

Experimental cells:

Porcine pgEpiSCs, i.e., porcine Pre-gastrulation epiblast stem cells (pre-gastrulation epiblast stem cells) that can be stably passaged, are also referred to in this article as porcine stable epiblast stem cells.

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

(1) pgEpiSCs serum-free myogenic differentiation technology system

pgEpiSCs were differentiated into mature muscle cells using a feeder-free method. The culture system was changed at different stages as shown in Figure 1. The BM culture medium was DMEM/F12 supplemented with NEAA, β-mercaptoethanol, penicillin-streptomycin, KOSR and ascorbic acid.

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

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

②(Stage II) Use TryPLE to dissociate the cells in the MDM I stage into single cells, harvest the cells after centrifugation at 1000 rpm for 5 min, resuspend the cell pellet in MDM II myogenic differentiation medium, and then inoculate into a cell culture plate containing MDM II medium for 3 days. The MDM II medium is the above-mentioned BM medium supplemented with CHIR99021, LDN193189 and FGF2.

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.

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

⑤ (Stage V) After differentiation and culture in MDM V myogenic differentiation medium (i.e., the above BM medium supplemented with IGF-1 and HGF) for 20-25 days, the production of fibroblasts can be clearly observed.

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.

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

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

Table 2: Formulas of culture media for each differentiation induction stage

(2) Preparation of pgEpiSCs-derived CM (Cultured meat)

pgEpiSCs-MCs (MDM V, i.e., Stage V) were resuspended in culture medium and uniformly seeded into the scaffold (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 1.0×10 6 cells/mL, and the adapted culture medium was added 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.

① All three-dimensional cell cultures were performed in an incubator at 37°C and 5% _CO2 , and the medium was changed individually.

② Confocal microscopy was used to observe the three-dimensional differentiation of pgEpiSCs-MCs.

After inoculation, culture and differentiation, pgEpiSCs-MCs were washed once with DPBS and then fixed with 4% PFA at room temperature. The three-dimensional staining method was the same as immunofluorescence staining, and the expression of F-actin in cells was observed by staining with Actin-Tracker Red-594. The nuclei were stained with DAPI and rinsed with DPBS after staining. Images were taken using a laser scanning confocal microscope.

③ Detection of texture characteristics of pgEpiSCs-derived CM

The texture characteristics (Texture profile analysis, TPA) of pgEpiSCs-derived CM were measured by a texture analyzer. pgEpiSCs-MCs were inoculated on a three-dimensional edible scaffold for culture and differentiation, and the culture medium on the surface was gently aspirated with filter paper, followed by a double compression cycle test, with the highest compression to 50% of the original part height and a compression strain rate of 5mm s-1. The three-dimensional edible scaffold without cell inoculation was used as a negative control, and the pork tenderloin purchased on the market was used as a positive control. The physical properties such as Springiness, Cohesiveness, Gumminess, Chewiness, Resilience and Hardness were measured to determine the similarity of pgEpiSCs-derived CM with real pork products.

④ Food coloring of pgEpiSCs-derived CMs

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

Experimental results:

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

We observed that the differentiated cells in the B27 group not only adhered better, but also showed greater random differentiation potential, down-regulated the expression of pluripotency-related genes OCT4, SOX2 and NANOG, and up-regulated the expression of three-germ layer differentiation-related genes PAX6 (ectoderm), T (mesoderm) and EOMES (endoderm). Therefore, we chose this medium for subsequent differentiation (as shown in Figure 2).

Muscle development originates from the paraxial mesoderm (PM), and early myogenic differentiation can be achieved by regulating the main signaling pathways for PSCs to differentiate into PM and muscle, 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), mainly due to the different culture medium components in each report. Therefore, based on the differentiation-based culture system obtained in the early screening, different small molecules were added to promote the differentiation of pgEpiSCs into the PM stage.

According to existing literature reports, this application optimized the system of early PM differentiation, namely WNT activation and BMP inhibition (CHIR+LDN), WNT activation and TGF-β inhibition (CHIR+SB), WNT activation and BMP inhibition and TGF-β inhibition (CHIR+LDN+SB), and differentiated pgEpiSCs into PM 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 appearance of the three cells (Figure 3A). In addition, all three differentiation systems can 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, there were significant differences in the expression of pluripotency and PM differentiation-related genes in differentiated cells, among which WNT activation and TGF-β inhibition (CHIR+SB) had the highest expression of PM differentiation-related genes compared with the other two groups (Figure 3E). The AP positivity of cells differentiated by this system weakened (Figure 3F), and they expressed the protein T associated with the development of the primitive streak stage (Figure 3G), indicating that pgEpiSCs gradually exited pluripotency and moved toward PM differentiation. Therefore, this system was selected for subsequent differentiation.

RT-PCR analysis found that mature muscle cells no longer expressed makers related to pluripotency (OCT4, NANOG), but expressed makers related to mature muscle cells (MYOG, MYMK, MYH2, etc.); in addition, the formation of extracellular matrix is another significant feature of mature muscle cells. The detection of extracellular matrix-related genes found that COL3A1, COL5A2, COL6A3, COL11A1, FBN1, LAMA4, FLN, etc. were highly expressed, and the karyotype of differentiated cells did not change. Transcriptomic analysis showed that the differentiated myoblasts had typical muscle-related characteristics, enriched in functions related to muscle development, and the gene expression was consistent with the RT-PCR results (as shown in Figures 4 and 5).

Immunofluorescence staining experiments showed ( FIG. 6 ) 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 ( FIG. 7 ).

We harvested the scaffolds after cell inoculation for testing. We performed immunostaining for F-actin to evaluate the maturity of myotubes differentiated from pgEpiSCs-derived myoblasts in three dimensions. Confocal results (Figure 8) showed that pgEpiSCs-derived myoblasts could achieve three-dimensional differentiation in the three-dimensional edible scaffolds, showing a highly connected and extended cytoskeleton morphology with a striped pattern of F-actin, which further indicated that pgEpiSCs-derived myoblasts had good adhesion, survival, extension, proliferation and differentiation capabilities on the three-dimensional edible scaffolds, and finally formed myotubes. In addition, the karyotype of pgEpiSCs-derived myoblasts did not change, and the normal number of 38 chromosomes was still maintained, indicating that the three-dimensional differentiation of cells on the three-dimensional edible scaffolds would not cause the cells to lose the number of chromosomes, which also reflected the safety of cells and scaffolds. Finally, the edible properties of pgEpiSCs-derived CM were evaluated. The simulated muscle tissue cultured for 15 days was colored with food coloring and fried, showing a round cake shape with a diameter of about 15 mm and a color similar to that of real meat products. The analysis of texture properties showed that the elasticity and cohesion of CM derived from pgEpiSCs were not significantly different from those of real meat products, and there were significant differences in other indicators. However, the relevant texture properties of CM derived from pgEpiSCs were significantly enhanced compared with those of scaffolds not inoculated with cells. Although the texture properties of CM derived from pgEpiSCs are still somewhat different from those of fresh pork, its large-sized block structure is still very eye-catching visually.

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, so 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 respectively. The results of live bacteria detection showed that Escherichia coli (5.82×10 ⁹ CFU), Staphylococcus aureus (6.14×10 ⁹ CFU) and Salmonella (5.52×10 ⁹ CFU) were detected, while the pgEpiSCs myogenic differentiation medium and homogenate after cell culture did not grow colonies. This result also clearly shows that the pgEpiSCs-derived meat-like tissue is cleaner in terms of microbial contamination.

Based on the above results, it can be shown that the muscle cells obtained by serum-free induced differentiation of pgEpiSCs have 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.

Example 2: Optimization of pgEpiSCs serum-free myogenic differentiation system

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

The CHIR99021 concentrations in the MDM I medium as shown in Table 2 were adjusted to 0.1 μM, 3 μM and 10 μM, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

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 0.1μΜ-10μM.

Furthermore, the above-mentioned groups of cells were further subjected to differentiation culture in Stage II and Stage III (the differentiation culture conditions in Stage II and Stage III were the same as those 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, and the results show that each group of cells can be differentiated 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.

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

The concentrations of SB431542 in the MDM I medium as shown in Table 2 were adjusted to 0.1 μM, 2 μM and 10 μM, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

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 0.1μΜ-10μM.

Furthermore, the above-mentioned groups of cells were further subjected to differentiation culture in Stage II and Stage III (the differentiation culture conditions in Stage II and Stage III were the same as those 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 13, and the results show that each group of cells can be differentiated into muscle progenitor cells (MPCs) stage, among which, the SB431542 concentration in MDM I medium is better at 10 μM, and the differentiation efficiency is higher.

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

The CHIR99021 concentrations in the MDM II medium as shown in Table 2 were adjusted to 0.1 μM, 3 μM and 10 μM, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring to Example 1, after the cells were cultured in MDM I medium for Stage I, they were further placed in MDM II medium containing different concentrations of CHIR99021 for Stage II culture. The cell morphology after Stage II culture is shown in Figure 14. The results showed that there was 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 0.1μΜ-10μM.

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

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

The concentrations of LDN193189 in the MDM II medium as shown in Table 2 were adjusted to 0.1 μM and 0.5 μM, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring to Example 1, after the cells were cultured in MDM I medium for Stage I, they were further placed in MDM II medium containing different concentrations of LDN193189 for Stage II culture. The cell morphology after Stage II culture is shown in Figure 16. The results showed that there was 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 0.1μΜ-0.5μM.

Furthermore, the above-mentioned groups of cells were further subjected to the differentiation culture of Stage III (the differentiation culture conditions of Stage III were the same as those of Example 1), and the marker protein PAX7 of each group of cells was immunofluorescently stained. The staining results are shown in Figure 17, and the results show that each group of cells can be differentiated to the muscle progenitor cell (MPCs) stage, among which, the effect is better when the concentration of LDN193189 in the MDM II culture medium is 0.1 μM, and the differentiation efficiency is higher.

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

The FGF2 concentration in the MDM II medium as shown in Table 2 was adjusted to 1 ng/mL, 20 ng/mL and 100 ng/mL, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring to Example 1, after the cells were cultured in MDM I medium for Stage I, they were further placed in MDM II medium containing different concentrations of FGF2 for Stage II culture. The cell morphology after Stage II culture is shown in Figure 18. The results showed that the cell morphology of each group was not much different, 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.

Furthermore, the above-mentioned groups of cells were further subjected to the differentiation culture of Stage III (the differentiation culture conditions of Stage III were the same as those of Example 1), and the marker protein PAX7 of each group of cells was immunofluorescently stained. The staining results are shown in Figure 19, and the results show that each group of cells can be differentiated to the muscle progenitor cell (MPCs) stage, among which, the effect is better when the FGF2 concentration in the MDM II culture medium is 100 ng/mL, and the differentiation efficiency is higher.

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

The FGF2 concentration in the MDM III medium as shown in Table 2 was adjusted to 1 ng/mL, 20 ng/mL and 100 ng/mL, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring 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 FGF2 to undergo Stage III culture. The cell morphology after Stage III culture is shown in Figure 20. The results show that the cell morphology of each group is not much different, indicating that FGF2 has little effect on the morphology of cells undergoing Stage III differentiation culture within the concentration range of 1ng/mL to 100ng/mL. Further, immunofluorescence staining was performed on the marker protein PAX7 of each group of cells. The staining results are shown in Figure 21, which shows that each group of cells can differentiate to the muscle progenitor cell (MPCs) stage, among which the effect is better when the FGF2 concentration in the MDM III culture medium is 100ng/mL, and the differentiation efficiency is higher.

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

The IGF-1 concentration in the MDM III medium as shown in Table 2 was adjusted to 1 ng/mL, 10 ng/mL and 100 ng/mL, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring 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 IGF-1 to culture in Stage III. The cell morphology after the Stage III culture is shown in Figure 22. The results show that the cell morphology of each group is not much different, indicating that IGF-1 has little effect on the morphology of cells undergoing Stage III differentiation culture within the 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 to the muscle progenitor cell (MPCs) stage, among which the IGF-1 concentration in the MDM III culture medium is better at 100 ng/mL, and the differentiation efficiency is higher.

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

The concentrations of LDN193189 in the MDM III medium as shown in Table 2 were adjusted to 0.1 μM and 0.5 μM, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring to Example 1, after the cells were cultured in Stage I and Stage II, they were further placed in MDM III medium containing different concentrations of LDN193189 to culture in Stage III. The cell morphology after the Stage III culture is shown in Figure 24. The results show that the cell morphology of each group is not much different, indicating that LDN193189 has little effect on the cell morphology undergoing Stage III differentiation culture 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, which show that each group of cells can differentiate to the muscle progenitor cell (MPCs) stage, among which the effect is better when the concentration of LDN193189 in the MDM III medium is 0.1 μM, and the differentiation efficiency is higher.

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

The HGF concentrations in the MDM III medium as shown in Table 2 were adjusted to 1 ng/mL, 10 ng/mL and 100 ng/mL, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring 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 undergo Stage III culture. The cell morphology after Stage III culture is shown in Figure 26. The results show that the cell morphology of each group is not much different, indicating that HGF has little effect on the morphology of cells undergoing Stage III differentiation culture within the concentration range of 1ng/mL to 100ng/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 to the muscle progenitor cell (MPCs) stage, among which the effect is better when the HGF concentration in the MDM III culture medium is 100ng/mL, and the differentiation efficiency is higher.

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

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

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 to undergo 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.

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

The IGF-1 concentrations in the MDM V medium as shown in Table 2 were adjusted to 1 ng/mL, 10 ng/mL and 100 ng/mL, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring to Example 1, after the cells were cultured from Stage I to Stage IV, they were further placed in MDM V culture medium containing different concentrations of IGF-1 to undergo 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.

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

The HGF concentrations in the MDM V medium as shown in Table 2 were adjusted to 1 ng/mL, 10 ng/mL and 100 ng/mL, respectively, and 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 induced differentiation of pgEpiSCs was performed with reference to Example 1.

Referring to Example 1, after the cells were cultured from Stage I to Stage IV, they were further placed in MDM V culture medium containing different concentrations of HGF to undergo 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 has little effect on the morphology of cells undergoing Stage V differentiation culture within the concentration range of 1 ng/mL to 100 ng/mL.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (13)

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. The method of claim 1 further comprises 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. The method of claim 1 or 2, wherein the first stage differentiation medium is a basal 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 non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out Serum Replacement) and ascorbic acid; (ii) the first stage differentiation medium does not contain serum; (iii) In the differentiation medium of the first stage, 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). The method of any one of claims 1 to 3, wherein the second stage differentiation medium is a basal 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 non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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). The method according to any one of claims 1 to 4, wherein the third stage differentiation medium is a basal 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 non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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). The method of any one of claims 1 to 5, wherein the fourth stage differentiation medium is a basal 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 non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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). The method according to any one of claims 1 to 6, wherein the fifth stage differentiation medium is a basal 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 non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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). The method of any one of claims 2 to 7, wherein the sixth stage differentiation medium is a basal 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 non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, KOSR (Knock Out 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%. The method of any one of claims 1 to 8, which has one or more selected from 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. 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. The method of claim 10, having 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. The method of claim 10 or 11, wherein the scaffold is a three-dimensional edible scaffold. Cell cultured meat prepared by the method according to any one of claims 10 to 12.