Complete feeder-free long-term culture system of bovine embryonic stem cells and its application

Abstract

This invention relates to the field of bovine embryonic stem cell culture technology, and particularly to a long-term culture system for morphological bovine embryonic stem cells completely without a feeder layer and its applications. After trying various culture methods, this invention establishes a feeder-free bovine morphological embryonic stem cell line (FF-bESCs). Bovine morphological embryonic stem cells cultured under the feeder-free conditions provided by this invention can maintain stable proliferation capacity and pluripotency over a long period. The bovine morphological embryonic stem cells provided by this invention can undergo gene editing and can be effectively induced to differentiate into primordial germ cell-like cells (PGCLCs). This invention lays an important foundation for the standardized and large-scale culture of bovine embryonic stem cells under feeder-free conditions, and for their application in stem cell breeding and other fields.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture technology, and in particular to a long-term culture system for bovine morphological embryonic stem cells without a feeder layer and its application. Background Technology

[0002] Embryonic stem cells (ESCs) originate from the inner cell mass (ICM) of the pre-implantation embryo and possess the ability to differentiate into all somatic cell lineages. The mouse ESC line was first established in 1981, and the human ESC line was successfully obtained in 1998. Mouse ESCs are in a primordial state (na). Human ESCs are in a pluripotent state (ve) while they are in a primed pluripotent state. In 2017, Austin Smith proposed a formative pluripotent state, which is intermediate between the two, and mouse, human, and horse ESCs with this characteristic have been successfully established in recent years. These cells possess both natriuretic and idioblastic properties. It possesses some molecular characteristics of ve and primed, and has the dual potential to form chimeras and efficiently induce primordial germ cells (PGCs).

[0003] Stable livestock embryonic stem cells are ideal cell sources for gene modification, cloning animal nuclear donors, cultured meat, and stem cell breeding. As an important livestock species, cattle suffer from a lack of stable and definitively pluripotent embryonic stem cells, coupled with a long reproductive cycle, hindering progress in stem cell breeding research. Establishing bovine embryonic stem cell lines with stable passage capacity and heritable manipulation capabilities is of significant scientific importance and practical value for overcoming technical bottlenecks in bovine genetic improvement, promoting the industrialization of cultured meat, and refining the theoretical framework of livestock pluripotency.

[0004] Conventional culture of embryonic stem cells relies on feeder layers such as mouse embryonic fibroblasts (MEFs), but this method suffers from problems such as cumbersome preparation, unclear composition, and large batch-to-batch variations, resulting in poor reproducibility and difficulty in standardization. Furthermore, the process of removing the feeder layer before downstream application is inefficient and may damage stem cells. Therefore, feeder-free culture systems have become a research hotspot. Currently, a long-term, stable, and functionally complete feeder-free culture method suitable for bovine morphological embryonic stem cells remains a bottleneck that urgently needs to be addressed in this field. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a completely feeder-free long-term culture system for bovine morphological embryonic stem cells (BESCs) and its applications. The transition from feeder-dependent systems to feeder-free systems with clearly defined chemical compositions has become an inevitable trend in pluripotent stem cell research and translational applications. This invention explores its feeder-free culture system and its applications, establishing a feeder-free bovine morphological embryonic stem cell line (FF-bESCs). FF-bESCs can be cultured long-term under feeder-free conditions while maintaining stable proliferation capacity and pluripotency. FF-bESCs can be gene-edited and effectively induced to differentiate into primordial germ cell-like cells (PGCLCs).

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a long-term culture system for morphological bovine embryonic stem cells without a feeder layer, comprising morphological embryonic stem cell culture medium and matrix proteins; The matrix protein includes Matrigel or Geltrex.

[0007] Preferably, the morphological embryonic stem cell culture medium comprises the following components: Neurobasal at a volume percentage of 48.00%, DMEM / F12 at a volume percentage of 48.00%, 0.1 mM 2-mercaptoethanol, 1 μM AR-A014418, 0.3 μM A-770041, 5 μM MSC2504877, and 50 μg / mL vitamin C.

[0008] Preferably, the volume ratio of the bEPSC culture medium to the matrix protein is 100:1~10.

[0009] Preferably, the volume ratio of the bEPSC culture medium to the matrix protein is 20:1.

[0010] The present invention also provides the application of the bovine morphological embryonic stem cell long-term culture system without feeder layer described above in supporting the stable expansion of bovine embryonic stem cells without feeder layer.

[0011] Preferably, the number of generations of amplification exceeds 60.

[0012] The present invention also provides the application of the bovine morphological embryonic stem cell long-term culture system without feeder layer described above in the culture of feeder-free bovine embryonic stem cells to achieve pluripotency.

[0013] Preferably, the pluripotency includes the formation of embryoid bodies in vitro and the generation of teratomas containing derivatives of three germ layers in vivo.

[0014] This invention also provides the long-term culture system of bovine morphological embryonic stem cells without feeder layer as described in the above technical solution, enabling the application of feeder-free bovine embryonic stem cells in stem cell breeding.

[0015] Preferably, it causes bovine embryonic stem cells without a feeder layer to differentiate into primitive germ cell-like cells.

[0016] The beneficial effects of this invention are: Bovine embryonic stem cells are a key cell source for the development of stem cell-cultured meat and the directed differentiation of germ cells in stem cell breeding. This invention develops a simplified feeder-free culture system: based on commercially available basal culture medium and combined with a Matrigel-simulated 3D microenvironment, a novel feeder-free bovine morphological embryonic stem cell line (FF-bESCs) has been successfully derived. This system has the following significant advantages: (1) Simple operation and stable amplification: FF-bESCs can be stably amplified for a long time without the need for ROCK inhibitors, and can be passaged for more than 60 generations while maintaining normal karyotype and high proliferative activity.

[0017] (2) Pluripotency is well maintained: FF-bESCs stably express core pluripotency markers; functional verification shows that they have true pluripotency, including the ability to form embryoid bodies in vitro and generate teratomas containing three germ layer derivatives in vivo.

[0018] (3) Clear pluripotency status: Transcriptome analysis confirmed that FF-bESCs are in a formative pluripotent state, possessing both na and omega-3 pluripotency. Some molecular characteristics of the ve and primed states.

[0019] (4) Excellent potential for directional differentiation: FF-bESCs can be efficiently induced to differentiate into primitive germ cell-like cells (PGCLCs), providing a reliable cell source for in vitro differentiation of germ cells.

[0020] The feeder-free culture system of this invention is easy to operate, has clearly defined components, and is highly reproducible. It lays a scalable technical foundation for promoting the large-scale application of bovine embryonic stem cells, including precise bovine genome editing, cultured meat production, and stem cell breeding based on in vitro gamete differentiation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1The clonal morphology and survival of bovine embryonic stem cells under BFF feeder layer and different feeder-free culture conditions; (A) BFF feeder layer; (B) Fibronectin; (C) Matrigel; (D) bEPSCM + Matrigel. bEPSCs were resuspended in bEPSCM containing low, medium and high concentrations of Matrigel and directly seeded into uncoated culture dishes; matrix proteins were diluted with pre-cooled DPBS or bEPSCM to three concentrations: low (1%, 1:100 dilution), medium (5%, 1:20 dilution), and high (10%, 1:10 dilution). Figure 2 For the long-term stable amplification detection of FF-bESCs; (A) Clonal morphology and alkaline phosphatase (AP) staining results of FF-bESCs during long-term passage; (B) Clonal morphology and AP staining results of FF-bESCs after being seeded into BFF or MEF feeder layers; (C) Doubling time of FF-bESCs; (D) Single-cell clonal formation efficiency of FF-bESCs; (H) Karyotype identification of FF-bESCs. Figure 3 (AC) Expression characteristics of pluripotency-related proteins and genes in FF-bESCs; (D) Immunofluorescence staining results of core pluripotency factors SOX2, OCT4, and NANOG in FF-bESCs; (E) Detection of core pluripotency genes and NANOG by RT-qPCR. mRNA expression levels of ve pluripotency genes and early differentiation marker genes of the three germ layers; Figure 4 (A) Schematic diagram of EB formation induced by FF-bESCs; (B) Morphological characteristics of EB in suspension culture and adherent culture stages; (C) Immunofluorescence staining to detect the expression of three germ layer markers in EB differentiated cells: ectoderm (β-III tubulin), endoderm (AFP), and mesoderm (SMA); (D) Morphology of teratomas (top) and formation efficiency statistics (bottom); (E) Immunofluorescence staining to detect the expression of three germ layer specific markers in teratoma tissue; Figure 5To analyze the overall gene expression profile of FF-bESCs based on RNA-seq; (A) Pearson correlation analysis between samples; (B) Principal component analysis (PCA) showing the relationship between bEPSCs, FF-bEPSCs, bEpiSCs and bESCs; (C) Volcano plot of differentially expressed genes; (D) Volcano plot of differentially expressed genes in FF-bESCs-P20 relative to bEPSCs; (E) KEGG pathway enrichment analysis of differentially expressed genes in FF-bESCs-P20 relative to bEPSCs; (G) Gene set enrichment analysis (GSEA) to analyze changes in the KEGG pathway in FF-bESCs-P20; (F) Selected Na+ in bEPSCs and FF-bESCs-P20. Relative expression of ve pluripotency marker genes; relative expression of selected formative pluripotency marker genes in (G)bEPSCs and FF-bESCs-P20; Figure 6 To identify the ability of FF-bESCs to differentiate into PGCLCs in vitro; (A) Schematic diagram of reporter gene targeted insertion into the TFAP2C gene site; the mCherry reporter gene was knocked into the TFAP2C gene stop codon via CRISPR / Cas9-mediated homology-directed repair (HDR); (B) Schematic diagram of the process of FF-bESCs differentiating into PGCLCs after pre-induction; (C) Bright-field and mCherry fluorescence images of FF-bESCs, pre-induction, and PGCLC aggregates; (D) mCherry obtained by flow cytometry sorting. + (E) Statistical graph of the proportion of positive PGCLCs and three biological replicates; (F) RT-qPCR detection of the expression level of primordial germ cell (PGC) related genes in PGCLCs; (F) Immunofluorescence staining detection of the expression of PGC-specific marker proteins in PGCLCs. Detailed Implementation

[0023] This invention provides a long-term culture system for morphological bovine embryonic stem cells without a feeder layer, comprising bEPSC medium and matrix proteins; the matrix proteins include Matrigel or Geltrex.

[0024] In this invention, the morphological embryonic stem cell culture medium comprises the following components: Neurobasal at a volume percentage of 48.00%, DMEM / F12 at a volume percentage of 48.00%, 0.1 mM 2-mercaptoethanol, 1 μM AR-A014418, 0.3 μM A-770041, 5 μM MSC2504877, and 50 μg / mL vitamin C.

[0025] In this invention, the volume ratio of the morphological embryonic stem cell culture medium to the matrix protein is preferably 100:1 to 10. In this invention, the volume ratio of the morphological embryonic stem cell culture medium to the matrix protein is preferably 20:1.

[0026] This invention also provides the application of the bovine morphological embryonic stem cell long-term culture system without a feeder layer, as described above, in supporting the stable expansion of bovine embryonic stem cells without a feeder layer. In this invention, the number of passages for expansion is preferably more than 60.

[0027] This invention also provides the application of the above-described bovine morphological embryonic stem cell long-term culture system without a feeder layer in culturing bovine embryonic stem cells without a feeder layer to achieve pluripotency. In this invention, the pluripotency preferably includes the formation of embryoid bodies in vitro and the generation of teratomas containing derivatives of three germ layers in vivo.

[0028] This invention also provides the application of the feeder-free bovine embryonic stem cell long-term culture system described in the above-mentioned technical solution in stem cell breeding. In this invention, it is preferable to differentiate the feeder-free bovine embryonic stem cells into primordial germ cell-like cells.

[0029] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1: Establishment of a long-term culture system for bovine morphological embryonic stem cells without feeder layer I. Experimental Design and Methods (1) Preliminary screening of bovine morphological embryonic stem cell culture system without feeder layer Bovine embryonic stem cell culture medium was prepared according to the following composition: 48.00% Neurobasal (v / v), 48.00% DMEM / F12 (v / v), 0.1 mM 2-mercaptoethanol, 1 μM AR-A014418, 0.3 μM A-770041, 5 μM MSC2504877, and 50 μg / mL vitamin C. After maintaining normal expansion on a bovine fetal fibroblast (BFF) feeder layer, bovine embryonic stem cells were transferred to various feeder-free culture systems based on different matrix proteins for adaptability testing. Specifically, culture dishes were coated with gelatin (STEMCELL Technologies, 7903), matrigel (Corning, 354230), fibronectin (Millipore, FC010), geltrex (Gibco, A1413202), and laminin521 (STEMCELL Technologies, 200-0117), respectively. During coating, except for gelatin, each substrate was diluted with pre-chilled DPBS to three specific v / v concentrations: low (1%, i.e., 1:100 dilution), medium (5%, i.e., 1:20 dilution), or high (10%, i.e., 1:10 dilution). Subsequently, 500 µL of the diluted substrate solution was added to each well of a 24-well plate. The culture dishes were incubated at 38.5 °C for 2 hours to promote substrate adsorption and stabilization. After incubation, the coating medium was aspirated, and 1 mL of bovine embryonic stem cell culture medium was seeded into each well. Simultaneously, two 3D-like culture conditions were tested: bovine embryonic stem cells were directly resuspended in pre-chilled medium supplemented with Matrigel or Geltrex (concentrations as above, low, medium, and high), and then seeded directly into uncoated 24-well plates at 1 mL per well. Cells were passaged every 3–4 days using Accutase (Gibco, A1110501) at a ratio of 1:3 to 1:5, and reseeded at the same density under all conditions. Culture progress was monitored daily using phase-contrast microscopy to assess cell adhesion, colony morphology, proliferation, spontaneous differentiation, and cell death. These morphological and growth-related indicators were used as the primary basis for preliminary screening of feasible feeder-free culture conditions.

[0031] (2) Determination of long-term culture, doubling time and single-cell cloning efficiency of FF-bESCs After initially screening the feeder culture system, FF-bESCs under this system were continuously passaged. For passage of FF-bESCs, clones were washed once with DPBS, digested with Accutase at 38.5℃ for 5 minutes to dissociate into single cells, collected by centrifugation at 1000 rpm for 5 minutes, and resuspended in Matrigel-containing bEPSCM, and cultured in a 38.5℃ and 5% CO2 incubator. When FF-bESCs reached approximately 80% confluence, cells were cryopreserved using cryopreservation solution (90% fetal bovine serum [FBS; VivaCell, CO4002] + 10% dimethyl sulfoxide [DMSO; Sigma, D2650]): first short-term storage overnight at –80℃, followed by long-term storage in liquid nitrogen. For doubling time determination, after Accutase digestion of FF-bESCs, the single cells obtained by Accutase digestion were passaged at 8 × 10⁻⁶ cells per cell. 4 Cells were seeded per well in 24-well plates, and cell counts were performed on days 2, 3, and 4 post-seeding to calculate doubling time. For single-cell colony formation efficiency assays, 1000 cells were seeded into 6-well plates, cultured for 3–4 days, and then stained with alkaline phosphatase (AP) to count the number of AP-positive colonies. Single-cell colony formation rate was defined as the percentage of AP-positive colonies out of the total number of seeded cells.

[0032] (3) Alkaline phosphatase activity detection The alkaline phosphatase (AP) activity of FF-bESCs was detected using the BCIP / NBT alkaline phosphatase colorimetric kit (Beyotime, C3206), strictly following the manufacturer's instructions. The specific steps were as follows: cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 5 minutes; washed 2–3 times with DPBS to remove the fixative; freshly prepared BCIP / NBT staining working solution was added and incubated at room temperature in the dark for 25 minutes; the staining solution was discarded, and the cells were gently washed 2–3 times with DPBS to terminate the colorimetric reaction; AP-positive clones were observed under a microscope and photographed.

[0033] (4) Chromosome karyotype analysis FF-bESCs at 50–60% confluence were treated with fresh medium containing 0.2 μg / mL colchicine (Beyotime, catalog number ST1173-1g) and incubated at 37 °C in a 5% CO2 incubator for 3 hours to arrest the cells at metaphase. Subsequently, the cells were digested with Accutase, collected by centrifugation, and hypotonic with 0.075 M KCl at 37 °C for 10 minutes. After hypotonic swelling, 1 mL of freshly prepared Carnoy's fixative (methanol:glacial acetic acid = 3:1, v / v) was added for pre-fixation. After centrifugation, the cell pellet was resuspended and fixed three times at 37 °C with 8 mL of Carnoy's fixative for 30 minutes each time. Finally, the cells were resuspended in 200 μL of pre-chilled Carnoy's fixative, gently pipetted to prepare a single-cell suspension, and dropped onto a pre-chilled glass slide. The slides were immediately dried rapidly over an alcohol lamp flame to promote chromosome spreading. They were then stained with Giemsa stain and allowed to air dry at room temperature. Metaphase cells with clear chromosome morphology and distinct banding were selected and photographed at high resolution under a 100× oil immersion microscope.

[0034] II. Experimental Results (1) Preliminary screening of bovine embryonic stem cell culture system without feeder layer To preliminarily screen effective systems for supporting feeder-free culture of bovine embryonic stem cells (BFF), this invention evaluated seven feeder-free culture protocols based on different matrix proteins and conducted preliminary evaluations based on cell clonal morphology and proliferation status. The results showed that tightly packed, dome-shaped, smooth-edged clones maintained on the BFF feeder (…). Figure 1 (A). After being transferred to culture conditions without a feeder layer, under five of the pre-coated culture dish conditions ( Figure 1 In the B–F generation, significant abnormalities appear after the third generation: cell volume increases, membrane boundaries become clearer, and differentiation characteristics are observed; or cells detach and die due to insufficient adhesion. In contrast, in bovine embryonic stem cell culture medium + Matrigel (… Figure 1 (G) and bovine embryonic stem cell culture medium + Geltrex ( Figure 1 In the two types of 3D culture systems (H and H), bovine embryonic stem cells were resuspended in bEPSCM containing high concentrations of Matrigel or Geltrex and directly seeded onto uncoated culture dishes. They were able to stably maintain a typical, dense and raised undifferentiated clonal morphology, indicating that these two conditions can effectively support self-renewal and morphological stability in a feeder-free environment. These bovine embryonic stem cells are defined as (FF-bESCs).

[0035] (2) Detection of stable amplification and undifferentiated state without feeder layer culture support This invention evaluated the long-term culture stability and cryopreservation recovery capability of FF-bESCs under feeder-free conditions. FF-bESCs were easily dissociated into single cells or clumps by Accutase enzyme and achieved stable passage. During more than 60 consecutive passages, the cells maintained the typical dense, raised stem cell clonal morphology and maintained high levels of alkaline phosphatase (AP) activity. Figure 2 AP (amylase activity) is a classic marker of pluripotency. Notably, when FF-bESCs were returned to MEF or BFF feeder cells, their clonal morphology and AP activity remained unchanged. Figure 2 (B) FF-bESCs were passaged every 3–4 days at a ratio of 1:3 to 1:5. Following standard cryopreservation protocols (90% fetal bovine serum + 10% DMSO) for short-term storage at -80°C or long-term cryopreservation in liquid nitrogen, FF-bESCs still exhibited efficient recovery and continued expansion. The doubling time of FF-bESCs was approximately 20 hours. Figure 2 In the middle (C), the single-cell clone formation efficiency reached 27% ( Figure 2 (D). Karyotype analysis confirmed that FF-bESCs maintained a stable and normal karyotype (2n = 60) during long-term passage. Figure 2 (E). The above indicates that FF-bESCs can be efficiently and stably expanded in a completely feeder-free system over a long period of time, exhibiting strong self-renewal capabilities and typical pluripotency-related phenotypic characteristics.

[0036] Example 2: Identification of pluripotency of bovine morphological embryonic stem cells cultured completely without feeder layer I. Experimental Design and Methods (1) Analysis of embryonic differentiation In in vitro differentiation experiments, FF-bESCs were dissociated from culture dishes by Accutase digestion. Cells were collected and resuspended in DMEM medium containing 10% FBS, and seeded at 5000 cells / well in ultra-low adsorption 96-well plates (Corning, 7007) to promote embryoid body (EB) formation. After culturing in suspension for 4 days, the formed EBs were transferred to 0.1% gelatin-coated tissue culture dishes and cultured adherently for another 8 days, followed by immunofluorescence staining analysis.

[0037] (2) Analysis of teratoma formation FF-bESCs were resuspended in DPBS containing 30% Matrigel (Corning, 354230) and 10 μM ROCK inhibitor Y-27632 (Selleck, S1049). 5 × 10⁻⁶ cells were then collected. 6 FF-bESCs were subcutaneously injected at a volume of 100 μL into the bilateral dorsal regions of 8-week-old female BALB / c-nu nude mice (Speford (Beijing) Biotechnology Co., Ltd.). Within 8–10 weeks post-injection, FF-bESCs formed macroscopically visible teratomas. When the teratoma reached approximately 1 cm in size… 3 The tissue was completely peeled off and immediately fixed overnight in 4% paraformaldehyde (PFA) at 4°C. Subsequently, the tissue was dehydrated, cleared, and embedded in paraffin to prepare serial sections for subsequent immunofluorescence staining analysis.

[0038] (3) Immunofluorescence staining FF-bESCs were washed with DPBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes or overnight at 4°C. After fixation, the cells were washed three times with DPBS. Subsequently, the cells were permeabilized and blocked with DPBS containing 3% normal donkey serum (NDS) and 0.3% Triton X-100, and incubated at room temperature for 30 minutes. The primary antibody was diluted with 1% NDS + 0.3% Triton X-100 in DPBS, and the cells were placed in the primary antibody solution prepared in this solution and incubated overnight at 4°C. The cells were washed three times with DPBS containing 0.3% Triton X-100 for 10 minutes each time. After removing the washings, a fluorescently labeled secondary antibody solution (diluted with 1% NDS + 0.3% Triton X-100 in DPBS) was added and incubated at room temperature in the dark for 1 hour. Wash three times with 0.3% Triton X-100 / DPBS, 10 minutes each time. For FF-bESCs, add DAPI-containing DPBS solution, incubate at room temperature in the dark for 10 minutes, wash with DPBS, and observe and photograph using an inverted fluorescence microscope. The primary antibody information used in this invention is as follows: OCT4 (Abcam, ab27985), SOX2 (Invitrogen, 14-9811-82), NANOG (Abcam, ab80892), TuJ-1 (R&D Systems, MAB1195), AFP (R&D Systems, MAB1368), SMA (R&D Systems, MAB1420), PRDM1 (Invitrogen, 14596380), SOX17 (R&D Systems, AF1924), TFAP2C (Santa Cruz Biotechnology, sc-12762).

[0039] (4) RT-qPCR detection Total RNA was extracted from cells using TRIzol reagent (Ambion, 15596018). cDNA synthesis was performed using Novizan reverse transcription reagent (Vazyme, R323-01). T-qPCR was performed using a Taq Pro Universal SYBR qPCR MasterMix (Vazyme, Q712-03) on a Bio-Rad CFX96 Touch™ system (Bio-Rad Laboratories, Inc. Hercules, CA, USA). Gene expression levels were normalized to relative quantification using GAPDH as an internal reference gene.

[0040] II. Experimental Results (1) Analysis of the multipotentiality index of FF-bESCs This invention first detected the expression levels of pluripotency markers using immunofluorescence staining and RT-qPCR. The results showed that even after long-term passage, FF-bESCs stably expressed the core pluripotency marker proteins SOX2, OCT4, and NANOG (…). Figure 3 (A). RT-qPCR analysis further showed that these cells highly expressed OCT4, SOX2, and NANOG, Na... The expression of the vegetal pluripotency gene KLF4 was decreased, while genes related to early differentiation of the three germ layers (PAX6, TBXT, and FOXA2) were either underexpressed or undetectable. Figure 3 (B)

[0041] This invention further investigated in vitro embryoid body (EB) formation and differentiation experiments. FF-bESCs can efficiently form EBs under ultra-low adsorption conditions. Figure 4 (A), and then adhered to the culture dish coated with gelatin, spontaneously differentiating into a monolayer of cells ( Figure 4 (Middle B). Immunofluorescence staining showed that differentiated cells expressed the ectoderm marker Tuj1, the mesodermal marker SMA, and the endoderm marker AFP (…). Figure 4 (C). Furthermore, subcutaneous injection of FF-bESCs into immunodeficient BALB / c-nu nude mice resulted in a volume of approximately 1 cm³ after about 6 weeks. 3 Teratoma ( Figure 4 (D). Immunofluorescence analysis of tissue sections confirmed that the teratoma contained cells derived from three germ layers (D). Figure 4 (Middle E). These results demonstrate that FF-bESC possesses the ability to differentiate into EB and form teratomas in vitro, fully proving its true pluripotency.

[0042] Example 3: Identification of developmental stages of bovine morphological embryonic stem cells cultured completely without feeder layer I. Experimental Design and Methods (1) Transcriptome sequencing (RNA-seq) and data analysis Total RNA was extracted using TRIzol and RNA quality was assessed. After RNA samples passed quality control, standard transcriptome library construction was performed, with 1 μg of RNA used per sample for initial library construction. Sequencing was then performed using Illumina NovaSeq 6000 after library quality control. Raw FASTQ data (raw reads) underwent quality control and filtering using FASTP, including adapter removal, removal of low-quality reads, and removal of reads with excessive nitrogen. All subsequent analyses were based on the clean reads after quality control. Hisat2 was used to align the clean reads to a reference genome, and featureCounts was used to count the number of reads aligned to each gene, ultimately generating a gene-level raw read count matrix for each sample, used for subsequent differential expression analysis. Differential expression analysis was performed using DESeq2. The Benjamini-Hochberg method was used to correct p-values ​​to control the false discovery rate (FDR). Genes with |log2 FoldChange| > 1 and FDR < 0.05 were finally identified as differentially expressed genes (DEGs). KEGG enrichment analysis of DEGs was performed using KOBAS. II. Experimental Results (1) Transcriptome analysis of FF-bESCs revealed their stable formative pluripotency. To thoroughly evaluate the gene expression characteristics and pluripotency status of FF-bESCs, we performed transcriptome sequencing (RNA-seq) on FF-bESCs from generations 5 and 20, as well as bEPSCs cultured on BFF feeder layers. Figure 5 (A). Inter-sample correlation analysis showed that FF-bESCs of generation 5 (P5) and generation 20 (P20) were highly correlated, while the correlation between FF-bESCs and bEPSCs was relatively weak. Figure 5 Principal component analysis (PCA) showed that FF-bESCs in the 5th generation (P5) and the 20th generation (P20) clustered tightly together with very little separation, which confirmed the high stability of their whole-genome expression during long-term culture. Figure 5 (B). Notably, FF-bESCs exhibited transcriptomic features highly consistent with formal bEpiSCs, and the two groups of cells clustered together. In contrast, FF-bESCs were significantly different from both primed bEPSCs and conventionally primed bESCs. Figure 5(B). Furthermore, although FF-bESCs share a formalistic identity with bEpiSCs, they are distinct from another form of cell line, JY-bESCs, which occupies a unique position ( Figure 5 (B). These results indicate that, under conditions without a feeder layer, FF-bESCs can rapidly and stably transition from the bEPSC state to a specific formative pluripotent state similar to bEpiSCs.

[0043] Next, this invention performed differentially expressed genes (DEGs) analysis on FF-bESCs-P5, FF-bESCs-P20, and bEPSCs. Volcano plots showed that compared to bEPSCs, FF-bESCs-P5 identified 1416 DEGs, of which 1348 genes were significantly downregulated and 68 genes were significantly upregulated; compared to bEPSCs, FF-bESCs-P20 identified 1495 DEGs, of which 1434 genes were downregulated and 61 genes were upregulated. Figure 5 (C). It is worth noting that there are only 13 DEGs between FF-bESCs-P5 and P20. Figure 5 The C-axis indicates that the transcriptome of FF-bESCs underwent significant changes compared to bEPSCs, but the transcriptome of FF-bESCs itself remained relatively stable during long-term passage. KEGG pathway enrichment analysis was performed on DEGs. The results showed that the differentially enriched KEGG pathways between FF-bESCs-P5 or FF-bESCs-P20 and bEPSCs were highly similar, mainly involving cell adhesion, PI3K-Akt signaling pathway, MAPK signaling pathway, Wnt signaling pathway, and Jak-STAT signaling pathway, etc. Figure 5 (D). Further gene set enrichment analysis (GSEA) showed that, compared with bEPSCs, FF-bESCs exhibited inhibition of the JAK / STAT, WNT, and MAPK signaling pathways (D). Figure 5 (E).

[0044] To further determine the pluripotency status of FF-bESCs, gene expression heatmaps showed that, compared to bEPSCas, FF-bESCs contained higher levels of Na+. VE pluripotency genes (e.g.) KLF2, KLF4, TBX3 ) are generally downregulated, Formative pluripotency genes (such as OTX2, DNMT3A, DNMT3B ) generally increased ( Figure 5(F). Further evaluation of the quality of formative FF-bESCs revealed that FF-bESCs exhibited a similar expression pattern to bEpiSCs, with both cell lines showing the presence of formative-related genes (such as...). OTX2, DNMT3A / B and ZIC2 / 5 The upregulation of FF-bESCs was observed, a stark contrast to the lower expression levels seen in JY-bESCs. Notably, the number of upregulated formic genes in FF-bESCs even exceeded that in bEpiSCs. Figure 5 (G). Among them, OTX2 is a marker for protoform pluripotent stem cells. In pluripotency genes, the expression levels of de novo DNA methyltransferase genes DNMT3A and DNMT3B are elevated in FF-bESCs (G). Figure 5 The presence of F indicates that FF-bESCs have a higher overall DNA methylation level. In summary, the feeder-free culture system drives the formation of formative pluripotency by regulating key signaling pathways, and on this basis, effectively maintains the stable self-renewal capacity of FF-bESCs.

[0045] Example 4: Application of FF-bESCs in stem cell breeding: Directed differentiation into germ cells I. Experimental Design and Methods (1) Inducing FF-bESCs to differentiate into germ cell-like cells (PGCLCs) 5×10 5FF-bESCs were seeded in 6-well plates and cultured for 48 hours. The maintenance medium was then replaced with pre-induction medium, and the cells were cultured for another 24 hours. The pre-induction medium consisted of N2B27 medium containing 5% KSR (Gibco, 10828028) supplemented with 100 ng / ml BMP4 (R&D Systems, 314-BP), 6 μM CHIR99021 (Selleck, S2924), 2.5 μM WR1 (Selleck, S7086), and 10 μM Y-27632. After 24 hours of pre-induction culture, the cells were digested with Accutase, resuspended in PGCLC differentiation medium, and seeded at 6000 cells / well in 96-well ultra-low adsorption U-shaped plates (Corning, 7007) for suspension culture for 4 days. The differentiation medium for PGCLCs was GK15 medium supplemented with 200 ng / ml BMP4, 100 ng / ml SCF (R&D Systems, 455-MC), 50 ng / ml EGF (R&D Systems, 236-EG), 10 ng / ml LIF (Millipore, LIF1010), 6 μM CHIR99021, 2.5 μM IWR1, and 10 μM Y27632.

[0046] (2) Fluorescence-activated cell sorting (FACS) Aggregates of PGCLCs cultured in suspension for 4 days were collected, washed with DPBS, and digested at 37°C for 6 minutes with 0.25% Trypsin-EDTA. The digestion reaction was then terminated by adding GK15 medium. Cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected and resuspended in FACS buffer (DPBS containing 0.1% BSA). After filtering through a 70 μm cell sieve to remove cell clumps, the cells were analyzed or sorted using a FACSAria Fusion flow cytometer (BD Biosciences). FACS data were analyzed using FlowJo software.

[0047] II. Experimental Results (1) Induction of differentiation of FF-bESCs into PGCLCs This invention precisely knocks in the mCherry fluorescent reporter gene (mCherry) before the TFAP2C gene stop codon via CRISPR / Cas9-mediated homology-directed repair (HDR). Figure 6 (A). This design ensures that mCherry expression is regulated by the endogenous TFAP2C promoter, thereby specifically labeling bPGCLCs.

[0048] FF-bESCs exhibited a flattened epithelial-like cell morphology one day after pre-induction; subsequently, after four days of suspension culture, they formed embryoid bodies with smooth edges, compact structure, and regular round shape. Figure 6 (B). During the differentiation of bPGCLCs, mCherry red fluorescence signal can be observed ( Figure 6 The presence of bPGCLCs (b-PGCLCs) indicates successful introduction of the reporter gene and effective differentiation into PGCLCs. Flow cytometry analysis showed that the differentiation efficiency of bPGCLCs was approximately 34%. Figure 6 (C). RT-qPCR results further confirmed that the primordial germ cell marker genes of differentiated bPGCLCs were significantly upregulated, including PRDM1, TFAP2C, NANOS3 and SOX17 ( Figure 6 (Middle D); Immunofluorescence staining also detected high expression of OCT4, PRDM1, TFAP2C, and SOX17 proteins ( Figure 6 (E).

[0049] The above results demonstrate that FF-bESCs can effectively integrate and stably express exogenous genes, which not only verifies their feasibility as gene editing vectors, but also successfully achieves efficient induction and visual tracking of bPGCLCs, laying a key technological foundation for stem cell-based livestock breeding.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A long-term culture system for bovine morphological embryonic stem cells without a feeder layer, characterized in that, The medium and matrix protein for forming the embryonic stem cells comprise the following components: Neurobasal at 48.00% by volume, DMEM / F12 at 48.00% by volume, 0.1 mM 2-mercaptoethanol, 1 μM AR-A014418, 0.3 μM A-770041, 5 μM MSC2504877, and 50 μg / mL vitamin C. The matrix protein comprises Matrigel or Geltrex.

2. The completely feeder-free long-term culture system of bovine formed state embryonic stem cells according to claim 1, characterized in that, The medium for forming the embryonic stem cells comprises the following components: Neurobasal at 48.00% by volume, DMEM / F12 at 48.00% by volume, 0.1 mM 2-mercaptoethanol, 1 μM AR-A014418, 0.3 μM A-770041, 5 μM MSC2504877, and 50 μg / mL vitamin C.

3. The completely feeder-free long-term culture system of bovine embryonic stem cells in the formation stage according to claim 1, characterized in that, The volume ratio of the medium for forming the embryonic stem cells to the matrix protein is 100:1-10.

4. The completely feeder-free long-term culture system of formed state bovine embryonic stem cells according to claim 1 or 3, characterized in that, The volume ratio of the medium for forming the embryonic stem cells to the matrix protein is 20:

1.

5. Use of the complete feeder-free long-term culture system of bovine forming embryonic stem cells according to any one of claims 1-4 in supporting stable expansion of bovine embryonic stem cells under complete feeder-free condition.

6. Use according to claim 5, characterized in that, The expanded generations exceed 60 generations.

7. Use of the complete feeder-free long-term culture system of bovine forming embryonic stem cells according to any one of claims 1-4 in endowing the bovine embryonic stem cells with pluripotency.

8. Use according to claim 7, characterized in that, The pluripotency comprises forming embryoid bodies in vitro and generating teratomas containing derivatives of three germ layers in vivo.

9. Use of the complete feeder-free long-term culture system of bovine forming embryonic stem cells according to any one of claims 1-4 in breeding bovine embryonic stem cells.

10. The use according to claim 9, characterized in that, The feeder-free bovine embryonic stem cells are differentiated into primordial germ cell-like cells.

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