Line establishment method of porcine formational pluripotent embryonic stem cells

Abstract

The invention discloses a line establishment method of porcine formation state pluripotent embryonic stem cells, and belongs to the technical field of cell biology. The invention aims to obtain a stable passage pig formation pluripotent embryonic stem cell line. The invention provides a line building method of a pig formation state pluripotent embryonic stem cell, which comprises the following steps of: inoculating a pig parthenogenetic activated blastocyst, a pig in-vitro fertilized blastocyst or a pig in-vivo blastocyst into a pFESCM culture medium on which feeder layer cells are paved in advance, replacing the culture medium once every two days until the diameter of a growth halo reaches 100-500 microns, blowing away the cells into a single cell or a small block mass with 3-4 cells, and culturing the small block mass in the form of the pig formation state pluripotent embryonic stem cell. The method comprises the following steps: inoculating the porcine embryonic stem cells into a pFESCM culture medium which is pre-paved with feeder layer cells, continuously culturing for 2-4 days to obtain first-generation porcine formational pluripotent embryonic stem cells, and carrying out passage to eight generations to obtain a stably inherited cell line. The prepared embryonic stem cells are used for breeding, gene editing, animal cloning, medical models, drug development carriers and induced production of reproductive gametes.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a method for establishing porcine morphologic pluripotent embryonic stem cells. Background Technology

[0002] Embryonic stem cells (ESCs) possess the ability to self-renew and proliferate indefinitely under in vitro culture conditions, while maintaining the potential for multi-lineage differentiation. Initially, ESCs were classified into two pluripotent states: the naïve state, represented by mouse ESCs (mESCs), and the primed state, represented by human ESCs (hESCs). The naïve state exhibits comprehensive differentiation capacity and germline chimerism potential, while the primed state has relatively limited differentiation capacity and chimerism contribution potential. During embryonic development, the transition between pluripotent states is gradual, and the division between the naïve and primed stages does not precisely correspond to specific stages of embryonic development. The formative state is considered part of the developmental continuity between the naïve and primed stages.

[0003] The research team has successfully constructed formative embryonic stem cell lines in horses and mice, demonstrating through experiments and multi-omics data analysis that these cell lines are indeed intermediate between naïve and primed, possessing certain characteristics of both. These cell lines can directly transform from a naïve state and transition directly to a primed state. Crucially, these cell lines exhibit highly efficient chimerism and can directly respond to BMP4 signaling, transforming into primordial germ cells. Furthermore, formative ESCs possess a certain inner cell mass (ICM) colonization capacity and contribute to late-stage embryonic chimeras. While significant progress has been made in mouse and human embryonic stem cell (ESC) research in recent decades, research on porcine embryonic stem cells faces major challenges. The pluripotency regulation mechanisms of porcine ESCs exhibit significant species differences in transcription factor networks, signaling pathway dependence, epigenetic status, and developmental stage specificity. Currently, researchers are dedicated to elucidating the mechanisms behind these differences, hoping to bring new breakthroughs to related research fields.

[0004] The isolation of porcine morphological stem cells (ESCs) relies on the precise regulation of specific signaling pathways (such as Wnt, FGF / Erk, and TGFβ) and is highly sensitive to culture conditions; traditional two-dimensional culture easily leads to cell differentiation or death. Although reported porcine ESCs can differentiate efficiently into the three germ layers, the cell types they contribute in in vivo chimerism experiments remain limited. The establishment of a novel "Formative" porcine embryonic stem cell culture system has significant theoretical implications for research on ESCs in pigs and other large animals, and also provides new research pathways for the development of stem cell-based functional products, genetic improvement of livestock, and the construction of human disease models. Summary of the Invention

[0005] The purpose of this invention is to obtain porcine morphological pluripotent embryonic stem cell lines that can be stably passaged.

[0006] This invention provides a method for establishing porcine morphologic pluripotent embryonic stem cells, the method of which is as follows: Step 1: Take porcine parthenogenetic activated blastocysts, porcine in vitro fertilized blastocysts, or porcine in vivo blastocysts and inoculate them into pFESCM medium pre-coated with feeder cells. Change the medium every two days until the growth halo diameter reaches 100-500 μm. Disperse the cells into single cells or small clumps of 3-4 cells and re-inoculate them into pFESCM medium pre-coated with feeder cells. Continue culturing for 2-4 days to obtain first-generation porcine morphological pluripotent embryonic stem cells. Step 2: The first-generation porcine morphological pluripotent embryonic stem cells obtained in Step 1 were cultured in pFESCM medium pre-coated with feeder cells. During the culture process, the pFESCM medium needed to be changed every 1-2 days. When the cell colony reached a diameter of 100 µm, the upper culture medium was removed, and the cells were digested with cell digestion solution for 3 minutes. Before the end of the digestion process, the cells were gently pipetted 3-4 times. Then, the digestion reaction was terminated with an equal volume of pFESCM medium. The cells were pipetted into a single-cell suspension, centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, the cell pellet was resuspended in culture medium, and seeded into a new culture dish pre-coated with feeder cells at a passage ratio of 1:2 to 1:5 to obtain the second-generation morphological pluripotent embryonic stem cells. Step 3: Repeat step 3 six times to obtain eighth-generation morphological pluripotent embryonic stem cells.

[0007] To further define, porcine parthenogenetic activated blastocysts are porcine blastocysts that have developed in vitro for 5.5 to 7.5 days.

[0008] To further define, porcine in vitro fertilization blastocysts are porcine blastocysts that have developed in vitro for 5.5 to 7.5 days.

[0009] To further define, the in vivo blastocyst is a porcine blastocyst that has developed in vivo for 4.5 to 8.5 days.

[0010] Further specify the method for pre-laying feeder cells in pFESCM medium: Inoculate the prepared feeder cells into a four-well plate containing 15% FBS DMEM medium, with a density of 85-90% of the bottom area of ​​the well. On the day of inoculation of parthenogenetic blastocysts, replace the 15% FBS DMEM medium for the feeder cells with 300 μl of pFESCM medium in advance.

[0011] Further specifying, the mouse strains used to prepare feeder cells are ICR, CD-1, or C57 strains.

[0012] Further specify the components of the pFESCM culture medium: 200-300 μL N2 (50×), 400-600 μL B27 (100×), 10-20 mg L-glutamine, 400-600 μL NEAA, 100 μL β-mercaptoethanol (100×), 500 μL penicillin-antibody, 25 µg / mL sodium pyruvate, 50 μL LC, 50 μL human LIF, 15 μL CHIR 99021, 10 μL Activin A, 15 μL IGF-2, 2.5 μL SAG, 5 µL DOR, 2.5 μL SB590885, and 7.8 μL XAV939. KO-DMEM was then added to bring the total volume to 50 mL.

[0013] Further restrictions were placed on the culture environment, which was 37°C, 5% CO2, and saturated humidity.

[0014] This invention provides a culture medium for culturing porcine morphological pluripotent embryonic stem cells. The culture medium contains 200-300 μL of 50×N2, 400-600 μL of 100×B27, 10-20 mg of L-glutamine, 400-600 μL of NEAA, 100 μL of 100×β-mercaptoethanol, 500 μL of penicillin-3 antibody, 25 µg / mL of sodium pyruvate, 50 μL of LC, 50 μL of human LIF, 15 μL of CHIR 99021, 10 μL of Activin A, 15 μL of IGF-2, 2.5 μL of SAG, 5 µL of DOR, 2.5 μL of SB590885, and 7.8 μL of XAV939, and is supplemented with KO-DMEM to a total volume of 50 mL.

[0015] Furthermore, the culture medium also contains mouse fibroblasts, and the density of feeder cells prepared from mouse fibroblasts accounts for 85-90% of the bottom area of ​​the well.

[0016] Beneficial effects: (1) The line-building system proposed in this invention can efficiently isolate and induce porcine formative pluripotent embryonic stem cell lines (pFESCs) from pre-implantation porcine embryos. These cell lines can be stably passaged for more than 80 generations in vitro.

[0017] (2) pFESCs exhibit significant potential for spontaneous and induced differentiation in vitro, and can spontaneously differentiate and express marker genes of embryonic cells. Under specific induction systems, pFESCs can be directed to differentiate into three germ layer-like cells.

[0018] (3) A significant feature of pFESCs is their ability to be cultured in suspension. Under suspension culture conditions, pFESCs not only maintain the expression of pluripotency marker genes, but also express the marker genes of each germ layer cell. In this state, pFESCs are ready to enter the differentiation process.

[0019] (4) After suspension culture, pFESCs can directly induce PGCLC expressing PGC marker genes without the need for any intermediate cells (such as epiblast-like cells or mesodermal precursor cells) to transition. Attached Figure Description

[0020] Figure 1 This is the clonal morphology of pFESCs embryonic stem cells described in this invention; Figure 2 These are images of alkaline phosphatase (AP) staining of pFESCs embryonic stem cells as described in this invention; Figure 3 These are karyotype images of pFESCs embryonic stem cells described in this invention; Figure 4 This involves using real-time quantitative polymerase chain reaction (RT-qPCR) technology to detect the expression of key genes in the WNT, TGFβ, and FGF / ERK signaling pathways of pFESCs embryonic stem cells. Figure 5 The heatmap analysis shows the gene expression of key nodes in the FGF / ERK, TGFβ / SMAD, and WNT-βcatenin signaling pathways in pFESCs embryonic stem cells. Figure 6 The results of spontaneous differentiation of pFESCs embryonic stem cells showed that they expressed markers of the three germ layers and trophectoderm markers by immunofluorescence. Figure 7 These are images showing the spherical morphology of pFESCs embryonic stem cells after suspension culture, as described in this invention. Figure 8It uses real-time quantitative polymerase chain reaction (RT-qPCR) technology to detect the expression of marker genes in pFESCs-induced primordial germ-like cells; Figure 9 The method involves using immunofluorescence staining to detect the colocalization of SOX2 and E-Cadherin with the red fluorescence of pFESCs in chimeric blastocysts. Figure 10 These are images showing the morphological differentiation of pFESCs embryonic stem cells into myofibroblast-like cells as described in this invention. Figure 11 The immunofluorescence staining technique was used to detect myogenic markers in pFESC-MCs cells; Figure 12 The real-time quantitative polymerase chain reaction (RT-qPCR) technique is used to detect the expression of marker genes in pFESCs-induced muscle fibrous cells; Figure 13 The immunofluorescence staining technique is used to detect the markers for the directional differentiation of pFESCs embryonic stem cells into the three germ layers; Detailed Implementation Example 1. Preparation of pFESCM medium The preparation method for 50 mL of pFESCM culture medium is as follows: First, add 200-300 μL of N2 (50×, Gibco 17502-048), 400-600 μL of B27 (100×, Gibco17504-044), 10-20 mg of L-glutamine (Sigma G8540-100G), 400-600 μL of NEAA (Invitrogen11140-050), 100 μL of β-mercaptoethanol (100×), 500 μL of penicillin-dextrin antibiotics, 25 µg / mL of sodium pyruvate, 50 μL of LC (Gibco 11905-031), 50 μL of human LIF (Millipore LIF 1010), 15 μL of CHIR 99021 (Stemgent04-0004), and 10 μL of Activin. The following solutions were added: A (R&D 338-AC-10), 15 μL of IGF-2 (R&D 292-G2-050), 2.5 μL of SAG (MCEHY-12848), 5 μL of DOR (MCEHY-13418A), 2.5 μL of SB590885 (R&D 2650), and 7.8 μL of XAV939 (Sigma X3004). Finally, KO-DMEM (Gibco10829-018) was used to bring the total volume to 50 mL.

[0021] Example 2. Preparation and culture of feeder cells (1) Primary culture of CD-1 mouse fetal fibroblasts: Pregnant CD-1 mice that had been naturally mated for 13.5 days were euthanized by cervical dislocation. The mouse fur and experimental instruments were then strictly disinfected with 75% alcohol. Next, a fenestration was performed on the abdomen of the pregnant mice to obtain the uterus. The obtained uterus was placed in a saline environment containing antibiotics. The uterus was repeatedly rinsed with saline in a 10 cm low-adhesion culture dish until no blood was visible in the rinsing solution. During rinsing, the saline containing antibiotics was changed frequently. Then, the uterine wall was dissected with forceps, and the complete fetal mouse, including the placenta and fetal membranes, was removed. The fetal mouse was washed in saline containing antibiotics (DPBS) to remove excess blood vessels until the washing solution was colorless. Next, the placenta and fetal membranes were removed with forceps, and the fetus was detached into a large dish containing antibiotic saline (DPBS). During this process, it was ensured that the extraembryonic tissue did not come into contact with the fresh saline. After simple washing, the head, limbs, tail, and organs of the fetal mice were dissected with forceps, leaving only the spine. The spine was washed 2-3 times with DPBS, then placed in a new high-adhesion culture dish and minced to ensure the tissue pieces were approximately 1 cubic millimeter in size. The minced tissue pieces were digested for 5 minutes with 1.6 mg / mL collagenase IV containing Dnase I, followed by stopping the digestion with twice the volume of 15% FBS culture medium. After repeated pipetting using a 1000 µL pipette tip, the suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1500 rpm / min for 5 minutes. After centrifugation, the suspended cells and tissue pieces were resuspended in fresh 15% FBS culture medium and seeded into high-adhesion culture dishes. The culture dishes were then placed in a 37°C CO2 incubator, with the culture medium changed every other day. After 2 days, when the cell density reached over 90%, the cells were passaged. At passage P3, feeder cells were created.

[0022] (2) Preparation of CD-1 mouse feeder cells: For P3 generation mouse fetal fibroblasts with a density of 90%, treatment with mitomycin C at a final concentration of 10 µg / mL was performed for 2–3 hours. After treatment, the cells were washed three times with DPBS to completely remove mitomycin C. Then, 0.25% trypsin was added for digestion for 1–2 minutes until 50% of the cells detached from the bottom of the dish. At this point, twice the amount of culture medium was added to terminate the digestion. The digested cell suspension was centrifuged (1500 rpm / min, 3 minutes), the supernatant was discarded, and an appropriate amount of cryopreservation buffer (taken from 4°C) was added at a rate of 1 × 10⁻⁶ cells per tube. 6The cryovials were frozen at the specified density. The cryovials were placed in cryovial boxes, and after the experiment, the cryovial boxes were stored in a -80°C freezer (if no cryovial box was available for the experiment, the cryovials were first placed in a 4°C environment for 20 minutes, and then placed in a -20°C environment for 2 hours).

[0023] This method can efficiently obtain feeder cells that support stable culture of porcine embryonic stem cells.

[0024] Example 3. Establishment and passage of porcine morphological pluripotent stem cell lines derived from parthenogenetic activated blastocysts I. Activating the blastocyst using parthenogenesis at 7.5 days 1. pFESCM medium pre-seeded with feeder cells: One day in advance, seed the prepared feeder cells (the feeder cells obtained in Example 2) into four-well plates containing 15% FBS in DMEM medium, with a density of 85-90% of the bottom area of ​​the wells. On the day of seeding parthenogenetic blastocyst activation, replace the 15% FBS DMEM medium for the feeder cells with 300 μl pFESCM medium in advance.

[0025] 2. Take parthenogenetic activated blastocysts cultured for 7.5 days. In preheated embryo manipulation medium (37°C), repeatedly blow and aspirate the zona pellucida using a pipette with an unburned tip slightly smaller than the embryo's diameter to remove the zona pellucida. On day 1 post-inoculation, add 200 μl of pFESCM medium to the wells containing the blastocysts. Starting on day 2 post-inoculation, discard the original culture medium from the wells and replace it with 500 μl of pFESCM medium until the growth halo appears.

[0026] Once the growth halo reaches a diameter of 300 μm, its size will affect the efficiency of subsequent cell line establishment. If it is too large or too small, the efficiency of cell line establishment will be reduced. The halo is picked up with a glass needle, digested in Accutase enzyme drops, and blown back and forth with a pipette to break it into single cells or small clumps of 3-4 cells. The cells are then transferred to pFESCM medium pre-coated with feeder cells to obtain P1 generation porcine morphological pluripotent stem cells.

[0027] 3. Under a culture environment of 37℃, 5% CO2, and saturated humidity, the P1 cells described above can be cultured in pFESCM medium containing feeder cells. MEF cells were used as the feeder cells, with a density covering 90% of the bottom area of ​​the wells. The pFESCM medium needed to be changed every day during the culture process. When the cell colony reached a diameter of 100 µm, the supernatant was removed, and the cells were digested with Accutase for 3 minutes. Before the digestion process was completed, the cells were gently pipetted 4 times, and then the digestion reaction was terminated with an equal volume of pFESCM medium. Next, the cells were pipetted into a single-cell suspension, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The cell pellet was then resuspended in culture medium and seeded into new culture dishes at an appropriate cell density. This generation of cells was labeled P2. This cell line was passaged to the P8 generation (step 3 was repeated 6 times to obtain the P8 generation), forming a stable porcine morphological embryonic stem cell line that maintained a stable clonal morphology during further passages. Figure 1 ).

[0028] II. Establishment and passage of porcine morphological pluripotent stem cell lines derived from blastocysts in vivo MEF feeder cells were precultured in four-well plates to a cell density of approximately 90% of the well bottom area. On the day of inoculation, 10% FBS in the feeder cell culture medium was replaced with 300 μl of pFESCM.

[0029] A blastocyst on day 5 was selected, and its inner cell mass (ICM) was isolated. The method for ICM isolation included: a) injecting 0.05% trypsin into the blastocyst using micromanipulation techniques to detach the ICM; b) repeatedly blowing and aspirating the zona pellucida using an unburnt pipette with a diameter slightly smaller than the embryo in a preheated MAN at 37°C, and then separating the ICM using a glass needle with a heated tip. The isolated ICM was then transferred to pEPSCM medium pre-coated with feeder cells.

[0030] When the growth halo reaches 200 μm in diameter, it is picked up with a glass needle, digested in Accutase drops, and then repeatedly blown and aspirated with a pipette to break down the growth halo into single cells or small clusters of 3-4 cells. The cells are then transferred to pFESCM culture medium containing feeder cells for further culture to obtain P1 generation porcine morphological pluripotent stem cells. This cell line can be passaged to P8 generation to form a stable porcine morphological embryonic stem cell line.

[0031] Example 4. Alkaline phosphatase staining (AP) of porcine morphologic pluripotent stem cell lines Porcine morphological embryonic stem cells (pFESCs) in good adherent culture were washed three times with DPBS. Fixation was performed with 4% (w / v) paraformaldehyde (PFA) at room temperature for 10 minutes. After removing the PFA, the adherent cells were washed three times with DPBS. Staining was performed according to the instructions of the BCIP / NBT alkaline phosphatase chromogenic kit. Incubation was carried out at 37°C in the dark for 5 to 30 minutes, or longer, until the desired staining depth was achieved. After washing three times with DPBS, the samples were stored in DPBS solution and photographed under an upright microscope. AP staining was performed on pFESCs of passage P40 obtained from 7.5-day parthenogenetic activated blastocysts. Under these conditions, AP staining was strongly positive, and almost all clones showed staining. Figure 2 ).

[0032] Analysis: High AP expression is a typical characteristic of undifferentiated pluripotent stem cells, indicating that the cells have the potential to differentiate into multiple cell types. It also suggests that the established pFESCs proliferate actively and have strong self-renewal capacity.

[0033] Example 5. Karyotype analysis of porcine morphological pluripotent stem cells pFESCs were treated with colchicine at a concentration of 0.1 mg / mL for 6 hours; cell clones were digested into single-cell states using TrypLE™ Express enzyme, and the cells were placed on 0.1% gelatin for 8 minutes to remove feeder cells; then centrifuged and incubated at 37°C for 20 minutes with the addition of hypotonic KCl solution; the supernatant was discarded after centrifugation; the cells were treated on ice for 40 minutes with pre-chilled fixative (methanol and glacial acetic acid in a 3:1 ratio), centrifuged and the supernatant was discarded, and this step was repeated twice; the cells were resuspended in 200 μL of fixative and dropped onto slides; Giemsa staining was applied for 7 minutes, and chromosome morphology was observed under a microscope.

[0034] Results: Karyotype analysis of pFESCs cells from P80 generation obtained by parthenogenetic activation of blastocysts at 7.5 days showed that the cells had a normal karyotype and contained 38 chromosomes. Figure 3 ).

[0035] Example 6. Detection of gene expression at key nodes of the WNT, TGFβ, and FGF / ERK signaling pathways in porcine pluripotent stem cells using real-time quantitative polymerase chain reaction (RT-qPCR). 1. Extraction of total RNA from cells (using an RNA extraction kit, Invitrogen 12183018A) Collect stably cultured pFESCs cells (P70 generation): Collect cell samples with the feeder layer removed into 1.5 ml EP tubes, wash twice with DPBS, and aspirate as much residual liquid as possible.

[0036] Add 350 μL of Lysis buffer to the precipitate and vortex for 2 min.

[0037] Then add 350 μL of 75% ethanol (prepared fresh for use), mix, add to the column, and centrifuge at 12000 g / min for 20 s.

[0038] After centrifugation, add 350 μL of Wash buffer I and centrifuge at 12000 g / min for 20 s using a four-degree centrifuge.

[0039] Add 80 μL of DNase enzyme (dissolve 1500 U of DNase I dry powder in 550 μL of RNase-FreeddH2O, mix gently, aliquot and store at -20℃), and incubate in the dark for 15 min.

[0040] Add 350 μL Wash buffer I and centrifuge at 12000 g / min for 20 s using a four-degree centrifuge.

[0041] Add 500 μL Wash buffer II and centrifuge at 12000 g / min for 20 s. This step needs to be repeated once.

[0042] Replace the bottom column and centrifuge at 12000 g / min for 2 min in a four-degree centrifuge.

[0043] After standing for 1 min, when no alcohol odor was detected, the sample was eluted with 30 μL ddH2O and centrifuged at 12000 g / min for 2 min using a four-degree centrifuge. The concentration was then measured, and the sample was stored at -80℃.

[0044] 2. Reverse transcription of RNA (using a reverse transcription kit, ABI4368814) The total volume of the reverse transcription system is 20 μL. Table 1

[0045] Reverse transcription reaction program: a) 25℃ for 10 min; b) 37℃ for 120 min; c) 85℃ for 5 s; d) 4℃ for 10 min. 3. Real-time quantitative PCR (using the Real-Time kit, Takara DRR066A) The total volume of the real-time quantitative PCR reaction system is 25 μL. Table 2

[0046] Real-time quantitative PCR reaction procedure: three-step method, 40 cycles in total: a) 95℃ pre-denaturation for 1 min; b) 95℃ denaturation for 30 s; c) 55℃ annealing for 30 s; d) 72℃ extension for 30 s. Each sample test needs to be repeated three times, and the data analysis is performed using the 2-ΔΔCt relative quantitative analysis method.

[0047] The research results indicate that ( Figure 4 The stable maintenance of porcine primordial germ cell (pFESCs) status depends on the precise regulation of signaling pathways such as Wnt, FGF / ERK, and TGFβ. Analysis using real-time quantitative PCR revealed that, compared to control cells, the Wnt signaling pathway... GSK3β, CTNNB1 and TCF3 Genes in the FGF / ERK signaling pathway FGFR1, RAF and ERK Genes, and in the TGFβ signaling pathway SMAD1, SMAD2 and TGFβ All genes showed significantly high expression levels. This result reveals that pFESCs possess the characteristics of maintaining the state of porcine primordial germ cells in terms of signaling pathway expression.

[0048] To further verify the expression status of the above signaling pathways in the pFESC cell line, the expression of key genes in the signaling pathways in pFESC was analyzed based on previous transcriptome sequencing data, and presented in the form of a heatmap. Figure 5 Key genes in the WNT / β-catenin pathway, such as TCF3, FZD7, and SRC, were highly expressed in pFESCs, indicating that this pathway is active in cells. Genes in the FGF / ERK pathway, such as SRC, ERBB3, and MAPK1, were also upregulated. Several core regulators of the TGFβ / BMP / SMAD pathway, including SMAD1, SMAD2, SMAD4, and SMAD7, also showed significant high expression in pFESCs. These results indicate that pFESCs simultaneously exhibit active expression of multiple key signaling pathways, including WNT, FGF / ERK, and TGFβ, providing an important molecular basis for the establishment and maintenance of their morphological pluripotency.

[0049] Example 7. Formation and spontaneous differentiation of porcine morphological pluripotent stem cells into embryoid bodies and their detection and analysis. pFESCs cells (passage P30) cultured on feeder cells were digested with 0.05% trypsin until they dissociated into single cells. The cells were then transferred to matrix-free medium containing 15% fetal bovine serum (FBS). Embryo bodies (EBs) were observed to form after 4–7 days of culture, and samples were collected to detect the expression of three germ layer markers. EBs were seeded into gelatin-treated 24-well plates, and the medium was changed daily with fresh medium containing 15% FBS. Immunofluorescence was performed on day 7 post-seeding to assess the spontaneous differentiation of EBs.

[0050] Results: pFESCs exhibited highly efficient embryoid body (EB) formation ability in vitro, characterized by a solid spherical structure. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed that EBs showed significantly upregulated expression levels of the three germ layer marker genes compared to pFESCs cells. When EBs were cultured adherently, their cell proliferation rate was significantly increased, and during spontaneous differentiation, they expressed not only three germ layer-related markers but also trophectoderm markers (see...). Figure 6 ).

[0051] Example 8. Suspension culture of porcine morphologic pluripotent stem cells and induction into primordial germ-like cells (PGCLC) Remove the supernatant from pFESCs (passage P30) stably adherent to the feeder cells and digest them with Accutase for 3 minutes. Near the end of digestion, gently pipette four times, then terminate the digestion with an equal volume of pFESCM medium. Next, pipette the cells into a single-cell suspension, centrifuge at 1200 rpm for 3 minutes, and discard the supernatant. Resuspend the cell pellet in culture medium and seed at an appropriate cell density into non-adhesive culture dishes without feeder cells for suspension culture. Replace the pFESCM medium daily using a half-change method.

[0052] Results: pFESCs cells were able to undergo suspension culture under conditions without a feeder layer and without attachment. After suspension culture, the cells spontaneously aggregated into smooth, spherical structures. Figure 7 Suspension-cultured pFESCs cells can be directly induced into primordial germ-like cells in primordial germ cell induction medium. RT-qPCR results showed that, compared with pFESCs cells, pFESCs-PGCLC contained more PGC marker genes. BLIMP1, NANOS3, TFAP2C, STELLA, DND1 and PRDM14 The expression of all of them was significantly increased ( Figure 8 ) Example 9. Chimerism experiment of porcine morphologic pluripotent stem cells in early blastocysts First, cells (passage P20) were labeled with a red fluorescent dye (Texas Red), and then the labeled cells were injected into porcine morula embryos, with eight cells injected into each embryo. Three days later, a significant increase in the number of red fluorescent cells in the blastocysts was observed (see...). Figure 9 Immunofluorescence assays showed that red fluorescence co-localized with the inner cell mass (ICM) marker gene SOX2 and the intercellular matrix E-Cadherin of pluripotent cells.

[0053] The results showed that pFESCs cells could proliferate moderately in early pig embryos and exhibited a relatively wide distribution pattern.

[0054] Example 10. Study on the induction of porcine morphological pluripotent stem cells into muscle cell-like cells Remove the supernatant from pFESCs stably adhered to the feeder layer and digest them with Accutase for 3 minutes. Near the end of digestion, gently pipette several times to fully dissociate the cells into single cells, and terminate the reaction with an equal volume of culture medium. Subsequently, centrifuge the cells at 1200 rpm for 3 minutes, discard the supernatant, resuspend the cells, and seed them at an appropriate density into gelatin-coated plates. Culture in pFESC basal medium containing Y27632 for 24 hours. The medium is then replaced with muscle-inducing medium the following day, and the cells are cultured for 25 days, with fresh medium every 2 days.

[0055] Results: In the optimized induction system, pFESCs were able to adhere stably and gradually form slender muscle fiber-like cells. Figure 10 Immunofluorescence assays showed that key myogenic regulatory factors MYOD, MYOG, and MYH1, as well as the muscle stem cell-related marker PAX7, were detected in pFESC-MCs cells. Figure 11 RT-qPCR results showed that ( Figure 12 After differentiation, the expression levels of muscle lineage-related genes such as MYOD, MYOG, MYH1 and PAX7 in the cells were significantly higher than those in the undifferentiated pFESCs, indicating that pFESCs have the potential to differentiate into muscle lineages.

[0056] Example 11. Study on the induction of porcine morphologic pluripotent stem cells into three germ layers The supernatant was removed from pFESCs stably adherent to the feeder layer. Cells were digested with Accutase to detach into single cells. After digestion, the cells were centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and the cells were resuspended and seeded at an appropriate density into gelatin-coated plates. The plates were cultured in pFESC basal medium for 36 hours, followed by differentiation culture using ectoderm, mesoderm, and endoderm induction media, respectively.

[0057] Results: Under the respective induction conditions, pFESCs could effectively differentiate into cells of all three germ layers. Immunofluorescence results showed that after induction, the cells were positively expressed for ectoderm marker protein β-tublin, mesodermal marker protein T (Brachyury protein), and endoderm marker protein GATA6. Figure 13 This indicates that pFESCs possess good trilaminar differentiation potential, consistent with the characteristics of morphological pluripotent stem cells.

Claims

1. A method for establishing porcine morphologic pluripotent embryonic stem cells, characterized in that, The method for establishing the system is as follows: Step 1: Take porcine parthenogenetic activated blastocysts, porcine in vitro fertilized blastocysts, or porcine in vivo blastocysts and inoculate them into pFESCM medium pre-coated with feeder cells. Change the medium every two days until the growth halo diameter reaches 100-500 μm. Disperse the cells into single cells or small clumps of 3-4 cells and re-inoculate them into pFESCM medium pre-coated with feeder cells. Continue culturing for 2-4 days to obtain first-generation porcine morphological pluripotent embryonic stem cells. Step 2: The first-generation porcine morphological pluripotent embryonic stem cells obtained in Step 1 were cultured in pFESCM medium pre-coated with feeder cells. During the culture process, the pFESCM medium needed to be changed every 1-2 days. When the cell colony reached a diameter of 100 µm, the upper culture medium was removed, and the cells were digested with cell digestion solution for 3 minutes. Before the end of the digestion process, the cells were gently pipetted 3-4 times. Then, the digestion reaction was terminated with an equal volume of pFESCM medium. The cells were pipetted into a single-cell suspension, centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, the cell pellet was resuspended in culture medium, and seeded into a new culture dish pre-coated with feeder cells at a passage ratio of 1:2 to 1:5 to obtain the second-generation morphological pluripotent embryonic stem cells. Step 3: Repeat step 3 six times to obtain eighth-generation morphological pluripotent embryonic stem cells.

2. The method for establishing a system according to claim 1, characterized in that, The porcine parthenogenetic activated blastocyst is a porcine blastocyst that has developed in vitro for 5.5 to 7.5 days.

3. The method for establishing a system according to claim 1, characterized in that, Porcine in vitro fertilization blastocysts are porcine blastocysts that have developed in vitro for 5.5 to 7.5 days.

4. The method for establishing a system according to claim 1, characterized in that, The in vivo blastocyst is a porcine blastocyst that has developed in vivo for 4.5 to 8.5 days.

5. The method for establishing a system according to claim 1, characterized in that, Method for pre-laying feeder cells in pFESCM medium: Inoculate the prepared feeder cells into a four-well plate containing 15% FBS DMEM medium, with a density of 85-90% of the bottom area of ​​the well. On the day of inoculation of parthenogenetic blastocysts, replace the 15% FBS DMEM medium for the feeder cells with 300 μl of pFESCM medium in advance.

6. The method for establishing a system according to claim 1, characterized in that, The mouse strains used to prepare feeder cells are ICR, CD-1, or C57 strains.

7. The method for establishing a system according to claim 1, characterized in that, The pFESCM culture medium consisted of 200–300 μL of N2 (50×), 400–600 μL of B27 (100×), 10–20 mg of L-glutamine, 400–600 μL of NEAA, 100 μL of β-mercaptoethanol (100×), 500 μL of penicillin-dextrose antibody, 25 µg / mL of sodium pyruvate, 50 μL of LC, 50 μL of human LIF, 15 μL of CHIR 99021, 10 μL of Activin A, 15 μL of IGF-2, 2.5 μL of SAG, 5 µL of DOR, 2.5 μL of SB590885, and 7.8 μL of XAV939. KO-DMEM was then added to bring the total volume to 50 mL.

8. The method for establishing a system according to claim 1, characterized in that, The culture environment was 37℃, 5% CO2 and saturated humidity.

9. A culture medium for culturing porcine morphologic pluripotent embryonic stem cells, characterized in that, The culture medium contained 200–300 μL of 50×N2, 400–600 μL of 100×B27, 10–20 mg of L-glutamine, 400–600 μL of NEAA, 100 μL of 100×β-mercaptoethanol, 500 μL of penicillin-3 antibody, 25 µg / mL of sodium pyruvate, 50 μL of LC, 50 μL of human LIF, 15 μL of CHIR 99021, 10 μL of Activin A, 15 μL of IGF-2, 2.5 μL of SAG, 5 µL of DOR, 2.5 μL of SB590885, and 7.8 μL of XAV939, and was supplemented with KO-DMEM to a total volume of 50 mL.

10. The culture medium according to claim 9, characterized in that, The culture medium also contains mouse fibroblasts, and the density of the feeder layer cells prepared from mouse fibroblasts accounts for 85-90% of the bottom area of ​​the well.

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