Method for preparing multi-tissue cell culture meat by multi-directional differentiation of porcine pluripotent stem cells

Abstract

The application discloses a method for preparing multi-tissue cell culture meat by multi-directional differentiation of pig pluripotent stem cells, and belongs to the technical field of biotechnology. The method comprises the following steps: co-culturing fat progenitor cells derived from pig gastrulated epiblast stem cells, muscle progenitor cells derived from pig gastrulated epiblast stem cells and vascular endothelial progenitor cells derived from pig gastrulated epiblast stem cells in a suspension mode, so that the three kinds of cells are differentiated, and multi-tissue cell culture meat is obtained. The multi-tissue culture meat obtained by the method reproduces the texture characteristics of traditionally produced pork and can realize nutritional regulation. The method combines scalable three-dimensional suspension culture technology with serum-free, species-specific stem cell multi-directional differentiation technology, overcomes the technical difficulties faced by the current cell culture meat technology field, and provides a new approach for the research and development of multi-tissue cell culture meat.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing multi-tissue cell cultured meat through the multi-directional differentiation of porcine pluripotent stem cells. Background Technology

[0002] Cultured meat (CM) is a food product made by culturing animal cells in vitro to replicate the sensory and nutritional qualities of traditional meat. It holds immense potential in areas such as sustainability, animal welfare, public health, and related fields. Currently, research and development of CM still face many challenges, including the development of serum-free differentiation systems, multi-tissue culture, and large-scale cell expansion. Furthermore, the sensory characteristics and nutritional value of cultured meat are increasingly attracting the attention of researchers and consumers.

[0003] Adult stem cells (ASCs), such as muscle stem cells (MuSCs), adipose-derived stem cells (ADSCs), fibroadipose-derived progenitor cells (FAPs), and fibroblasts, are the more commonly used cell sources for CM generation. However, ASCs invariably lose their proliferative and differentiation capabilities during long-term culture, requiring continuous extraction from animal sources, and immortalized cell lines may pose potential safety risks. Pluripotent stem cells (PSCs), with their potential for multi-lineage differentiation, enable them to generate all types of cells that make up meat. Furthermore, due to their inherently safe and controllable self-renewal capacity, PSCs provide a virtually unlimited cell source, potentially enabling the creation of cell banks and ultimately eliminating the need for animal tissue biopsies as a material source. These characteristics make them ideal candidates for chassis seed cells in CM production.

[0004] Natural meat contains a wide variety of cells and tissues, including muscle, fat, blood vessels, nerves, fibrous tissue, and potential immune cells. To effectively mimic natural meat, it is necessary to develop various tissues and appropriate nutritional profiles. Three-dimensional (3D) scaffolds and bioprinting have been investigated to replicate meat structures by assembling various cell types. However, isolating cells from animals to establish cultures and implementing scaffolds involves costs and complex procedures, hindering large-scale expansion. Furthermore, microcarrier technology has been used in the development of CMs, but the maximum achievable cell density and cost remain limited.

[0005] Therefore, there is an urgent need to develop a method for large-scale culture of multi-tissue cell-based meat that can differentiate over a long period of time. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for preparing multi-tissue cultured meat through the multi-lineage differentiation of porcine pluripotent stem cells. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] This invention claims protection for a method for preparing multi-tissue cultured meat through multi-lineage differentiation of porcine pluripotent stem cells, comprising co-culturing three types of cells—adipocyte progenitor cells derived from porcine gastrulation epiblast stem cells, muscle progenitor cells derived from porcine gastrulation epiblast stem cells, and vascular endothelial progenitor cells derived from porcine gastrulation epiblast stem cells—in suspension to allow the three types of cells to differentiate, thereby obtaining multi-tissue cultured meat; wherein the adipocyte progenitor cells are prepared according to M1, the muscle progenitor cells according to M2, and the vascular endothelial progenitor cells according to M3; M1 is a method for culturing 2D-adipocyte progenitor cells, the method comprising: inducing porcine gastrulation epiblast stem cells to differentiate into adipocyte progenitor cells, thereby obtaining the adipocyte progenitor cells; M2 is a method for culturing 2D-muscle progenitor cells, the method comprising: inducing porcine gastrulation epiblast stem cells to differentiate into muscle progenitor cells, thereby obtaining the muscle progenitor cells; The M3 is a method for culturing 2D-vascular endothelial progenitor cells, the method comprising: inducing porcine gastrulation epiblast stem cells to differentiate into vascular endothelial progenitor cells, thereby obtaining the vascular endothelial progenitor cells.

[0009] The porcine gastrulation epiblast stem cells were pluripotent stem cell lines isolated and cultured from the epiblast before the formation of porcine E10 gastrulation, according to the method described in Part III of the Material Methods section of the non-patent literature “Zhi, ML et al. Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res. 32, 383-400 (2022).”.

[0010] The adipocytes are undifferentiated mesenchymal cells originating from the mesoderm and possessing adipogenic differentiation potential; they are the "seed cells" for the regeneration and expansion of adipose tissue.

[0011] The adipocyte progenitor cells derived from porcine gastrulation epiblast stem cells are pgEpiSCs-derived adipocyte progenitor cells (pgAPCs). They express surface markers such as PDGFRA and CD29, but do not express hematopoietic or endothelial markers (such as CD45 and CD31). Under appropriate stimulation, they can differentiate into mature adipocytes (PPARγ). + FABP4 + ADIPOQ + ).

[0012] The muscle progenitor cells mentioned are progenitor cells with the potential to develop into muscle cells, specifically Pax7 cells residing in skeletal muscle. + Precursor cells, which can be activated, proliferate and differentiate into myotubes after injury, are the core driving force for muscle regeneration and repair.

[0013] The muscle progenitor cells derived from porcine gastrulation epiblast stem cells are pgEpiSCs-derived muscle progenitor cells (pgMPCs). They express MYOD and Pax7 genes and are capable of proliferating and differentiating into muscle cells (MYOG). + MYMK + ).

[0014] The vascular endothelial progenitor cells are progenitor cells residing in the bone marrow or tissues and possessing the potential to differentiate into mature vascular endothelial cells.

[0015] The vascular endothelial progenitor cells derived from porcine gastrulation epiblast stem cells are pgEpiSCs-derived vascular endothelial progenitor cells (pgVPCs). They are a heterogeneous group of cells capable of proliferating and differentiating into vascular cell types (endothelial cells and pericytes). All three types of cells are isolated cells.

[0016] Multi-tissue cultured meat refers to cultured meat prepared by culturing adipocyte progenitor cells, muscle progenitor cells, and endothelial progenitor cells derived from porcine gastrulation epiblast stem cells in a 3D suspension mixture, resulting in aggregated spheres containing multiple tissues including muscle, fat, and endothelium.

[0017] In the above method, the cell ratio of muscle progenitor cells (hereinafter referred to as pgMPCs), adipocyte progenitor cells (hereinafter referred to as pgAPCs) and vascular endothelial progenitor cells (hereinafter referred to as pgVCs) is (2-0.5):(2-0.5):1.

[0018] In one specific embodiment of the present invention, the ratio of the number of adipocytes, muscle progenitors and vascular endothelial progenitors is 1:1:1.

[0019] In one specific embodiment of the present invention, the ratio of the number of adipocytes, muscle progenitors and vascular endothelial progenitors is 2:2:1.

[0020] In one specific embodiment of the present invention, the ratio of the number of adipocytes, muscle progenitors and vascular endothelial progenitors is 1:1:2.

[0021] In the above method, the co-culture includes the following steps: A1) Use MADVM I medium for co-culture from day 1 to day 5; A2) Use MADVM II medium during co-culture from day 6 to day 14; A3) Use MADVM III medium during co-culture from day 15 to day 30; The MADVM I medium is a liquid culture medium containing composition I; the MADVM II medium is a liquid culture medium containing composition II; the MADVM III medium is a liquid culture medium containing composition III. Composition I consists of IGF-1, FGF2, hEGF and VEGF; composition II consists of IGF-1, HGF, IBMX, Dex, insulin and VEGF; composition III consists of IGF-1, HGF, insulin and VEGF.

[0022] In the above method, the MADVM I culture medium contains 10 ng / ml IGF-1, 10 ng / ml FGF2, 10 ng / ml hEGF, and 50 ng / ml VEGF. The MADVM II medium contained IGF-1 at 10 ng / ml, HGF at 10 ng / ml, IBMX at 500 μM, Dex at 1 μM, insulin at 10 ng / ml, and VEGF at 50 ng / mL. The MADVM III culture medium contained 10 ng / ml IGF-1, 10 ng / ml HGF, 10 ng / ml insulin, and 25 ng / ml vascular endothelial growth factor (VEGF).

[0023] The MADVM I, MADVM II, and MADM III media, excluding the aforementioned compositions I, II, and III, contain the remaining portion of MADM basal medium (MADM BM). The composition of the MADM basal medium is as follows: DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 15% knockout serum substitute, and 200 μM ascorbic acid.

[0024] In the above method, the suspension method is provided by a non-adsorbent cell culture dish and a rotating device, and the parameters of the rotating device during the co-culture process are 70 rpm / min.

[0025] In one specific embodiment of the present invention, the rotating device is a horizontal shaking table of model TS-100 from Zhicheng Company.

[0026] In the above method, the induction of porcine gastrulation epiblast stem cells into adipocyte progenitor cells in M1 includes the following steps: culturing porcine gastrulation epiblast stem cells in ADM I medium, then in ADM II medium, and then in ADM III medium to obtain the adipocyte progenitor cells.

[0027] In a specific embodiment of the present invention, the induction of porcine gastrulation epiblast stem cells into adipocyte progenitor cells in M1 includes the following steps: culturing porcine gastrulation epiblast stem cells in ADM I medium for 3 days, then in ADM II medium for 9 days, and then in ADM III medium for 6 days to obtain the adipocyte progenitor cells; wherein ADM I medium is a liquid culture medium containing composition one, wherein composition one includes B27, CHIR99021 and SB431542; wherein ADM II medium is a liquid culture medium containing composition two, wherein composition two contains CHIR99021, LDN193189 and FGF2; and wherein ADM III medium is a culture medium containing composition three, wherein composition three contains FGF2 and hEGF.

[0028] Except for composition one, the remaining amount of the ADM I medium is ADM basal medium (ADM BM).

[0029] Except for composition one, the remaining amount of the ADM II medium is ADM basal medium (ADM BM).

[0030] Except for composition one, the remaining amount of the ADM III medium is ADM basal medium (ADM BM).

[0031] The ADM basal medium (ADM BM) consists of the following: DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 15% knockout serum substitute and 200 μM ascorbic acid.

[0032] In the above method, the induction of porcine gastrulation epiblast stem cells into vascular endothelial progenitor cells in M3 includes the following steps: culturing porcine gastrulation epiblast stem cells in VDM I medium, and then replacing it with VDM II medium to obtain vascular endothelial progenitor cells.

[0033] In a specific embodiment of the present invention, the induction of porcine gastrulation epiblast stem cells into vascular endothelial progenitor cells in M3 includes the following steps: culturing porcine gastrulation epiblast stem cells in VDM I medium for 1 day, and then culturing them in VDM II medium for 5 days to obtain vascular endothelial progenitor cells; The VDM I medium is a liquid culture medium containing composition A, which includes B27, CHIR99021, Activin A, BMP4 and VEGF; the VDM II medium is a liquid culture medium containing composition B, which includes SB431542 and VEGF.

[0034] Except for composition A, the remaining portion of the VDM I medium is VDM basal medium (VDM BM).

[0035] Except for composition B, the remaining portion of the VDM II medium is VDM basal medium (VDM BM).

[0036] The composition of the VDM basal medium (VDM BM) is as follows: DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 15% knockout serum substitute and 200 μM ascorbic acid.

[0037] The method of inducing porcine gastrulation epiblast stem cells to differentiate into muscle progenitor cells includes the following steps: culturing porcine gastrulation epiblast stem cells in MDM I medium for 3 days, then changing to MDM II medium for 3 days, and then culturing in MDM III medium for 2 days to obtain vascular endothelial progenitor cells; The MDM I medium is a liquid culture medium containing composition A, which includes B27, CHIR99021, and SB431542; the MDM II medium is a liquid culture medium containing composition B, which includes CHIR99021, LDN193189, and FGF2; and the MDM III medium is a liquid culture medium containing composition C, which includes VEGF and FGF2.

[0038] Except for composition A, the remaining amount of the MDM I medium is MDM basal medium (VDM BM).

[0039] Except for composition B, the remaining portion of the MDM II medium is MDM basal medium (VDM BM).

[0040] Except for composition C, the remaining portion of the MDM III medium is MDM basal medium (VDM BM).

[0041] The composition of the MDM basal medium (VDM BM) is as follows: DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 15% knockout serum substitute and 200 μM ascorbic acid.

[0042] In some specific embodiments of the present invention, the B27 is a Thermo Fisher Scientific product with part number Cat# 12587-010 and name B-27 TM Supplement (50×).

[0043] In some specific embodiments of the present invention, CHIR99021 is a product of Selleckchem with the product number Cat#S1263.

[0044] In some specific embodiments of the present invention, SB431542 is a product of Selleckchem with the product number Cat#S1067.

[0045] In some specific embodiments of the present invention, the LDN193189 is a product of Stemgent Corporation with part number Cat#04-0074.

[0046] In some specific embodiments of the present invention, the FGF2 is a product of Stemgent Corporation with part number Cat# 04-0074.

[0047] In some specific embodiments of the present invention, the hEGF is a PeproTech product with catalog number Cat# 100-39H and name Recombinant Human HGF.

[0048] In some specific embodiments of the present invention, the non-essential amino acid is a Thermo Fisher Scientific product with catalog number Cat# 1140-050 and name MEM Non-Essential Amino Acids Solution (100×).

[0049] In some specific embodiments of the present invention, the β-mercaptoethanol is a product of Thermo Fisher Scientific with catalog number Cat# 21985-023.

[0050] In some specific embodiments of the present invention, the knockout serum substitute is a product of Thermo Fisher Scientific with catalog number Cat# A3181502.

[0051] In some specific embodiments of the present invention, the Activin A is a product of PeproTechg with part number Cat#120-14E and name Human / Murine / Rat Activin A.

[0052] In some specific embodiments of the present invention, the BMP-4 is a PeproTech product with part number Cat# 315-2 and name Recombinant Murine BMP-4.

[0053] In some specific embodiments of the present invention, the VEGF is a product of MCE Corporation with catalog number Cat# HY-P78229 and name VEGF165 Protein Human.

[0054] This invention also claims protection for any of the following methods: B1) Method for culturing 2D-adipocyte progenitor cells: The method described in M1 above; B2) A method for culturing 2D-adipocytes, wherein the first step of the method is the aforementioned M1, and the second step of the method is to culture the adipocyte progenitor cells obtained by M1 in a medium containing composition ADM-IV for 6 days, and then culture them in a medium containing composition ADM-V for 4-7 days to obtain adipocytes derived from porcine gastrulation epiblast stem cells, wherein composition ADM-IV includes insulin-transferrin-selenium, rosiglitazone, isobutylmethylxanthine, dexamethasone and insulin, and composition ADM-V includes insulin; B3) Method for culturing 2D-vascular endothelial progenitor cells: The method is the aforementioned M2; B4) Method for culturing 2D-vascular type cells: The first step of the method is the aforementioned M3, and the second step of the method is to culture the vascular endothelial progenitor cells obtained by the M3 in a medium containing the composition VDM-III for 3 days to obtain vascular type cells derived from porcine gastrulation epiblast stem cells, wherein the composition VDM-III contains VEGF and EGF2. B5) Method for co-culturing muscle cells and adipocytes in 2D: Adipocyte precursor cells prepared by M1 and myoblasts prepared by M2 are mixed to obtain hybrid cells, which are then cultured according to B51) or B52): B51) The hybrid cells are cultured in the medium containing composition ADM-IV for 6 days, followed by culture in the medium containing composition ADM-V for 3 days; B52) The hybrid cells are cultured in a first mixed medium (the medium containing composition ADM-IV and the medium containing composition MDM-V (volume ratio 1:1)) for 6 days, followed by culture in a second mixed medium (the medium containing composition ADM-V and the medium containing composition N2 (volume ratio 1:1)) for 4 days; the composition MDM-V includes HGF and IGF-1, and the composition N2 contains knockout serum substitute and N2; B6) Method for culturing 3D-adipocyte spheroids: Adipocyte progenitor cells prepared by M1 as described above were cultured in a rotating apparatus for 5 days in a medium containing composition ADM III, then cultured in a rotating apparatus for 6 days in ADM IV medium, and finally cultured in a rotating apparatus with ADM V medium until day 30 to obtain adipocyte spheroids derived from porcine gastrulation epiblast stem cells; the composition ADM III contains FGF2 and hEGF; B7) Method for culturing 3D-muscle cell spheroids: Muscle progenitor cells prepared by M2 as described above are cultured in a rotating apparatus for 2 days in a medium containing composition MDM-III, then cultured in a rotating apparatus for 4 days in a medium containing composition MDM-IV, and finally cultured in a rotating apparatus for 30 days in a medium containing composition MDM-V to obtain muscle cell spheroids derived from porcine gastrulation epiblast stem cells; the composition MDM-III includes HGF, IGF-1, FGF2, and LDN193189, the composition MDM-IV includes IGF-1, and the composition MDM-V includes HGF and IGF-1; B8) Method for culturing 3D-vascular cell spheroids: Vascular endothelial progenitor cells prepared by the aforementioned M3 are cultured in a rotating device for 2 days in a medium containing composition VDM-II, and then cultured in the medium containing composition VDM-III until day 30 to obtain vascular cell spheroids derived from porcine gastrulation epiblast stem cells; the composition VDM-II contains SB431542 and VEGF; B9) Method for culturing 3D-muscle / adipocyte spheroids: Adipocyte progenitor cells prepared by M1 and muscle progenitor cells prepared by M2 are mixed to obtain hybrid cells. The hybrid cells are cultured in a rotating apparatus for 5 days in a medium containing composition MADM-I, then in a rotating apparatus for 9 days in a medium containing composition MADM-II, and finally in a rotating apparatus for 30 days in a medium containing composition MADM-III to obtain muscle-adipocyte aggregate spheroids. Composition MADM-I contains IGF-1, FGF2, and hEGF; composition MADM-II contains IGF-1, hEGF, isobutylmethylxanthine, dexamethasone, and insulin.

[0055] In some specific embodiments of the present invention, the ratio of the number of adipose precursor cells to myoblasts in B5) is 1:1.

[0056] The culture medium containing composition ADM-III contains, apart from composition ADM-III, the remainder being the aforementioned ADM basal culture medium.

[0057] The culture medium containing composition ADM-IV contains, except for composition ADM-IV, the remaining amount of which is the aforementioned ADM basal culture medium.

[0058] The culture medium containing composition ADM-V contains, apart from composition ADM-V, the remaining amount of which is the aforementioned ADM basal culture medium.

[0059] The medium containing composition VDM-II contains VDM-II, with the remainder being the aforementioned VDM basal medium.

[0060] The medium containing composition VDM-III contains VDM-III, with the remainder being the aforementioned VDM basal medium.

[0061] The culture medium containing composition MDM-IV contains, apart from composition MDM-IV, the remainder being the aforementioned MDM basal culture medium.

[0062] The medium containing composition MDM-V contains, apart from composition MDM-V, the remainder being the aforementioned MDM basal medium.

[0063] The N2 medium containing the composition N2 consists of N2 medium in addition to the composition N2. The N2 medium is composed of the following: DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 15% knockout serum substitute and 200 μM ascorbic acid.

[0064] The culture medium containing the MADM-I composition, excluding the MADM-I composition, contains MADM basal culture medium as the remainder.

[0065] The remaining portion of the MADM-II-containing culture medium is MADM basal culture medium, excluding the MADM-II composition.

[0066] The medium containing composition MADM-III contains MADM basal medium, with the remainder being MADM basal medium, excluding composition MADM-III.

[0067] The MADM basal medium composition is as follows: DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 15% knockout serum substitute and 200 μM ascorbic acid.

[0068] The present invention also claims protection for compositions that are any of the following: M1) A composition for preparing multi-tissue cell cultured meat, said composition being any of the following: M1-1) includes the aforementioned composition I, composition II and composition III; M1-2) includes the preparation of the adipocytes, muscle progenitors, and vascular endothelial progenitors by the aforementioned methods; M1-3) is composed of M1-1) and M1-2); M2) A composition for culturing 2D-adipocyte progenitor cells, said composition comprising the aforementioned composition one, composition two and composition three; M3) A composition for culturing 2D-adipocytes, said composition comprising any of the following: M3-1) includes the aforementioned Composition 1, Composition 2 and Composition 3; M3-2) includes adipocyte progenitor cells prepared from the aforementioned M1; M3-3) is composed of M3-1) and M3-2); M4) A composition for culturing 2D-vascular endothelial progenitor cells, said composition being the aforementioned composition A and composition B; M5) A composition for culturing 2D-vascular type cells, said composition being any of the following: M5-1) includes the aforementioned Composition 1, Composition 2 and Composition 3; M5-2) includes vascular endothelial progenitor cells prepared from the aforementioned M3; M5-3) is composed of M5-1) and M5-2); M6) A composition for 2D co-culture of muscle and fat, said composition being any of the following: M6-1 consists of adipocyte progenitor cells prepared by M1, muscle progenitor cells prepared by M2, ADM IV medium, and ADM V medium. M6-2) consists of adipose progenitor cells prepared by M1, muscle progenitor cells prepared by M2, a first mixed culture medium and a second mixed culture medium. The first mixed culture medium is a mixture of ADM IV culture medium and MDM V culture medium in equal proportions, and the second mixed culture medium is a mixture of ADM V culture medium and N2 culture medium in equal proportions. M7) is a composition for culturing 3D-APC spheres, consisting of adipose progenitor cells prepared by M1 above, ADM III medium, ADM IV medium, and ADM V medium; M8) is a composition for culturing 3D-MPCs spheres, consisting of muscle progenitor cells prepared by M2 as described above, MDM III medium, MDM IV medium, and MDM V medium; M9) is a composition for culturing 3D-VPCs spheres, consisting of vascular endothelial progenitor cells prepared by M3 as described above, VDM II medium and VDM III medium; M10) is a composition for culturing 3D muscle / adipocyte spheroids, consisting of adipocyte progenitor cells prepared by M1, muscle progenitor cells prepared by M2, MADM I medium, and MADM II medium.

[0069] This invention also claims protection for a product, which is any one of P1)-P9): P1) Multi-tissue cultured meat prepared according to the aforementioned method for preparing multi-tissue cultured meat through multi-directional differentiation of porcine pluripotent stem cells; P2) The adipocyte progenitor cells prepared according to M1 above; P3) The adipocytes prepared according to the aforementioned method for culturing 2D adipocytes; P4) The vascular endothelial progenitor cells prepared according to M2 above; P5) The vascular type cells prepared according to the aforementioned method for culturing 2D-vascular type cells; P6) The adipocyte spheroids obtained by the aforementioned method for culturing 3D-APCs spheroids; P7) The muscle cell spheroids obtained by the aforementioned method for culturing 3D-MPCs spheroids; P8) The vascular cell spheroids obtained by the aforementioned method for culturing 3D-VPCs spheroids; P9) The muscle-fat aggregate spheres obtained by the aforementioned method for culturing 3D-muscle / fat cell spheres.

[0070] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 9:1.

[0071] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 8:2.

[0072] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 7:3.

[0073] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 5:5.

[0074] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 3:7.

[0075] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 2:8.

[0076] In one specific embodiment of the present invention, the ratio of muscle progenitor cells (hereinafter referred to as pgMPCs) to adipocyte progenitor cells (hereinafter referred to as pgAPCs) in the above-described method, product, or composition is 1:9.

[0077] This invention successfully induced porcine gastrulated epiblast stem cells (pgEpiSCs) to differentiate into muscle, fat, and vascular endothelial cells through directed lineage-specific differentiation. A scalable 3D suspension culture platform was also established, facilitating cell recognition and co-culture via scaffold-free self-assembly. The multi-tissue meat produced by this invention is similar to real meat in texture and nutritional value. Furthermore, the multi-tissue cultured meat exhibits advantages in elasticity and tenderness and contains relatively high levels of polyunsaturated fatty acids (PUFAs). This invention achieves multi-lineage differentiation of pluripotent stem cells and enables scalable 3D suspension culture for CM production through self-assembly. This advancement opens the possibility of creating nutritionally customized and ecologically sustainable meat products. Attached Figure Description

[0078] Figure 1 This is a schematic diagram of the protocol for differentiating pgEpiSCs into adipocytes in a serum-free culture system. a, Schematic diagram of pgEpiSCs differentiating into adipocytes. b, Immunostaining of NANOG and T proteins under WNT activation and TGF-β inhibition conditions. Scale bar, 20 μm. c, Gene expression associated with APC differentiation from day 0 to day 18. d, Immunostaining of PDGFRA, CD29, and CD45 in pgAPCs. Scale bar, 50 μm. e, Genes related to MSC characteristics expressed by pgAPCs after 12 days. f, Cell morphology and Oil Red O staining results of pgAPCs before and after adipogenic differentiation. Scale bar, 50 μm. g, Compared with early ( PPARG , CEBPA ) and late ( FABP4 , LPL , ADIPOQ , PLIN1 , LEPGene expression related to adipogenesis differentiation. h, Fluorescent staining images of FABP4 and BODIPY in pgADs. Scale bar, 50 μm. i, Heatmaps of each cell population during in vitro adipogenesis in pgEpiSCs, showing similar expression patterns of specific gene clusters. j, Gene ontology (GO) enrichment terms of representative clusters with high q values ​​in different cell populations during adipogenesis differentiation in pgEpiSCs. Note: For (c, e, g), error bars represent ± standard deviation, n = 3. p<0.05, p<0.01, p<0.001, p < 0.0001, ns means p ≥ 0.05. Similar results were obtained for (bh) in three independent experiments.

[0079] Figure 2 Schematic diagram of a protocol for inducing vascular endothelial cell differentiation from pgEpiSCs in a serum-free culture system. a, Schematic diagram of pgEpiSCs directed induction into vascular cell types. b, Observation of cell morphology during differentiation induction. Scale bar, 50 μm. c, d, with pluripotency ( OCT4, SOX2, NANOG ), ectoderm ( SOX1 ), mesoderm ( T Gene, Brachyury ) and endoderm ( GATA6e) Gene expression related to vascular endothelial cells (ECs, PECAM1, CDH5) and pericytes (PDGFRB). f, g) Immunostaining results of pgECs (CD31 and eNOS) and pgPericytes (αSMA). Scale bar, 50 μm. pgECs: vascular endothelial cells derived from pgEpiSC; pgPericytes: pericytes derived from pgEpiSC. h) Tubular structures formed by pgVCs. Scale bar, 50 μm. i) Assessment of the uptake capacity of pgVCs for LDL. Scale bar, 50 μm. j) Representative images of cell staining for survival (Calcein-AM, green) and death (PI, red) of pgVCs. Scale bar, 200 μm. k) Flow cytometry detection of pgVC survival. l, Karyotype analysis of pgEpiSCs after differentiation into angiocytes, maintaining a normal chromosome number (n=38). m, Volcano plot of differential gene expression between pgEpiSCs and pgVCs. n, Gene Ontology (GO) terminology for representative clusters with high q values ​​in different cell populations during pgEpiSCs differentiation into angiocytes. Note: For (ce), error bars represent mean ± standard deviation, n=3. p<0.05, p<0.01, p < 0.0001, ns indicates p > 0.05. Similar results were obtained for (bl) in three independent experiments.

[0080] Figure 3 Schematic diagrams of co-culturing pgEpiSCs-derived myogenic and adipogenic cells in 2D and 3D systems. a, Schematic diagram of the directed induction process of co-culturing myoblasts and preadipocytes derived from pgEpiSCs. Cells were first proliferated for 2 days, then cultured in differentiation medium for 6 days, and continued to be cultured in maintenance medium for 4 days. b, c, Bright field and Oil Red O staining results of co-cultured cells. Scale bar, 50 μm. d, e, Immunostaining results of myotubes and lipid droplets (LDs) in co-cultured cells. Images were taken using confocal microscopy after immunofluorescence staining of myosin or actin in mature smooth muscle fibers (SMFs), BODIPY in lipid droplets (LDs), and DAPI in cell nuclei. Scale bar, left side 50 μm, right side 20 μm. f, g, 2D co-cultured cells with myogenic differentiated ( MYOG, MYH2, MYH3 ) and adipogenesis differentiation ( ADIPOQ, PLIN1, LEPExpression of related maturation marker genes. M1, medium 1; M2, medium 2. h, Schematic diagram of pgEpiSCs differentiated into pgEpiSC-derived myoblasts and preadipocytes, seeded on plant-based edible 3D scaffolds. i, Cells stained with Calcein-AM (green) and PI (red) after 24 hours of scaffold culture. Scale bar, 100 μm. j, Flow cytometry results of co-cultured cell viability after 24 hours of culture on 3D scaffolds. k, Scanning electron microscopy (SEM) observation of empty scaffolds and scaffolds seeded with co-cultured cells for 12 days. Scale bar, left side 200 μm, right side 20 μm. l, Immunostaining results of co-cultured cells and myoblasts and lipid droplets in the scaffold. Scale bar, 50 μm. m, Oil Red O staining results of co-cultured cells and lipid droplets (LDs) in the scaffold. Scale bar, 50 μm. n, Co-cultured cells and lipid droplets (LDs) in the scaffold, related to myogenic differentiation (… MYOG, MYMK ) and adipogenesis differentiation ( ADIPOQ, LEP The expression of related maturation marker genes. Note: For (f, g, n), the error bar represents the mean ± standard deviation, n=3. p<0.05, p<0.01, p<0.001, p < 0.0001. Similar results were obtained in three independent experiments for (bh) and (in).

[0081] Figure 4Schematic diagram illustrating the long-term expansion of pgEpiSC-derived progenitor cells in three-dimensional suspension culture. a, Schematic diagram of pgMPCs and pgAPCs forming spheres and undergoing 3D suspension differentiation via a non-adsorption suspension oscillation system. b, Bright-field microscopy images of spheres formed by pgMPCs from day 1 to day 30. c, Bright-field images of spheres formed by pgAPCs during 3D adipogenesis differentiation from day 0 to day 15. d, e, Statistical diameter of spheres differentiated by three-dimensional suspension of pgMPCs and pgAPCs. f, Myospheric spheres showing smooth surface features via scanning electron microscopy (SEM). g, SEM showing rough surface and relatively loose cell arrangement of fat spheres. h, Representative image of myospheric spheres stained with hematoxylin and eosin (H&E). i, Fluorescent image of cytoskeletal protein (F-actin) and mature myofibrils (myosin) in myospheric spheres on day 30. j, Representative image of fat spheres stained with Oil Red O. k, Fluorescence images of lipid droplets (BODIPY) and adipocytes (FABP4) in fat globules on day 15. l, Volcano plot of differential gene expression in pgEpiSCs between 2D and 3D muscle differentiation. m, Gene Ontology (GO) terminology of representative clusters with high q values ​​in pgMCs during 2D and 3D differentiation. n, Volcano plot of differential gene expression in pgEpiSCs between 2D and 3D adipocyte differentiation. o, Gene Ontology (GO) terminology of representative clusters with high q values ​​in pgADs during 2D and 3D differentiation. Note: Scale bar is 50 μm for (b, c). Scale bar is 100 μm for (f, g, h, j). Scale bar is 50 μm for (i, k), top plot is 50 μm, bottom plot is 20 μm. For (d, e), n=50, violin plot is truncated at minimum and maximum values, dashed lines are quartiles, dotted lines are medians, and dots represent individual data points. For (bk), similar results were obtained in three independent experiments.

[0082] Figure 5Schematic diagram of cell-cultured meat generated from multi-tissue aggregates, showing similarity in nutrition and texture to that from pork. a, Schematic diagram of 3D aggregate models of pgMPCs, pgAPCs, and pgVPCs. b, Bright-field microscopy images of aggregates formed from different cell sources from day 1 to day 30. Scale bar, 100 μm. c, Changes in diameter of 3D suspension differentiated spheres. d, Changes in cell density within 3D suspension differentiated spheres. ef, Representative images of multi-tissue spheres observed by (e) hematoxylin-eosin (HE) staining and (f) Oil RedO staining. Scale bar, 100 μm. g, Immunofluorescence staining image of aggregates on day 25; MF20, BODIPY, and CD31 are markers for myofibrils, adipocytes, and vascular endothelial cells (ECs), respectively. Scale bar, top image 100 μm, bottom image 50 μm. h. The heatmap shows the expression of T3_3D-specific genes and genes shared with the pgMCs_3D or pgADs_3D groups. T3_3D represents spheres formed from three different cell types. i. Overview of the essential amino acid (EAA) and non-essential amino acid (NEAA) composition in spheres, pgEpiSCs, and conventional pork. j. Overview of the saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) composition in spheres, pgEpiSCs, and conventional pork. k. Appearance of sausages made from multi-tissue spheres and pork.

[0083] Figure 6 This diagram illustrates the optimization of the culture system and identification of cell characteristics of pgEpiSCs during adipogenesis. a) Cell morphology images at different stages of adipocyte differentiation. b, c) During differentiation into mesoderm from day 1 to day 4, the relationship between pluripotency (…). OCT4, SOX2, NANOG ) and ectoderm ( PAX6 ), mesoderm ( T Gene, Brachyury ), endoderm ( GATA6 d. Alkaline phosphatase (AP) staining under WNT activation and TGF-β inhibition conditions. e. Tracking the expression of mesenchymal stem cell (MSC) related genes ITGB1, THY1, and PTPRC by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). f. Tracking the expression of adipogenesis-related genes in the early stages of adipogenesis induction by real-time quantitative PCR (qRT-PCR). PPARG, CEBPA ) or late stage ( LPL, FABP4Expression changes during the process. g, Adipogenic differentiation observed using the pgEpiSCs-NLS-GFP cell line. Nile red staining reveals GFP cells, where GFP represents the nucleus and can be observed in single cells. h, Karyotype analysis of pgEpiSCs after adipogenic differentiation, maintaining a normal chromosome number (n=38). Note: For (a, d, g), the scale bar is 50 μm. For (b, c, e, f), the error bars represent the mean ± standard deviation, n=3. p<0.05, p<0.01, p<0.001, p < 0.0001, ns means p ≥ 0.05. Similar results were obtained in three independent experiments for (ag).

[0084] Figure 7 Schematic diagram for identifying the differentiation potential of porcine fibroblast / adipocyte precursor cells (pFAPs). a, Schematic diagram of pFAP isolation. b, Cell morphology and Oil Red O staining results of pFAPs during proliferation and differentiation at different passage numbers. Scale bar, 50 μm. c, BODIPY-stained lipid droplets (LDs) and immunostaining results of FABP4 after adipocyte differentiation (P3 and P13). Green represents BODIPY, red represents FABP4, and blue represents DAPI. Scale bar, 50 μm. d, Detection of adipocyte differentiation-related cells in P3 and P13 by real-time quantitative PCR (qRT-PCR). PPARG, CEBPA, FABP4, ADIPOQ, PLIN1 and LEP Gene expression status. Error bars represent mean ± standard deviation, n=3. p<0.01, p<0.001, p < 0.0001. e, Principal component analysis (PCA) plot of adipogenesis in pgEpiSCs and ADs derived from p3-pFAPs. Different colors represent different cell populations. f, Heatmap of differentially expressed genes (DEGs) between pFAPs (isolated in vivo) and pgAPCs and ADs. Note: For (bd), similar results were obtained in three independent experiments.

[0085] Figure 8 Schematic diagram showing the co-culture of myoblasts and preadipocytes differentiated from pgEpiSCs at different ratios. a) The content of lipid droplets and myofibrils in co-cultured cells with different myoblast to preadipocyte ratios was assessed by BODIPY and Myosin fluorescence staining. Scale bar, 50 μm. b, c) Muscle-related genes were analyzed by qRT-PCR. MYOG, MYMK, MYH3, MYH11 ) and adipocyte-related genes ( ADIPOQ, PLIN1, LEP, FABP4 The relative mRNA expression levels of ). Error bars represent mean ± standard deviation, n=3. p<0.05, p<0.01, p < 0.001, ns (p > 0.05). Similar results were obtained for (ac) in three independent experiments.

[0086] Figure 9 Schematic diagram illustrating the characteristics of aggregates of monocellular progenitor cells. a, Immunofluorescence staining of MYOD in pgMPCs-derived spheroid sections, day 3 of culture. b, Immunofluorescence staining of PDGFRA in pgAPCs-derived spheroids, day 3 of culture. c, Expression analysis of PAX7 and MYOD in pgMPCs spheroids, day 3 of culture. d, Expression analysis of ITGB1 and PDGFRA in pgAPCs spheroids, day 3 of culture. e, f, After 3D suspension culture, muscle spheroids (day 30) or fat spheroids (day 15) stained with Calcein-AM (green) and PI (red). Scale bar, 100 μm. g, h, Representative fluorescent images of collagen (COL3A1) formation in muscle or fat spheroids. Scale bar, 100 μm. i, Quantitative analysis of myogenic factors in 2D myogenic differentiation and 3D suspension myogenic differentiation by qPCR. MYOG, MYH3 ) and extracellular matrix (ECM) formation ( COL3A1 The expression levels of related mRNAs were compared using qPCR. Specifically, the expression levels of adipogenesis (mRNA) in 2D adipogenesis differentiation and 3D suspension adipogenesis differentiation were compared with those in adipogenesis. PPARG, LPL ) and ECM formation ( COL3A1Related gene expression. k, Bright-field image of pgEpiSCs-derived vascular cell spheroids during 30 days of culture. l, Statistical analysis of the diameter of pgEpiSCs-derived vascular cell spheroids during 30 days of culture. m, Representative scanning electron microscopy (SEM) image of pgEpiSCs-derived vascular cell spheroids. n, Staining of pgEpiSCs-derived vascular cell spheroids with Calcein-AM (green) and PI (red) after 30 days of culture. o, Analysis of pgEpiSCs-derived vascular cell spheroids by qPCR. PECAM1, CDH5, PDGFRB and COL3A1 Gene expression. Note: For (c, d, o), the scale bar is 50 μm. For (e, h, k, m, n), the scale bar is 100 μm. For (c, d, i, j, n), the error bars represent the mean ± standard deviation, n=3. p<0.1, p<0.01, p<0.001, p < 0.0001. Similar results were obtained for (ap) in three independent experiments.

[0087] Figure 10 A schematic diagram illustrating the characteristics of pgMPCs and pgAPCs self-assembling muscle-fat globules. a, 3D aggregation scheme of pgMPCs and pgAPCs. Initial cell density was 5 × 10⁻⁶. 5a) Cells / mL, cultured for 30 days. b) Bright-field microscopy images of muscle-fat globules from day 1 to day 30. Scale bar: 100 μm. c) Diameter statistics of 3D suspension differentiated muscle-fat globules, n=50, violin plot truncated at minimum and maximum values, dashed lines represent quartiles, dotted lines represent medians, and dots represent individual data points. d) Scanning electron microscopy (SEM) observations of globules formed on day 30. Scale bar: 100 μm. e, f) Hematoxylin-eosin (HE) staining and Oil Red O staining results of muscle-fat globules. Scale bar: left side 50 μm, right side 20 μm. g, h) Representative fluorescence images of myosin, body droplets, and cytoskeleton (F-actin) in globule sections on day 30. Scale bar: top image 50 μm, bottom image 20 μm. i. The heatmap shows the differential expression of genes related to pluripotency, myogenesis, adipogenesis, and extracellular matrix (ECM) formation in myosomal, fat globules, and myo-fat globules. j. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was used to analyze the relationship between myogenesis and differentiation. MYOG, MYH2, MYH3 ), adipogenesis differentiation ( ADIPOQ, PLIN1, LEP ) and collagen formation ( COL3A1, COL5A2 Related mRNA expression levels. Error bars represent mean ± standard deviation (n = 3). p<0.1, p<0.01, p<0.001, p<0.0001.

[0088] Figure 11Schematic diagrams illustrating the formation of muscle-fat globules from mixed cultures of pgMPCs and pgAPCs at different ratios. a. Bright-field images of muscle-fat globules composed of different ratios of pgMPCs and pgAPCs. Scale bar: 200 μm. b. Evaluation of lipid droplet and myosin content in muscle-fat globules with different pgMPC / pgAPC ratios using BODIPY and myosin fluorescence staining. Scale bar: 200 μm. c. Relative area ratios of BODIPY and myosin in muscle-fat globules with different pgMPC / pgAPC ratios. Error bars represent mean ± standard deviation, n = 3. Different letters indicate significant differences of p < 0.05; similar results were obtained in three independent experiments. In the figure, 9:1, 8:2, 7:3, 5:5, 3:7, 2:8, and 1:9 represent muscle fat globules composed of pgMPCs and pgAPCs in cell number ratios of 9:1, 8:2, 7:3, 5:5, 3:7, 2:8, and 1:9, respectively.

[0089] Figure 12 Schematic diagram of the tissue and nutrient composition of multi-tissue spheres. a. Bright-field images of muscle-fat-vascular spheres composed of different proportions of pgMPCs, pgAPCs, and pgVPCs. Scale bar, 200 μm. b. Fluorescent staining with BODIPY, myosin, and CD31 to assess the content of lipid droplets, myofibrils, and endothelial cells in muscle-fat-vascular spheres with different proportions of pgMPCs, pgAPCs, and pgVPCs. Scale bar, 200 μm. c. Relative area ratios of BODIPY, myosin, and CD31 in muscle-fat-vascular spheres with different seeding cell ratios. Error bars represent mean ± standard deviation, n = 3, different letters indicate significant differences when p < 0.05. In the figure, 2:2:1, 1:1:1, and 1:1:2 represent muscle-fat-vascular spheres composed of pgMPCs, pgAPCs, and pgVCs in cell number ratios of 2:2:1, 1:1:1, and 1:1:2, respectively. d. Overview of essential (EAA) and non-essential (NEAA) amino acids in spheres and conventional pork. e. Comparative analysis of amino acid composition in pgEpiSCs, multi-tissue spheres, and conventional pork. f. Overview of saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids in spheres and conventional pork. g. Detailed comparison of fatty acid composition in pgEpiSCs, multi-tissue spheres, and conventional pork. h. Composition of flavor compounds in pgCM and cultured pork sausages. For d-h, error bars represent mean ± standard deviation (n = 3). p<0.1, p<0.001, p < 0.0001, ns means p ≥ 0.05. Detailed Implementation

[0090] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are all commercially available. The quantitative experiments in the following embodiments are all repeated three times, and the results are averaged. The following embodiments use GraphPadPrism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test is used, and P < 0.05 ( () indicates a significant difference. The reagents or primers involved in the following examples are: Chemicals, peptides, and recombinant proteins: CHIR99021, Selleckchem, Cat# S1263; IWR-1-endo, Selleckchem, Cat# S7086; Y-27632, SelleckchemCat# S1049; WH-4-023, Selleckchem, Cat# S7565; Recombinant, Human LIF, PeproTech, Cat# 300-05; Human / Murine / Rat Activin A, PeproTech, Cat# 120-14E; Recombinant Human FGF-2, PeproTech, Cat# 100-18B; Recombinant Human IGF-1, PeproTech, Cat# 100-11; Recombinant Human HGF, PeproTech, Cat# 100-39H; Recombinant Murine BMP-4, PeproTech, Cat# 315-27; SB431,542, Selleckchem, Cat# S1067; LDN193189, Stemgent, Cat# 04-0074; VEGF165 Protein Human, MCE, Cat# HY-P78229; Rosiglitazone, Sigma-Aldrich, Cat# R2408; 3-Isobutyl-1-methylxanthine (IBMX), Sigma-Aldrich, Cat# I5879; Dexamethasone (Dex), Sigma-Aldrich, Cat# D4902; Bovine Insulin, Sigma-Aldrich, Cat# I6634; Bovine Serum Albumin (BSA), Sigma-Aldrich, Cat# A1470; Ascorbic Acid (Vc), Sigma-Aldrich, Cat# A4544; Knockout Serum Replacement, Thermo Fisher Scientific, Cat# A3181502; NeurobasalTM Medium, Thermo Fisher Scientific, Cat# 21103-049; DMEM / F12, GlutaMAXTM supplement, Thermo Fisher Scientific, Cat# 10565-018; N-2 Supplement (100×), Thermo Fisher Scientific, Cat# 17502-048; B-27 TM Supplement (50×), minus vitamin A, Thermo Fisher Scientific, Cat# 12587-010; Insulin–transferrin–selenium(ITS), Thermo Fisher Scientific, Cat#41400-045; GlutaMAXTM Supplement, Thermo Fisher Scientific, Cat# 35050-061; β-Mercaptoethanol, Thermo Fisher Scientific, Cat# 21985-023; MEM Non-Essential Amino Acids Solution(100×), Thermo FisherScientific, Cat# 1140-050; Penicillin-Streptomycin(10000 U / mL), Thermo Fisher Scientific, Cat#15140-122; Gelatin (0.1% in water), Stem Cell Technologies, Cat# 07903; Trypsin-EDTA(0.05%), phenol red, Gibco, Cat# 25300120; DMEM, high glucose, no glutamine, Gibco, Cat# 11960-044; Fetal bovine serum(FBS), Gibco, Cat# 16000-044; Accutase cell dissociation reagent, Gibco,Cat# A11105-01; TrypLETM Express, Gibco, Cat# 12605010; Collagenase II, Coolaber, Cat# CC3791G; Mitomycin C, Selleckchem, Cat# S8146; Red Blood Cell Lysis Buffer, Solarbio, Cat# R1010; Dulbecco's phosphate-buffered saline(DPBS),Gibco,Cat# C14190500CP; Hanks' Balanced Salt Solution(HBSS, with Ca2+&Mg2+),Beyotime,Cat#C0219; Oil Red O Solution,Sigma-Aldrich,Cat# O1391; KaryoMAX Colcemid Solution,Gibco,Cat# 15210-040; Tween 20,Solarbio,Cat# T8220; Triton X-100,Solarbio,Cat# T8200; Matrigel,Corning,Cat# BD354230; Sakura Tissue-Tek® O.C.T. Compound,Sakura,Cat# 4853; Haematoxylin,Sigma-Aldrich,Cat# MHS16; Eosin,Sigma-Aldrich,Cat# HT110116; RNApre pure Cell / Bacteria Kit,TIANGEN,Cat# DP430; Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR(gDNAdigester plus),YEASEN,Cat# 11141ES60; 2 × RealStar Green Power Mixture,GenStar,Cat# A311-05; Alkaline Phosphatase Detection Kit,Millipore,Cat# SCR004; Rapid Giemsa Staining kit,BBI Life Science,Cat# E6073141; Calcein / PI Cell Activity and Cytotoxicity Assay Kit, Beyotime, Cat#C2015M; TIANSeq mRNA capture kit, TIANGEN, Cat# NR105.

[0091] Antibodies: Rabbit polyclonal anti - human Nanog, PeproTech, 500 - P236, 1:500; Goat polyclonal anti - Brachyury, Santa Cruz Biotechnology, sc17743, 1:50; Rabbit polyclonal anti - MYOD1, Proteintech, 18943 - 1 - AP, 1:200; Mouse monoclonal anti - Skeletal Myosin (fast), Sigma - Aldrich, M4276,1:300; Mouse monoclonal anti - Myosin heavy chain (MyHc), DSHB, MF20 - S, 1:200; Mouse PDGFR alpha, R&D Systems, AF1062 - SP, 1:300; Rabbit polyclonal Integrin Bera 1 (CD29), Proteintech, 12594 - 1 - AP, 1:300; Mouse monoclonal CD45 (35 - Z6), Santa Cruz Biotechnology, sc - 1178, 1:300; Rabbit FABP4 Polyclonal, Proteintech, 12802 - 1 - AP, 1:300; Rabbit polyclonal eNOS, MCE, HY - P80656, 1:300; Rabbit polyclonal Anti - CD31, Abcam, ab28364, 1:300; Rabbit polyclonal anti-alpha smooth muscle Actin, Abcam, ab5694, 1:250; Rabbit COL4A4 Polyclonal antibody, Proteintech, 19674-1-AP, 1:300; Human DiI-Acetylated Low Density Lipoprotein (Human DiI-Ac-LDL), YEASEN, 20606ES76, 1:200; Donkey anti-Rabbit IgG (H+L) highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, Invitrogen, A-21207; Donkey anti-Mouse IgG (H+L) highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, Invitrogen, A-21203; Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, Invitrogen, A11058; Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen, A-21202; Donkey anti-Mouse IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, Invitrogen, A32787。

[0092] Primers: OCT4 F:5'-CAAACTGAGGTGCCTGCCCTTC-3', R:5'--ATTGAACTTCACCTTCCCTCCA-3'; SOX2 F:5'-CATCAACGGTACACTGCCTCTC-3',R:5'-ACTCTCCTCCCATTTCCCTCTT-3'; NANOG F:5'-CATCTGCTGAGACCCTCGAC-3',R:5'-GGGCTTGTGGAAGAATCAGG-3'; T F:5'-GCCAGATCATGCTGAACTCCTTA-3',R:5'-ATAAGCCGTCACCGCTATGAAC-3'; PAX6 F:5'-TGTCCAACGGATGTGTGAGT-3',R:5'-TCTGTCTCGGATTTCCCAA-3'; SOX1 F:5'-AGAGGGTACCATTTGCACGG-3',R:5'-AGGGCCAGCCTACAGAACTA-3'; GATA6 F:5'-CACTACTTGTGCAACCGCTG-3',R:5'-TTCTGCGGCTTTATGAGGGG-3'; PAX7 F:5'-GTGCCCTCAGTGAGTTCGATTA-3',R:5'-TTCCCTTTGTCGCCCAAGAT-3'; MYOD F:5'-CGCTTGAGCAAAGTCAACGA-3',R:5'-GCTATAATCCATCATGCCGTCG-3'; MYOG F:5'-CCAGGGGATCATCTGCTCACA-3',R:5'-TGGGCATGGTTTCATCTGGG-3'; MYMK F:5'-CTTCCTCCCCACGGTCAG-3',R:5'-TACTCCAGGATGTCAAGGCG-3'; MYH2 F:5'-GGGCTCAAACTGGTGAAGC-3',R:5'-AGATGCGGATGCCCTCCA-3'; MYH3 F:5'-GCCGACGCTGACAGCGGAAA-3',R:5'-AGATGCGGATGCCCTCCA-3'; MYH11 F:5′-GAGCGCCACATCTCAACTCT-3′,R:5′-CTCCTCGGCCAACAACTGAT-3′ PDGFRA F:5'-ATCGTGGAGAATCTGCTGCCTG-3',R:5'-GATGATGTAGCCGCTGTCTG-3' VIM F:5′-GCCCGTCACCTTCGTGAATA-3′,R:5′-GTCCATCTCTGGTCTCAACCG-3′ DLK1 F:5'-GTCTGTGCAAGCCCAAGTTC-3',R:5'-GTTGTAGCGGAGGTTGGACA-3' ZNF423 F:5'-AAGCTTCTAAGCGAGCAGGC-3′,R:5′-CCTCCCAGCTCGATGGTTTT-3′ ITGB1 F:5′-CCAAATGGGACACGGGTGA-3′,R:5′-AGCTACCTCACTGTGACTGC-3′ THY1 F:5′-GGCATCGCTCTCTTGCTAAC-3′,R:5′-GGACCTTGATGTCGTACTTGC-3′ ENG F:5′-ATGCTGTCGTAGCAACCCAA-3′,R:5′-GCCGGACCTCTTCTGTTCTC-3′ PTPRC F:5′-TGATGATTGCTGCTCAGGGG-3′,R:5′-CTCTTCCCGCATTCCAGTGGT-3′ CEBPA F:5'-GGCCAGCACACACACATTAGA-3',R:5'-CCCCCAAAGAAGAGAACCAAG-3' PPARG F:5′-GAGGGCGATCTTGACAGGAA-3′,R:5′-GCCACCTCTTTGCTCTGCTC-3′ FABP4F: 5'-TGAAAGAAGTGGGAGTGGGC-3', R: 5'-CTGGCCCAATTTGAAGGCAA-3'; ADIPOQ F: 5'-TTGAAGGTCCCCGAGGTTTC-3', R: 5'-CCACACTGAATGCTGAACGG-3'; LEP F: 5'-TACGGTTGAATGCCCGTTGA-3', R: 5'-TCCATTAGTCTCACGGCAGC-3'; PLIN1 F: 5'-CGATCTCCCTCGTGACTTGG-3', R: 5'-ACGTTGTCAGTAACGCCCTT-3'; PDGFRB F: 5'-TTCTCCCAGCTGAGCCAATC-3', R: 5'-CCAAGTGGCTCACAAAACGG-3'; CDH5 F: 5'-AAGAACATCGCCCGTGTCAT-3', R: 5'-CACTGAGCCGATCCAAGGTT-3'; PECAM1 F: 5'-CACCGAGGTCTGGGAACAAA-3', R: 5'-GGGAGCCTTCCGTTCTAGAATATC-3'; COL5A2 F: 5'-AAACTGGGCAGAAGCAAGAC-3', R: 5'-ATTTCTTCACCATATCCTTCATCCT-3'; COL3A1 F: 5'-CTAGCCGAGCTTCCCAGAAC-3', R: 5'-TCCCCAGTGTGTTTAGTGCAA-3'; EF1A F: 5'-AATGCGGTGGGATCGACAAA-3', R: 5'-CACGCTCACGTTCAGCCTTT-3'.

[0093] Table 1

[0094]

[0095]

[0096] The percentages in the above culture medium are volume percentages, and the content of each component is the final content of the component in the culture medium.

[0097] The non-adsorbent cell culture dishes used in the following examples are products of Zhejiang Baimeite Medical Plastics Co., Ltd., item number WD9.

[0098] The horizontal shaking table used in the following examples is a product of Zhicheng Company, model TS-100.

[0099] The experimental methods involved in the following embodiments: Animal handling and ethical statement: All mouse and pig experiments were conducted with the approval of the Laboratory Animal Ethics Committee of China Agricultural University (Approval No.: AW52114202-3-01).

[0100] Animals: ICR mice used for isolating mouse embryonic fibroblasts (MEFs) were purchased from Beijing Spaford Biotechnology Co., Ltd. (Beijing, China). MEFs for the feeder layer of porcine embryonic pluripotent stem cells (pgEpiSCs) were prepared by treatment with mitomycin C (Selleckchem, S8146). One-week-old Nongda Xiang pigs were obtained from the experimental mini-pig farm of China Agricultural University for isolating porcine adipose-derived stem cells (pFAPs).

[0101] BODIPY staining: Cell samples were washed with DPBS (Gibco, C14190500CP) and fixed with 4% formaldehyde for 1 hour at room temperature. Subsequently, cells were incubated with 1 µg / mL BODIPY 493 / 503 (Duofluor, P10051-100) for 1 hour, followed by washing three times with DPBS. Finally, the nuclei were stained with DAPI for 3 minutes, and the cells were immediately observed and photographed under a fluorescence microscope.

[0102] Oil Red O staining: After adipocyte induction, cells were washed twice with DPBS (Gibco, C14190500CP) and fixed with 4% paraformaldehyde for 1 hour at room temperature. They were then washed three times with double-distilled water (ddH2O). Next, 2 mL of 60% isopropanol was added, and the cells were incubated at room temperature for 2 minutes. After isopropanol removal, 1 mL of Oil Red O working solution (Oil Red O staining solution (Sigma-Aldrich, O1391) and ddH2O were added, and the cells were incubated at room temperature for 10 minutes, followed by washing with ddH2O until colorless. Finally, the formation of lipid droplets (LDs) was observed and recorded under an inverted microscope.

[0103] Nile Red staining: Fixed cells were washed three times with DPBS, then stained with Nile Red (Sigma-Aldrich, 19123) in the dark. After staining, the cells were washed three times with DPBS, and the nuclei were stained with DAPI for 3 minutes. Immediately afterwards, the cells were observed under a fluorescence microscope.

[0104] In vitro tube formation assay: 50 µL of Matrigel was added to a 96-well plate and incubated at 37°C for 30–60 minutes to allow the gel to solidify. Subsequently, cells were introduced at a rate of 1 × 10⁻⁶ cells / well. 4 The cells were seeded at a density of cells / well in Matrigel-coated 96-well plates. After incubation at 37°C for 4–6 hours, each well was imaged and observed using an inverted microscope.

[0105] Dil-Ac-LDL uptake assay: Cells were seeded in VDM III medium, and 20 µg / mL of human Di-Acetylated low-density lipoprotein (Human Di-Ac-LDL, YEASEN, 20606ES76) was added. The cells were incubated at 37°C for 4 hours. Subsequently, the cells were washed three times with PBS and observed under a fluorescence microscope.

[0106] Alkaline phosphatase (AP) staining: Differentiated cells were fixed with 4% paraformaldehyde (PFA) for 5 minutes, followed by washing with DPBS (Gibco, Cat# C14190500CP). Staining was performed according to the recommended protocol of the AP staining kit (Millipore, Cat# SCR004), followed by observation.

[0107] Karyotype analysis: During the active proliferation phase, the culture medium was replaced with fresh medium containing 15% (v / v) KaryoMAX cytochalasin solution (Gibco, 15210-040) and cultured for 2 hours. Subsequently, cells were dissociated into single-cell suspensions using TrypLE™ Express (Gibco, Cat No. 12,605,010) and collected by centrifugation at 1000 rpm for 5 minutes. The cells were then resuspended in hypotonic 0.075 M KCl (Sigma, P5405) solution and incubated at 37°C for 30 minutes. The hypotonic cells were then fixed with pre-chilled fixative (methanol:glacial acetic acid = 3:1) and centrifuged at 1000 rpm for 8 minutes; this process was repeated three times. The cell suspension was dropped onto a pre-cooled glass slide and placed in a 37°C oven overnight to ensure complete drying. It was then stained with a rapid Giemsa staining kit (BBI Life Science, E6073141) for 15 minutes, dried, and observed under a microscope.

[0108] Quantitative RT-PCR: Total RNA was extracted using an RNA prep pure Cell / Bacteria Kit (TIANGEN, DP430) and then reverse transcribed into cDNA using a Hifair® III 1st Strand cDNA synthesis ultramixer (YEASEN, Cat# 11141ES60). PCR was then performed using a 2× RealStar Green Power Mixture (GenStar, A311-05) on a LightCycler 480 II real-time quantitative PCR system (Roche). Data were analyzed using the comparative CT method, with ΔCT calculated using EF1A as an internal reference gene. All experiments were performed in triplicate. Primer information for qRT-PCR is provided above under "Primers". The CT formula is as follows:

[0109] Immunofluorescence staining and imaging: Cells were fixed with 4% PFA solution at room temperature for 30 minutes. After washing, they were washed three times with elution buffer (DPBS containing 0.1% Tween 20 (Solarbio, T8220) and 0.01% Triton X-100 (Solarbio, T8200)), incubated with 0.05% Triton X-100 for 15 minutes, washed three more times with elution buffer, and blocked with 3% BSA (Sigma, Cat# A1470) at room temperature for 1 hour. Primary antibody was then added, and the cells were incubated overnight at 4°C, followed by three washes with elution buffer. Secondary antibody was incubated at room temperature for 1 hour, followed by three washes with the same elution buffer. Finally, cell nuclei were stained with DAPI (Roche Life Science, 10236276001) for 3 minutes, and then observed and photographed under a fluorescence microscope. Information on the antibodies used is provided in the "Antibodies" section above.

[0110] Cell viability assay: For both 2D adherent and 3D scaffold-cultured cells, cell viability and cytotoxicity were assessed using the Calcein / PI Cell Viability and Cytotoxicity Assay Kit (Beyotime, C2015M). Cells were double-stained with Calcein-AM (AM) and propidium iodide (PI), observed under a fluorescence microscope, and cell viability was quantified using flow cytometry (BD FACSVerse). For 3D suspension cultured spheroids, Calcein / PI staining was performed every 5 days, and the staining results were observed under a fluorescence microscope.

[0111] Fixation and Freeze-Sectioning: Before fixation, discard the cell culture medium and wash the sample with PBS. After incubating the spheres in 30% sucrose solution for 30 minutes, fix them with 4% PFA for 1 hour. For freeze-sectioning, the spheres were embedded in a mold using Sakura Tissue-Tek® OCT embedding compound (Sakura, 4853). Subsequently, the bottom of the embedding cassette was rapidly frozen in liquid nitrogen, and the sample was cooled to -80°C and sectioned to a thickness of 8µm using a cryostat (MEV, SLEE) at a chamber temperature of -20°C.

[0112] Hematoxylin-eosin (H&E) staining: Tissue sections were washed twice with double-distilled water (ddH2O), then stained with hematoxylin (Sigma-Aldrich, MHS16) and eosin (Sigma-Aldrich, HT110116), and finally observed under a microscope (Leica, DM5500B).

[0113] Scanning electron microscopy (SEM) imaging: Scaffolds and spheres seeded with differentiated cells were fixed with 4% PFA for 1 hour, followed by washing three times with DPBS, and then dehydrating sequentially with 30%, 50%, 70%, 80%, 90%, and 100% ethanol, 30 minutes each, at room temperature. After dehydration, Pt / Pd was sputtered onto the SEM (HITACHI, TM-4000 plus) stage, followed by scanning imaging with an accelerating voltage of 5 kV.

[0114] Laser scanning confocal microscopy observation: After seeding cells onto a scaffold, they were washed with Hank's balanced salt solution (HBSS, Beyotime, C0219) and fixed with 4% paraformaldehyde (PFA) at room temperature for 1 hour. The staining method for 3D samples was the same as for immunofluorescence staining, followed by washing with HBSS. Images were acquired using a laser scanning confocal microscope (Nikon, A1 HD25).

[0115] Transcriptome sequencing: PolyA mRNA was enriched from total RNA using Oligo(dT) magnetic beads and fragmented to approximately 300 bp by ionic shearing. One-stranded and two-stranded cDNAs were synthesized using random hexameric primers and reverse transcriptase. After library construction, fragments of approximately 450 bp were amplified by PCR and selected. Library quality and concentration were assessed using an Agilent 2100 bioanalyzer. Barcoded libraries were mixed, diluted to 2 nM, denatured, and then sequenced using an Illumina platform for paired-end sequencing.

[0116] RNA sequencing data analysis: Raw sequencing reads were quality checked using FastQC (v0.11.9), and adapters and low-quality bases were removed using Trimmomatic (v0.39) with default parameters. Processed reads were aligned to a porcine reference transcriptome (Sus scrofa, Ensembl version 113, cDNA sequence: Sus_scrofa.Sscrofa11.1.cdna.all.fa.gz, downloaded from https: / / ftp.ensembl.org / pub / release-113 / fasta / sus_scrofa / cdna / ) using Kallisto (v0.46.0). The gene-level read counts and transcript per million (TPM) values ​​were summarized using the tximport package (v1.18.0) in R in conjunction with the gene annotation file (Sus scrofa, Ensembl version 113, GTF file: Sus_scrofa.Sscrofa11.1.113.gtf.gz, downloaded from https: / / ftp.ensembl.org / pub / release-113 / gtf / sus_scrofa / ).

[0117] Principal Component Analysis: To explore sample variability and assess data quality, principal component analysis (PCA) was performed using variance-stabilized read counts generated by DESeq2 (v1.30.0). The top 50 genes with the highest variance across samples were selected for PCA calculations. Results were visualized using ggplot2 (v3.4.0) in R.

[0118] Differential gene expression analysis: Differential gene expression analysis was performed using DESeq2 (v1.30.0) 52. The Wald test was used to identify differentially expressed genes (DEGs) between different sample groups. The Benjamini-Hochberg method was used to perform multiple tests to correct for p-values, with FDR < 0.05 and |log2 fold change| ≥ 1 as the criteria for significant differential expression. A heatmap of DEGs was generated using pheatmap (v1.0.12) in R to visualize expression patterns among samples.

[0119] Gene functional enrichment analysis: Functional enrichment analysis of DEGs was performed using the Metascape53 and Gene Ontology Bioprocesses (GO-BP) databases. Significant enrichment criteria were set as follows: minimum number of genes ≥ 3, corrected p-value < 0.01, and enrichment factor ≥ 1.5. Related terms were grouped based on similarity. Key enriched pathways and terms were visualized using bar charts generated by R.

[0120] Amino acid composition analysis: Accurately weigh 50 mg of solid sample and transfer it to a hydrolysis tube. Add 10 mL of analytical grade hydrochloric acid (approximately 6 M, 1:1 mixture). After purging the sample tube with nitrogen for 30 seconds to remove oxygen, seal the tube tightly. Then, place the sealed tube in an oil bath and hydrolyze at 110°C for 22-24 hours. After the tube cools to room temperature, filter the hydrolysis product through a 0.45 µm membrane into a 50 mL volumetric flask. Transfer 2 mL of the diluted sample to a rotary evaporator and evaporate at 45°C until the sample is nearly dry, leaving a small amount of solid residue or trace moisture at the bottom of the tube. Then, add 2 mL of sample buffer to the tube to completely dissolve the residue. After filtration, the sample is ready for analysis using an amino acid analyzer (LA8080, Hitachi, Tokyo, Japan).

[0121] Fatty acid composition analysis: Accurately weigh 30-50 mg of sample, dissolve in ethanol, and then dilute to a volume of 25 mL. Transfer 1 mL of the prepared solution to a 15 mL centrifuge tube, and add 2 mL of 5% hydrochloric acid-methanol solution, 3 mL of chloroform-methanol mixture (1:1, v / v), and 100 µL of methyloctadecanoic acid (internal standard). Incubate the centrifuge tube in an 85°C water bath for 1 hour. After the reaction, allow the tube to cool to room temperature, add 1 mL of n-hexane, and vortex for 2 minutes to extract the target compound. Let the mixture stand for 1 hour to complete phase separation. Take 100 µL of the upper organic phase (n-hexane phase) from the mixture and dilute to a total volume of 1 mL with n-hexane. After filtration, the sample is ready for analysis.

[0122] This method employed a TG-5MS (30 m × 0.25 mm × 0.25 µm) capillary column for separation, with elution of the target compounds optimized using a programmed temperature gradient. The specific temperature program was as follows: 80 °C for 1 minute, then increased to 200 °C at 10 °C / min, followed by 250 °C at 5 °C / min, and finally to 270 °C at 2 °C / min, held for 3 minutes. The gas chromatograph was coupled with a mass spectrometer in electron ionization (EI) mode, with both the ion source and transfer line temperatures set to 270 °C. A scan range of 30–400 amu ensured comprehensive detection of the target compounds, while a 5-minute solvent delay was included to prevent detector overload due to solvent peaks.

[0123] The content of each fatty acid in the sample is calculated using the following formula:

[0124] in: X i : The content of each fatty acid in the sample (mg / kg).

[0125] A si Peak area of ​​each fatty acid in the sample test solution.

[0126] m skli Mass (mg) of standard (fatty acid triglycerides) in the standard working solution.

[0127] F j : A conversion factor that converts fatty acid triglycerides into fatty acids.

[0128] A stdi Peak area of ​​each fatty acid in the standard test solution.

[0129] m: Mass of the sample taken (kg).

[0130] Gas chromatography-mass spectrometry (GC-MS): Transfer 1 g of sample to a 20 mL extraction vial and add 10 µL of 50 µg / mL 2-methyl-3-heptanone solution (internal standard). Quickly seal the vial to prevent the release of volatile compounds. Place the SPME fiber in the GC-MS inlet and age it at 250 °C until no interfering peaks appear. Place the sealed sample vial on the SPME apparatus and set the temperature to 60 °C. Insert the SPME fiber through the stopper into the headspace, with the fiber tip approximately 1.0 cm from the sample surface. Expose the fiber for 30 minutes to adsorb volatile compounds. After extraction, remove the fiber and immediately insert it into the GC-MS inlet, exposing it at 250 °C for 3 minutes to desorb the target compound. This method uses a DB-5MS (30 m × 250 µm, 0.25 µm) capillary column, and optimizes the temperature gradient to achieve efficient elution of the target compound. The specific temperature ramping procedure was as follows: hold at 40℃ for 1 minute, ramp to 100℃ at 6℃ / min, then ramp to 230℃ at 10℃ / min and hold for 3 minutes. The gas chromatograph was coupled with a mass spectrometer in electron ionization (EI) mode, with the ion source temperature, transfer line temperature, and quadrupole temperature set to 230℃, 230℃, and 150℃, respectively. The mass spectrometry scan range was set to 40-550 m / z to achieve comprehensive detection. Qualitative analysis utilized the NIST17 database and retention times, while quantitative analysis was based on peak area normalization of the total ion chromatogram.

[0131] Texture Analysis (TPA): TPA was measured using a texture analyzer (TA.XTplus, Stable Micro Systems Ltd, TPA mode, P / 75 probe). A cylindrical sausage sample with a diameter of 15 mm and a height of 20 mm was placed in the center of the test platform, and then TPA analysis was performed. The test parameters were set as follows: pre-test speed 1 mm / s, test speed 1 mm / s, post-test speed 1 mm / s; trigger force 0.005 kg, trigger mode, automatic, pressure level 50%, interval time 5 seconds, repeated twice.

[0132] Example 1: pgEpiSCs were differentiated into pgEpiSCs-derived adipocyte progenitor cells (pgAPCs) and pgEpiSCs-derived adipocytes (pgADs) through 2D culture. 1. Preparation of MEF feeder cells Neutralization medium composition: DMEM medium with 10% fetal bovine serum (FBS, Thermo Fisher, 16000044).

[0133] MEF medium composition: DMEM medium supplemented with 10% FBS, 1% non-essential amino acids (NEAAs, Thermo Fisher, 11140050), 1% sodium pyruvate (Thermo Fisher, 11360070), 1% glutathione (GlutaMax, Thermo Fisher, 35050061) and 1% penicillin-streptomycin (Thermo Fisher, 15140122), all by volume percentage.

[0134] Pregnant female CD1 mice at 12.5 days post-mating (dpc) were euthanized by cervical dislocation, and embryos were isolated from the uterus and named E12.5 embryos. These E12.5 embryos were placed in cold DPBS containing 1% penicillin-streptomycin (Thermo Fisher, 15140122). The embryos were dissected under a stereomicroscope, and the fetal torsos were collected in cold DMEM medium (Thermo Fisher, 11965092). The torsos were placed in 50 mL tubes and repeatedly minced with fine scissors for 10 min. The cells were washed once with DPBS, digested with 0.25% trypsin-EDTA for 10 min, gently agitated in a 37°C water bath, and then quenched with neutralization medium to obtain mouse embryonic fibroblasts (MEFs). The mouse embryonic fibroblasts (MEFs) were resuspended in MEF medium and seeded in 10 cm culture dishes, approximately one dish per fetus, labeled P0 generation. Once the cells have reached 90% confluence, they can be frozen as primary cells or passaged into 15 cm culture dishes. Cells are expanded to generation P3 at a ratio of 1:2 or 1:3, using 13 µg / mL... -1 Treatment with mitomycin C (Selleck, S8146) for 2.5 hours, followed by washing twice with pre-warmed DPBS, digestion with 0.125% trypsin to form a single-cell suspension, neutralization, and counting. The cells were counted at a concentration of 2.4 × 10⁻⁶. 6 Cryopreserved at a density of 1 cell / mL for use as feeder cells in subsequent stem cell cultures.

[0135] 2. Culture of porcine gastrulation epiblast stem cells (pgEpiSCs) Porcine pregastrulation epiblast stem cells (pgEpiSCs): described in the non-patent literature "Zhi, ML et al. Generation and characterization of stable pig pregastrulation epiblast stemcell lines. Cell Res. 32, 383-400 (2022).", and referred to as pgEpiSCs in the aforementioned literature, are publicly available from China Agricultural University. This biomaterial is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0136] Culture method: Undifferentiated porcine gastrulated epiblast stem cells (pEpiSCs) were seeded onto MEF feeder cells obtained in "1. MEF feeder cell preparation" for maintenance growth. The culture medium used was 3i / LAF chemical medium (formula shown in Table 1). Fresh 3i / LAF chemical medium was added daily, and the cells were passaged every 2-3 days using Accutase cell dissociation reagent at a ratio of 1:3 to 1:5, with single-cell state at passage. The specific number of passages and dilution ratios should be adjusted according to the actual situation.

[0137] 3. Isolation and culture of porcine adipose-derived stem cells Take 1.5 grams of the longissimus dorsi muscle and wash it three times with phosphate-buffered saline (DPBS, Gibco, C14190500CP) containing 10% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122). Then, mince the tissue and add collagenase II (Coolaber, CC3791G) 10× stock solution, and digest it in a shaker incubator at 37°C and 90 rpm for 1 hour. After digestion, complete culture medium (DMEM / F12, Thermo Fisher Scientific, 10565-018), 1% penicillin-streptomycin (PS, Thermo Fisher Scientific, 15140-122), 1% non-essential amino acids (NEAA, Thermo Fisher Scientific, 1140-050), 10% fetal bovine serum (FBS, Gibco, 16000-044), and 5 ng / mL fibroblast growth factor 2 (FGF2, PeproTech, 100-18B) were added to terminate digestion. The solution was then filtered sequentially through 100 μm and 40 μm cell filters.

[0138] The cell suspension was centrifuged at 1000 rpm for 5 minutes, then resuspended in erythrocyte lysis buffer (Solarbio, R1010) and incubated on ice for 5 minutes. After centrifugation at 1000 rpm for 5 minutes again, the cells were resuspended and seeded into gelatin-coated (Stem Cell Technologies, 07903) T75 cell culture dishes and incubated in a cell culture incubator at 37°C and 5% CO2. Cells were isolated by differential adhesion; after 2 hours, the supernatant was removed and replaced with fresh culture medium. The adherent cells were identified as preadipocyte progenitor cells (FAPs). Cell morphology and growth status were observed using an inverted microscope. The pFAP growth medium (formula shown in Table 1) was changed every 2 days to continue culturing. When the cell confluence reached 90%, the medium was replaced with pFAP differentiation medium (formula shown in Table 1) and cultured for 6 days; subsequently, the medium was replaced with pFAP differentiation maintenance medium (formula shown in Table 1) and cultured for 3 days.

[0139] 4. The serum-free adipogenic differentiation method of pgEpiSCs provided in this embodiment The specific steps for serum-free adipogenic differentiation of pgEpiSCs are as follows: Remove the feeder layer cells, dissociate pgEpiSCs into single cells using Accutase cell dissociation reagent, and then... 5 Cells were seeded at a density of 10 cells / well in gelatin-coated 6-well plates using ADM I medium (formula shown in Table 1). After 3 days of culture, cells were harvested using TryPLE™ Express and seeded in ADM II medium (formula shown in Table 1) for 9 days. Subsequently, the medium was changed to ADM III medium (formula shown in Table 1) for 6 days to obtain pgEpiSCs-derived adipocyte progenitor cells (pgAPCs). The pgAPCs were then cultured in ADM IV medium (formula shown in Table 1) for 6 days. Cells cultured at this stage were defined as pgEpiSCs-derived adipocyte precursor cells. The medium was then changed to ADM V medium (formula shown in Table 1) for 4-7 days to obtain pgEpiSCs-derived adipocytes (pgADs).

[0140] The results are as follows: The process of pgEpiSCs differentiating into adipocytes is described in [see...]. Figure 1 Image a. Cell images at different time points are shown in [reference 1]. Figure 6 In the culture process of "4. The serum-free adipogenic differentiation method of pgEpiSCs provided in this embodiment", cells were collected at different time points. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine that the optimal time for mesodermal induction was 3 days. This was based on the maximum reduction in the expression of pluripotency markers (NANOG, OCT4, and SOX2) and the upregulation of the expression of the mesodermal-specific marker T (Brachyury). Figure 6 (b, c). Staining with alkaline phosphatase (AP) ( Figure 6 (d) and NANOG downgraded ( Figure 1 b) confirmed the loss of pluripotency, while T (Brachyury) immunostaining ( Figure 1 Figure b) confirms the success of mesodermal differentiation. Therefore, in the ADM I and ADM II media provided by this invention, the WNT / β-catenin pathway is activated by adding CHIR99021, and the TGF-β signaling pathway is inhibited by using SB431542 to initiate mesodermal differentiation.

[0141] During the culture process of "4. The serum-free adipogenic differentiation method of pgEpiSCs provided in this embodiment", from day 4 to day 12, the markers of adipogenic precursor cells (APCs) ( PDGFRA, ZNF423 and VIM The expression of ) increased, while that of adipogenesis inhibitory factor increased. DLK1 Decreased expression of [a specific substance] indicates directed differentiation of preadipocytes. Figure 1 c). pgEpiSC-derived adipocyte progenitor cells (pgAPCs) simultaneously express PDGFRA. + CD29 + CD45 - ( Figure 1 d) and express genes ITGB1, Thy1, ENG , without expressing PECAM1 and PTPRC ( Figure 1 The results (d, e) are consistent with in vivo experimental results. After 6 days of culture, the main regulator of adipogenesis (d, e) PPARG, CEBPA ) reaches peak expression, while mature genes ( FABP4, LPL The expression of ) gradually increased. Oil Red O, BODIPY, and Nile Red staining confirmed the large accumulation of lipid droplets (LD) in pgEpiSC-derived adipocytes (pgADs), and the adipocyte maturation marker FABP4 was positive. Figure 1 e, h and Figure 6 (f). Chromosomal stability is maintained throughout the differentiation process, which can be confirmed by normal karyotype analysis. Figure 6 (g).

[0142] To verify the adipogenesis differentiation process, pgADs were compared with adipocytes derived from porcine preadipocytes (pFAPs) isolated from muscle tissue. Figure 7 (a) After 13 generations of passage, pFAPs showed a decrease in adipogenic potential, manifested as a reduction in lipid droplet formation. Figure 7(middle bd). RNA-Seq analysis showed that, compared with the undifferentiated control group, key transcription factors ( ) in pgADs were higher. PPARG, CEBPA, ADD1 ) and lipid metabolism-related genes ( PLIN3, AGPAT2 The expression of ) was upregulated (p<0.01); Figure 1 (i) Gene Ontology (GO) enrichment analysis further highlighted the adipogenesis differentiation pathway (j) in Figure 1. Furthermore, compared to pFAPs, principal component analysis (PCA) revealed correlations among the four cell groups (i). Figure 7 Both pFAPs and pgEpiSC-derived adipocytes (pgADs) are enriched with functions related to adipose tissue development. These adipocyte groups exhibit similar gene expression profiles related to adipogenesis and development during differentiation. Figure 7 (f) indicates that pgEpiSC-derived adipocytes and pFAPs-derived adipocytes share certain characteristics. These results suggest that pgEpiSCs can serve as a serum-free, stable source for generating functional adipocytes, overcoming the limitations of pFAPs in long-term culture.

[0143] Example 2: pgEpiSCs were differentiated into pgEpiSCs-derived vascular endothelial progenitor cells (pgVPCs) and vascular type cells (pgVCs) through 2D culture. Specific operational steps for serum-free method of differentiating pgEpiSCs into vascular cell types: pgEpiSCs were cultured according to "2. Culture of porcine gastrulation epiblast stem cells (pgEpiSCs)" in Example 1. Feeder cells were removed, and pgEpiSCs were dissociated into small cell clusters using Accutase cell dissociation reagent. Then, they were cultured at 2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 10 cells / well in gelatin-coated 6-well plates using VDM I medium (formula shown in Table 1). After 1 day of culture, the medium was replaced with VDM II medium (formula shown in Table 1) and cultured for 5 days (to allow pgEpiSCs to specialize into vascular lineages), yielding pgEpiSCs-derived vascular endothelial progenitor cells (pgVPCs). Subsequently, the medium was replaced with VDM III medium (formula shown in Table 1), and the cells were cultured in VDM III medium for 3 days to obtain vascular type cells (pgVCs). The vascular type cells were then maintained in VDM III medium for subsequent experiments.

[0144] The results are as follows: The process of pgEpiSCs differentiating into vascular cells is described in [the following text is incomplete and likely refers to a separate document]. Figure 2 Image a. Cell images at different time points are shown in [reference 1]. Figure 2 b.

[0145] During the differentiation of pgEpiSCs into vascular cells, cells were collected at different time points. Time-series analysis of pluripotency genes and germ layer marker genes showed that the expression of mesodermal genes (T) peaked on day 1 of differentiation, confirming the success of mesodermal induction. Figure 2 (c, d). Subsequent optimization experiments determined that SB431542 and VEGF supplementation for 5 days was the optimal condition for vascular lineage specialization. Figure 2 (e). Further differentiation via VEGF and FGF2 yielded a heterogeneous cell population, exhibiting polygonal and spindle-shaped morphologies, corresponding to endothelial cells and pericytes, respectively. Figure 2 (b) Immunostaining results confirmed the presence of endothelial cells (CD31). + eNOS + ) and pericytes (αSMA) + Coexistence of ) Figure 2 (f, g). It can be seen that activating the WNT / β-catenin pathway with CHIR99021 and supplementing the culture medium with activin A, bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) can efficiently induce mesodermal formation, because these factors are crucial for vascular lineage specialization.

[0146] Functional validation showed that pgEpiSC-derived vascular cells (pgVCs) were able to form a structurally coherent tubular network after 6 hours of incubation on Matrigel thick layers. Figure 2 (h), and exhibits the ability to take up low-density lipoprotein (LDL), a hallmark of functional endothelial cells (h). Figure 2 (i). Furthermore, these cells maintained a survival rate of >96% throughout the differentiation process (as determined by calcein and propidium iodide staining) and retained a normal karyotype. Figure 2 (jl). Compared with pgEpiSCs, differential analysis showed that 3044 genes were upregulated after angiogenesis induction. Gene Ontology (GO) enrichment analysis revealed biological processes related to angiogenesis and vascular development. Figure 2 (m, n). Through this serum-free differentiation system, vascular cell types were successfully generated from pgEpiSCs, laying the foundation for subsequent development of multi-lineage aggregation and multi-tissue construction.

[0147] Example 3: 2D co-culture of pgEpiSCs-derived myoblasts and pgEpiSCs-derived adipocytes 1. A method for differentiating pgEpiSCs into pgEpiSCs-derived muscle progenitor cells (pgMPCs) via 2D culture is documented in the non-literature article "Zhu, GX et al. Generation of three-dimensional meat-like tissue from stable pig epiblast stem cells. Nat Commun 14 , 8163. (2023).。

[0148] Specific steps: Remove the feeder layer cells, dissociate pgEpiSCs into single cells using Accutase cell dissociation reagent, and then use 2×10 5 Cells were seeded at a density of 10 cells / well in gelatin-coated 6-well plates and cultured for 3 days in MDM I medium (formula in Table 1). Cells were then harvested using TryPLE and seeded in MDM II medium (formula in Table 1) for another 3 days. Subsequently, the medium was changed to MDM III medium (formula in Table 1) for 2 days to obtain pgEpiSC-derived myoblast progenitor cells (pgMPCs). The pgMPCs were then cultured in MDM IV medium (formula in Table 1) for 4 days, followed by MDM V medium (formula in Table 1) for 20-25 days. Cells cultured during this stage were defined as pgEpiSCs-derived myoblasts. Finally, the medium was changed to N2 medium for 7 days to obtain pgEpiSCs-derived myoblasts (pgMCs).

[0149] 2. 2D co-culture of pgEpiSCs-derived myoblasts and pgEpiSCs-derived adipocyte precursor cells The proliferation medium consisted of DMEM / F12 medium supplemented with 1% penicillin-streptomycin (P / S), 1% non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 15% KOSR, 200 μM ascorbic acid, 10 ng / mL LIFG-1, 10 ng / mL HGF, and 10 ng / mL FGF2.

[0150] N2 medium composition: DMEM / F12 medium supplemented with 15% knockout serum substitute, 1% N2, 1% penicillin / streptomycin, 1% non-essential amino acids and 10 μg / mL insulin.

[0151] Operating steps ( Figure 3 a) is as follows: The pgEpiSCs-derived myoblasts (i.e., the myoblasts described in section B5 of the invention) obtained by "1. Differentiating pgEpiSCs into pgMCs through 2D culture" and the pgEpiSCs-derived adipocyte precursor cells (i.e., the adipocyte precursor cells described in section B5 of the invention) obtained in Example 1 were mixed at cell ratios (0:10, 1:9, 3:7, 5:5, 7:3, 9:1, 10:0, with 5:5 being optimal) to prepare a cell suspension, which was then seeded into 12-well plates containing proliferation medium. When the cell confluence reached 90%, differentiation was induced by changing to differentiation medium. The two culture schemes were designed as follows: (I) Cells were cultured in ADM IV medium (formula shown in Table 1) for 6 days, followed by culture in ADM V medium (formula shown in Table 1) for 3 days. (II) Cells were cultured for 6 days in the first mixed medium (ADM IV medium (formula shown in Table 1) and MDM V medium (formula shown in Table 1) in a volume ratio of 1:1) and then for 4 days in the second mixed medium (ADM V medium (formula shown in Table 1) and N2 medium (volume ratio of 1:1)). All cultures were performed in a humidified incubator at 37°C and 5% CO2, with the medium changed every 2 days.

[0152] The results are as follows: In a two-dimensional monolayer co-culture, both cell types were first expanded in growth medium for 2 days until cell confluence and 90% confluence were achieved, followed by differentiation induction. Optimization of culture conditions showed that a 1:1 mixture of pgEpiSCs-derived myoblasts and pgEpiSCs-derived adipocyte precursor cells supported co-differentiation into mature cell types. Figure 8 (ac). Optical microscopy revealed a mixed distribution of adipocytes and skeletal muscle fibers (SMFs). Figure 3 (b) Oil Red O staining and immunofluorescence staining further confirmed the maturation of adipocytes and myocytes. Figure 3 (Ce). Real-time quantitative PCR analysis further confirmed that both cell types can co-differentiate into the mature form: skeletal muscle fibers ( MYOG, MYH2, MYH3 ()( Figure 3 (f) and adipocytes ( ADIPOQ, PLIN1, LEP ()( Figure 3 (g). Notably, the aforementioned scheme (II) performed better in supporting this differentiation process. These results demonstrate the compatibility of pgEpiSC-derived myoblasts and pgEpiSC-derived adipocyte precursor cells in two-dimensional co-culture.

[0153] Example 4: 3D suspension culture enables long-term expansion of pgEpiSC-derived progenitor cells. I. Experimental Methods: 1. 3D-Muscle Cell Spheroid Culture: pgMPCs obtained in Example 3, "1. Differentiation of pgEpiSCs into pgMPCs via 2D Culture," were digested using TrypLE™ Express. The cell pellet was collected, resuspended in MDM III medium (formula shown in Table 1), and then processed using a cell sieve (40 μm pore size) to form a single-cell suspension of pgMPCs. The cell density of the mixture was adjusted to 5.0 × 10⁶ cells / mL. 5 Cells / mL were seeded into 3.5 cm non-adsorbent cell culture dishes. The culture dishes were placed in a horizontal shaker and cultured at 37°C, 5% CO2, and 100% humidity at a rotation speed of 70 rpm / min for 2 days. Then, the medium was replaced with MDM IV medium (formula shown in Table 1) for 4 days, and finally replaced with MDM V medium (formula shown in Table 1) for 30 days. Except for medium changes, the culture was carried out in the aforementioned horizontal shaker throughout the entire process, resulting in muscle cell spheroids derived from porcine gastrulation epiblast stem cells.

[0154] 2. 3D-Adipocyte Spheroid Culture: pgAPCs obtained in Example 1, "4. Serum-free adipogenic differentiation method of pgEpiSCs provided in this example," were digested using TrypLE™ Express. The cell pellet was collected, resuspended in ADM III medium (formula shown in Table 1), and then processed using a cell sieve (40 μm pore size) to form a single-cell suspension of pgAPCs. The cell density of the mixture was adjusted to 5.0 × 10⁶ cells / mL. 5 Cells were seeded at a density of 100 cells / mL in 3.5 cm non-adsorbent cell culture dishes (day 0 was the day of seeding). The culture dishes were placed in a horizontal shaker and cultured at 37°C, 5% CO2, and 100% humidity at a rotation speed of 70 rpm / min for 5 days. The culture was then changed to ADM IV medium (formula shown in Table 1) for 6 days, and finally cultured in ADM V medium (formula shown in Table 1) until day 30. Except for medium changes, the cells were cultured in the aforementioned horizontal shaker throughout the entire process, yielding adipocyte spheroids derived from porcine gastrulation epiblast stem cells.

[0155] 3. 3D-vascular cell spheroid culture: pgVPCs obtained in Example 2 were digested using TrypLE™ Express. The cell pellet was collected, resuspended in VDM II medium (formula shown in Table 1), and then processed using a cell sieve (40 μm pore size) to obtain a pgVPCs single-cell suspension. The cell density of the mixture was adjusted to 5.0 × 10⁶ cells / mL. 5Cells / mL were seeded into 3.5 cm non-adsorbent cell culture dishes (day 0 was the day of seeding). The culture dishes were placed in a horizontal shaker and cultured at 37°C, 5% CO2, and 100% humidity at a rotation speed of 70 rpm / min for 2 days. Then, the culture was changed to VDM III medium (formula shown in Table 1) and cultured until day 30. The entire culture was carried out in the aforementioned horizontal shaker to obtain vascular cell spheroids derived from porcine gastrulation epiblast stem cells.

[0156] II. Experimental Results: pgMPCs (MYOD) were cultured separately. + PAX7+) and pgAPCs (CD29) + PDGFRA + ) and pgVPCs( Figure 10 (ad).

[0157] Cells were collected at various time points. From the first day of culture, the cells spontaneously aggregated into spheres, and their volume gradually increased over time. Figure 4 The spheres have a diameter of 800-1200 micrometers while maintaining a high survival rate. Figure 10 (e, f). Morphological analysis revealed a striking feature: muscle spheres ( Figure 4 The middle f) has a smooth surface and tightly packed cells, while the fat globules ( Figure 4 The middle g) has a rough, irregular surface and a loose cell arrangement.

[0158] Functionally, myosins form an internal structure similar to fibrous tissue and express markers of myofiber differentiation, such as myosin (…). Figure 4 In the middle h, i), while fat globules are mainly occupied by lipid droplets and express FABP4 ( Figure 4 Both types of spheres express COL3A1 (j,k). Figure 10 The presence of g and h in these spheres indicates that they contain extracellular matrix (ECM) components. Transcriptomic and real-time quantitative PCR analyses showed that, compared to two-dimensional conditions, muscle progenitor cells in three-dimensional suspension culture significantly upregulated genes associated with skeletal muscle development and ECM formation, such as key myogenic markers. MYOG and MYH3 Increased expression ( Figure 4 l, m and Figure 10 (i). Although compared to two-dimensional conditions, markers of adipogenesis in three-dimensional adipocytes ( PPARG, LPL The expression differences were not significant, but the three-dimensional system showed superior performance in tissue morphogenesis and cell recognition. Figure 4 n, o and Figure 10 (j).

[0159] The inventors further tested the feasibility of the three-dimensional suspension culture system in the culture of vascular progenitor cells (VPCs). Vascular spheres formed a rough surface and irregular cell arrangement, expressing endothelial cells (VPCs). PECAM1 and CDH5 ) and pericytes ( PDGFRB They are markers of [something]. However, after 10 days of culture, their proliferative capacity is limited, and they hardly expand thereafter.

[0160] These results indicate that the three-dimensional suspension culture system is superior to the two-dimensional system in supporting the long-term expansion, cell differentiation, or tissue morphogenesis of muscle and adipocyte progenitors.

[0161] Example 5: 3D dual-lineage co-culture can generate multi-tissue cell cultured meat with enhanced nutritional and structural properties. The specific operation of the muscle / adipocyte spheroid culture method provided in this embodiment is as follows: The pgAPCs obtained in "4. The serum-free adipogenic differentiation method of pgEpiSCs provided in this example" in Example 1 and the pgMPCs obtained in "1. Differentiation of pgEpiSCs into pgMPCs by 2D culture" in Example 3 were digested using TrypLE™ Express. Cell pellets were collected, resuspended in MADM I medium (formula shown in Table 1), and then processed using a cell sieve (40 μm pore size) to obtain pgMPCs single-cell suspensions and pgAPCs single-cell suspensions. The pgMPCs single-cell suspension and pgAPCs single-cell suspension were mixed at a cell ratio of 1:1 to obtain a mixture, and the cell density of the mixture was adjusted to 5.0 × 10⁻⁶. 5 Cells / mL were seeded into 3.5 cm non-adsorbent cell culture dishes. The dishes were placed in a horizontal shaker and cultured at 37°C, 5% CO2, and 100% humidity at a rotation speed of 70 rpm / min for 5 days. The culture was then replaced with MADM II medium (formula shown in Table 1) for 9 days, and finally supplemented with MADM III medium (formula shown in Table 1) for 30 days. Except for medium changes, the entire culture was performed in the aforementioned horizontal shaker.

[0162] Experimental results: pgAPCs and pgMPCs were mixed in a 1:1 ratio (5 × 10⁻⁶). 5 (cells / mL) were co-cultured in a three-dimensional suspension culture, and dual-lineage spheroids formed on day 1. Figure 11 (b) After 5 days of proliferation in differentiation medium, the spheres were transferred to terminal differentiation conditions (i.e., cultured in MADM II medium) and cultured for another 9 days, maintaining this condition until day 30. During this period, the sphere size increased to approximately 1000 micrometers while maintaining a high survival rate. Figure 11(c, a) Its structure is more compact and organized than the sphere of day 15, and it has clearer organized regions. Figure 12 Functional validation showed a high accumulation of lipid droplets (LD) in the spheres (BODIPY staining), accompanied by co-expression of myofibril-specific protein (myosin). Real-time quantitative PCR analysis confirmed its association with myogenic maturation (myotrophic lateral fibrosis). MYOG, MYH2, MYH3 ), Adipocyte development ( ADIPOQ, LEP, PLIN1 ) and ECM formation ( COL3A1, COL5A2 Upregulation of related genes Figure 12 (f). Furthermore, compared to spheres derived from a single cell type, the formation efficiency of muscle, fat, and ECM in co-cultured spheres was at an intermediate level.

[0163] Example 6: 3D multi-lineage co-culture can generate multi-tissue cell cultured meat with enhanced nutritional and structural properties. The specific operation of the muscle / fat / vascular cell spheroid culture method provided in this embodiment is as follows ( Figure 5 a, Figure 11 a) is as follows: Using TrypLE™ Express, the pgAPCs obtained in Example 1 ("4. Serum-free adipogenic differentiation method of pgEpiSCs provided in this example"), the pgVPCs obtained in Example 2, and the pgMPCs obtained in Example 3 ("1. Differentiation of pgEpiSCs into pgMPCs through 2D culture") were digested. Cell pellets were collected, resuspended in MADVM I medium (formula shown in Table 1), and then processed using a cell sieve (40 μm pore size) to prepare single-cell suspensions of pgMPCs, pgAPCs, and pgVPCs. These single-cell suspensions were then mixed at a cell ratio of 1:1:1 to obtain a mixture, and the cell density of the mixture was adjusted to 5.0 × 10⁻⁶. 5 Cells / mL were seeded into non-adsorbent cell culture dishes. The dishes were placed in a horizontal shaker and cultured at 37°C, 5% CO2, and 100% humidity at a rotation speed of 70 rpm / min for 5 days. Then, the medium was replaced with MADVM II medium (formula shown in Table 1) and cultured for 9 days. Finally, MADVM III medium (formula shown in Table 1) was added and cultured for 16 days to obtain multi-tissue cell culture meat. Except for medium changes, the entire process was carried out in the aforementioned horizontal shaker.

[0164] The results show that: Co-culturing MPCs, APCs, and VPCs in a 1:1:1 ratio further increased structural complexity. Notably, the spheres began forming internal chambers on day 10, subsequently expanding over time while maintaining high survival rates. Figure 5 (b) By day 30, the sphere diameter reached approximately 1000 micrometers, and the cell density reached 1.35 × 10⁻⁶. 8 cells / mL Figure 5 (c, d). Real-time quantitative PCR analysis showed that it was associated with vascular markers ( PECAM1, CDH5, PDGFRB ) and ECM-related genes ( COL3A1, COL5A2 Simultaneously, the levels were increased. Multiple tissue types were detected in the spheres. Figure 5 Immunostaining confirmed the coexistence of lipid droplets (BODIPY), myosin (myosin), and endothelial cells (CD31) within the sphere. Figure 5 (g), accompanied by potent ECM generation. Transcriptomic analysis showed that multi-tissue spheroids simultaneously exhibited characteristics of muscle, adipose, and vascular tissue. Notably, compared to spheroids derived from a single cell type, many of the components in multi-tissue spheroids were associated with muscle (g). DES, ACTN2, MYL3 ),Fat( CEBPD, LPL ) and ECM ( FN1, COL3A1 The expression levels of related marker genes are higher. Figure 5 (h). These results indicate that co-culturing of multiple cell types promotes their respective differentiation processes.

[0165] Nutritional and textural analysis of the spheres showed significant similarities to conventionally produced pork. Analysis of 17 amino acids (AAs) and 19 fatty acids (FAs) revealed proportions similar to natural meat, with increased polyunsaturated fatty acid (PUFAs) content and decreased saturated fatty acid (SFAs) and monounsaturated fatty acid (MUFAs) content, resulting in a ∑PUFA / ∑SFA ratio twice that of conventional pork. Figure 5 (i,j)

[0166] Example 7: Preparation of cultured meat sausage and traditional pork sausage Sausage preparation steps: mincing, weighing raw materials, chopping / mixing, filling / bottling, cooking, and cooling.

[0167] Cooking conditions: 82°C for 45 minutes, filling diameter 15 mm (using 15 mL centrifuge tubes as an alternative).

[0168] Table 2. Sausage Recipe

[0169] The results showed that the sausage derived from cell-cultured meat (prepared in Example 6) exhibited unique characteristics and was similar in appearance to ordinary pork sausage and pgEpiSCs sausage. Figure 5 (k). Cellular meat exhibits similar firmness, cohesiveness, adhesiveness, chewiness, and resilience to regular pork sausages, and is superior to pgEpiSCs sausages. Cellular meat also demonstrates greater elasticity. Figure 5 (l). This indicates that cell differentiation has a significant impact on the texture of cell-cultured meat. Furthermore, the relatively higher content of aldehydes and alcohols may contribute to the formation of a more complex and richer aroma. These results demonstrate that large-scale production of multi-tissue cultured meat with a texture similar to conventional meat can be achieved through three-dimensional suspension co-culture of pgEpiSCs-derived multi-lineage progenitor cells, and nutritional regulation can be achieved through inoculation of cells at different ratios.

[0170] In summary, this application successfully achieved multi-tissue directed differentiation of pgEpiSCs in a serum-free system and promoted the self-assembly of multi-tissue cultured meat through a three-dimensional suspension system, providing the possibility for the production of nutritionally customized cultured meat products.

[0171] Currently, the main cell types used for cultured meat production include muscle satellite cells, mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs), and fibroblasts. These cells are all monolineage cells and cannot maintain their differentiation capacity during continuous passage. Therefore, continuous isolation of cells from animal sources is necessary, raising significant concerns about animal welfare and sustainability principles. Pluripotent stem cells (PSCs), with their multilineage differentiation potential enabling them to differentiate into all cell types, and their self-renewal capacity allowing for unlimited expansion, are ideal candidate cells for cultured meat development. In this study, the inventors elucidated the diverse differentiation mechanisms of pgEpiSCs using stem cell resources and successfully induced pgEpiSCs to differentiate into muscle, adipose, and vascular endothelial cells in serum-free and animal-component-free differentiation systems.

[0172] Two-dimensional monolayer culture, due to its limited surface area and the need for continuous passaging, cannot effectively achieve large-scale cell expansion and is therefore not an effective method for cultured meat development. Consequently, three-dimensional culture systems have gained widespread attention in cultured meat research. In this study, the method combining cells with a three-dimensional scaffold for cultured meat preparation is the most commonly used approach. However, scaffold production costs and cell numbers remain limiting factors. Furthermore, microcarrier suspension culture may affect the cost of cell isolation and purification, the maximum achievable cell density, and the nutritional or sensory characteristics of the cultured meat product. In this study, the inventors developed a highly efficient three-dimensional suspension culture system. In this system, cells spontaneously aggregate into spheroids based on their own extracellular matrix, forming an organized multicellular structure, thereby effectively improving cell proliferation and differentiation efficiency. This scaffold-free strategy based on the spheroid method not only eliminates cell dependence on scaffolds but also promotes large-scale cell culture and expansion. Moreover, this system allows for the co-culture of different cell types.

[0173] While most cultured meat research focuses primarily on muscle cells and tissues, fat is also a crucial component of meat. Fat provides essential nutrients and significantly influences the flavor, texture, and tenderness of meat, properties that significantly impact consumer preferences. Furthermore, blood vessels are vital for maintaining tissue growth and structure by supplying oxygen and nutrients in vitro. In this study, the inventors explored the co-culture of multiple cell types and reported that different cell types can recognize each other and be cultured in varying proportions to form multi-tissue structures. Although the specific interaction mechanisms between different cell types are not fully understood, the inventors have provided a potential technical approach for co-culturing multiple cell types in a single system. The amino acid (AAs) and fatty acid (FAs) profiles of the cultured meat products in this study were consistent with those of conventional pork, and the ∑PUFA / ∑SFA ratio of the cultured meat was significantly higher than that of conventional pork. These results suggest that the cultured meat has a relatively high proportion of unsaturated fatty acids (PUFAs) and a low proportion of saturated fatty acids (SFAs), which may be more beneficial for cardiovascular health. The texture and tenderness of cultured meat may be related to factors such as muscle fibers, connective tissue, extracellular matrix proteins, and scaffold materials. In this study, the three-dimensionally cultured multi-tissue meat not only contained muscle fibers, adipose tissue, and microvessels, but also generated a large amount of extracellular matrix and collagen. After cooking, the cultured meat exhibited suitable tenderness and elasticity. In the future, by adjusting the proportion of different cell types, it is hoped that the nutritional composition, flavor, and texture characteristics of cultured meat can be customized.

[0174] In summary, the inventors successfully prepared multi-tissue cultured meat products by differentiating pgEpiSCs from multiple lineages and applying three-dimensional suspension culture technology, thereby endowing the cultured meat products with enhanced texture and nutritional value. The inventors hope that this research will provide a novel technical method for the development of cultured meat and enhance their understanding of the application of stem cells in the food industry.

[0175] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing multi-tissue cell cultured meat through multi-lineage differentiation of porcine pluripotent stem cells, comprising co-culturing three types of cells—adipocyte progenitor cells derived from porcine gastrulation epiblast stem cells, muscle progenitor cells derived from porcine gastrulation epiblast stem cells, and vascular endothelial progenitor cells derived from porcine gastrulation epiblast stem cells—in suspension to allow the three types of cells to differentiate and obtain multi-tissue cell cultured meat; wherein the adipocyte progenitor cells are prepared according to M1, the muscle progenitor cells are prepared according to M2, and the vascular endothelial progenitor cells are prepared according to M3; M1 is a method for culturing 2D adipocyte progenitor cells, the method comprising: Porcine gastrulated epiblast stem cells were induced to differentiate into adipocyte progenitor cells to obtain the adipocyte progenitor cells; M2 is a method for culturing 2D-muscle progenitor cells, the method comprising: inducing porcine gastrulation epiblast stem cells to differentiate into muscle progenitor cells, thereby obtaining the muscle progenitor cells; The M3 is a method for culturing 2D-vascular endothelial progenitor cells, the method comprising: inducing porcine gastrulation epiblast stem cells to differentiate into vascular endothelial progenitor cells, thereby obtaining the vascular endothelial progenitor cells.

2. The method according to claim 1, characterized in that, The ratio of the number of adipocytes, muscle progenitors and vascular endothelial progenitors is (2-0.5):(2-0.5):

1.

3. The method according to claim 1 or 2, characterized in that, The co-cultivation includes the following steps: A1) Use MADVM I medium for co-culture from day 1 to day 5; A2) Use MADVM II medium during co-culture from day 6 to day 14; A3) Use MADVM III medium during co-culture from day 15 to day 30; The MADVM I medium is a liquid culture medium containing composition I; the MADVM II medium is a liquid culture medium containing composition II; the MADVM III medium is a liquid culture medium containing composition III. Composition I consists of IGF-1, FGF2, hEGF, and VEGF; composition II consists of IGF-1, HGF, IBMX, dexamethasone, insulin, and VEGF; and composition III consists of IGF-1, HGF, insulin, and VEGF.

4. The method according to claim 3, characterized in that, The MADVM I culture medium contained 10 ng / ml IGF-1, 10 ng / ml FGF2, 10 ng / ml hEGF, and 50 ng / ml VEGF. The MADVM II medium contained IGF-1 at 10 ng / ml, HGF at 10 ng / ml, IBMX at 500 μM, Dex at 1 μM, insulin at 10 ng / ml, and VEGF at 50 ng / mL. The MADVM III culture medium contained 10 ng / ml IGF-1, 10 ng / ml HGF, 10 ng / ml insulin, and 25 ng / ml vascular endothelial growth factor (VEGF).

5. The method according to any one of claims 1-4, wherein the suspension method is provided by a non-adsorbent cell culture dish and a rotating device, and the parameters of the rotating device during the co-culture process are 70 rpm / min.

6. The method according to any one of claims 1-5, characterized in that, The induction of porcine gastrulation epiblast stem cells into adipocyte progenitor cells in M1 includes the following steps: culturing porcine gastrulation epiblast stem cells in ADM I medium, then in ADM II medium, and finally in ADM III medium to obtain the adipocyte progenitor cells; wherein ADM I medium is a liquid medium containing composition one, which includes B27, CHIR99021, and SB431542; wherein ADM I medium is a liquid medium containing composition two, which contains CHIR99021, LDN193189, and FGF2; and wherein ADM III medium is a medium containing composition three, which contains FGF2 and hEGF.

7. The method according to any one of claims 1-6, characterized in that, The M3 method for inducing porcine gastrulation epiblast stem cells to differentiate into vascular endothelial progenitor cells includes the following steps: culturing porcine gastrulation epiblast stem cells in VDM I medium, and then replacing it with VDM II medium to obtain vascular endothelial progenitor cells. The VDM I medium is a liquid culture medium containing composition A, which includes B27, CHIR99021, Activin A, BMP4 and VEGF; the VDM II medium is a liquid culture medium containing composition B, which includes SB431542 and VEGF.

8. The method, characterized in that, The method is any one of the following: B1) Method for culturing 2D-adipocyte progenitor cells: the method is M1 as described in any one of claims 1-7; B2) A method for culturing 2D-adipocytes, wherein the first step of the method is M1 as described in any one of claims 1-7, and the second step of the method is to culture the adipocyte progenitor cells obtained from M1 in a medium containing composition ADM-IV for 6 days, and then in a medium containing composition ADM-V for 4-7 days to obtain adipocytes derived from porcine gastrulation epiblast stem cells, wherein composition ADM-IV comprises insulin-transferrin-selenium, rosiglitazone, isobutylmethylxanthine, dexamethasone and insulin, and composition ADM-V comprises insulin; B3) Method for culturing 2D-vascular endothelial progenitor cells: the method is M2 as described in any one of claims 1-7; B4) Method for culturing 2D-vascular type cells: The first step of the method is M3 as described in any one of claims 1-7, and the second step of the method is to culture the vascular endothelial progenitor cells obtained by the M3 in a medium containing the composition VDM-III for 3 days to obtain vascular type cells derived from porcine gastrulation epiblast stem cells, wherein the composition VDM-III contains VEGF and EGF2. B5) A method for co-culturing muscle cells and adipocytes using 2D: adipocyte precursor cells prepared by M1 according to any one of claims 1-7 and myoblasts prepared by M2 according to any one of claims 1-7 are mixed to obtain mixed cells, which are then cultured according to B51) or B52): B51) The mixed cells are cultured in the medium containing composition ADM-IV for 6 days, followed by culture in the medium containing composition ADM-V for 3 days; B52) The mixed cells are cultured in a first mixed medium (the medium containing composition ADM-IV and the medium containing composition MDM-V (volume ratio 1:1)) for 6 days, followed by culture in a second mixed medium (the medium containing composition ADM-V and the medium containing composition N2 (volume ratio 1:1)) for 4 days; wherein composition MDM-V comprises HGF and IGF-1, and composition N2 contains a knockout serum substitute and N2; B6) Method for culturing 3D-adipocyte spheroids: Adipocyte progenitor cells prepared by M1 according to any one of claims 1-7 are cultured in a rotating apparatus for 5 days in a medium containing composition ADM III, then cultured in a rotating apparatus for 6 days in ADM IV medium, and finally cultured in a rotating apparatus with ADM V medium until day 30 to obtain adipocyte spheroids derived from porcine gastrulation epiblast stem cells; wherein composition ADM III contains FGF2 and hEGF; B7) Method for culturing 3D-muscle cell spheroids: Muscle progenitor cells prepared by M2 according to any one of claims 1-7 are cultured in a rotating apparatus for 2 days in a medium containing composition C, then cultured in a rotating apparatus for 4 days in a medium containing composition MDM-IV, and finally cultured in a rotating apparatus for 30 days in a medium containing composition MDM-V to obtain muscle cell spheroids derived from porcine gastrulation epiblast stem cells; wherein composition MDM-III includes HGF, IGF-1, FGF2, and LDN193189, composition MDM-IV includes IGF-1, and composition MDM-V includes HGF and IGF-1; composition C includes VEGF and FGF2; B8) Method for culturing 3D-vascular cell spheroids: Vascular endothelial progenitor cells prepared by M3 according to any one of claims 1-7 are cultured in a rotating device for 2 days in a medium containing composition VDM-II, and then cultured in the medium containing composition VDM-III until day 30 to obtain vascular cell spheroids derived from porcine gastrulation epiblast stem cells; wherein composition VDM-II comprises SB431542 and VEGF; B9) Method for culturing 3D-muscle / adipocyte spheroids: Adipocyte progenitor cells prepared by M1 according to any one of claims 1-7 and muscle progenitor cells prepared by M2 according to any one of claims 1-7 are mixed to obtain mixed cells. The mixed cells are cultured in a rotating device for 5 days in a medium containing composition MADM-I, then cultured in a rotating device for 9 days in a medium containing composition MADM-II, and finally cultured in a rotating device for 30 days in a medium containing composition MADM-III to obtain muscle-adipocyte aggregate spheroids. The composition MADM-I contains IGF-1, FGF2, and hEGF. The composition MADM-II contains IGF-1, hEGF, isobutylmethylxanthine, dexamethasone, and insulin.

9. A composition, wherein the composition is any one of the following: M1) A composition for preparing multi-tissue cell cultured meat, said composition being any of the following: M1-1) includes composition I, composition II and composition III as described in claim 3; M1-2) comprises the preparation of the adipocytes, muscle progenitors and vascular endothelial progenitors by the method of any one of claims 1-7; M1-3) is composed of M1-1) and M1-2); M2) A composition for culturing 2D-adipocyte progenitor cells, said composition comprising composition one, composition two and composition three as described in claim 6; M3) A composition for culturing 2D-adipocytes, said composition comprising any of the following: M3-1) includes composition one, composition two and composition three as described in claim 6; M3-2) includes adipocyte progenitor cells prepared according to any one of claims 1-7; M3-3) is composed of M3-1) and M3-2); M4) A composition for culturing 2D-vascular endothelial progenitor cells, said composition being composition A and composition B as described in claim 7; M5) A composition for culturing 2D-vascular type cells, said composition being any of the following: M5-1) includes composition one, composition two and composition three as described in claim 6; M5-2) includes vascular endothelial progenitor cells prepared from M3 as described in any one of claims 1-7; M5-3) is composed of M5-1) and M5-2); M6) A composition for 2D co-culture of muscle and fat, said composition being any of the following: M6-1) consists of adipocytes prepared by M1 as described in any one of claims 1-7, muscle progenitor cells prepared by M2 as described in any one of claims 1-7, ADM IV medium, and ADM V medium; M6-2) consists of adipocytes prepared by M1 as described in any one of claims 1-7, muscle progenitor cells prepared by M2 as described in any one of claims 1-7, a first mixed culture medium and a second mixed culture medium, wherein the first mixed culture medium is a mixture of ADM IV culture medium and MDM V culture medium in equal proportions, and the second mixed culture medium is a mixture of ADM V culture medium and N2 culture medium in equal proportions; M7) is a composition for culturing 3D-APC spheres, comprising adipocyte progenitor cells prepared by M1 according to any one of claims 1-7, ADM III medium, ADM IV medium, and ADM V medium; M8) is a composition for culturing 3D-MPCs spheres, consisting of muscle progenitor cells prepared by M2 according to any one of claims 1-7, MDM III medium, MDM IV medium, and MDM V medium; M9) is a composition for culturing 3D-VPCs spheres, comprising vascular endothelial progenitor cells prepared by M3 according to any one of claims 1-7, VDM II medium and VDM III medium; M10) is a composition for culturing 3D muscle / adipocyte spheroids, comprising adipocyte progenitor cells prepared by M1 according to any one of claims 1-7, muscle progenitor cells prepared by M2 according to any one of claims 1-7, MADM I medium, and MADM II medium.

10. A product, wherein the product is any one of the following: P1) Multi-tissue cell cultured meat prepared according to any one of claims 1-7; P2) The adipose progenitor cells prepared according to M1 as described in any one of claims 1-7; P3) The adipocytes prepared according to the method for culturing 2D-adipocytes in claim 8; P4) The vascular endothelial progenitor cells prepared according to M2 as described in any one of claims 1-7; P5) The vascular type cells prepared according to the method for culturing 2D-vascular type cells in claim 8; P6) The adipocyte spheroids obtained by the method for culturing 3D-APCs spheroids according to claim 8; P7) The muscle cell spheroids obtained by the method for culturing 3D-MPCs spheroids according to claim 8; P8) The vascular cell spheroids obtained by the method for culturing 3D-VPCs spheroids according to claim 8; P9) The muscle-fat aggregate spheres obtained by the method of culturing 3D-muscle / fat cell spheres according to claim 8.

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