Culture method of ovine excited-state stem cells, culture medium used and application

Abstract

The application discloses a culture method of sheep primed-state stem cells, a culture medium used in the culture method and application of the culture method. The application belongs to the technical field of biotechnology and particularly relates to a culture method of sheep primed-state stem cells, a culture medium used in the culture method and application of the culture method. The composition for culturing sheep primed-state pluripotent stem cells contains an N2B27 basic culture medium, cytokines and small molecules. The cytokines and the small molecules contain human Activin A, recombinant human FGF2 and IWR1. The addition concentration of the human Activin A is 25 ng / mL; the addition concentration of the recombinant human FGF2 is 12.5 ng / mL; and the addition concentration of the IWR1 is 5 muM. The culture medium prepared by using the above composition can obtain stable sheep primed-state pluripotent stem cells, and can be applied in construction of a sheep gene editing model animal.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for culturing sheep excited-state stem cells, the culture medium used, and its application. Background Technology

[0002] The pluripotency state of the embryonic epiblast undergoes a transition from Naïve to Formative and then to Primed, and it is also the source of pluripotent stem cells (PSCs). PSCs cultured in vitro can reflect the pluripotency state of the embryonic epiblast in vivo, and different culture conditions can help establish PSCs in different pluripotent states in vitro. To date, stable Naïve, Formative, and Primed PSCs have been successfully established in mice, humans, and monkeys, exhibiting differences in signaling pathways, gene regulatory networks, epigenetics, and metabolism. Primed PSCs possess molecular characteristics and pluripotency states similar to the post-implantation ectoderm, in addition to expressing core pluripotency marker genes. POU5F1 , SOX2 , NANOG In addition, it simultaneously activates Primed pluripotency-related transcription factors. OTX2 , POU3F1 , FGF5 Upregulation of cell adhesion factors and WNT signaling pathway-related genes. During the transition from Naïve to Primed pluripotency, upregulation of methyltransferases is accompanied by an increase in CpG genome-wide DNA methylation levels, and random inactivation of one X chromosome in female cells. POU5F1 The expression of these cells is regulated by proximal enhancer elements, and their metabolic pathway shifts to glycolysis. In terms of in vivo developmental capacity testing, Primed PSCs do not possess the ability to form germline chimeras. The establishment of livestock embryonic stem cells holds promise for promoting biomedical development and accelerating livestock breeding. Sheep are one of the most important livestock animals in my country's livestock production. The lack of elucidation of the regulatory pathways of preimplantation embryonic development and epiblast pluripotency in sheep limits the application of sheep pluripotent stem cells in agriculture and biomedicine. Excited-state stem cells with a stable and clearly defined pluripotency state have not yet been successfully established. Therefore, it is urgent to elucidate the regulatory mechanisms of epiblast pluripotency in early sheep embryos, optimize culture systems, and establish stable excited-state sheep PSCs. Summary of the Invention

[0003] The main problem this invention aims to solve is how to establish and culture sheep excited-state pluripotent stem cells.

[0004] To address the above problems, the present invention provides a composition for culturing sheep excited-state pluripotent stem cells.

[0005] The composition for culturing sheep excited-state pluripotent stem cells provided by the present invention contains N2B27 basal medium and cytokines, wherein the cytokines contain human Activin A, recombinant human FGF2, and IWR1.

[0006] In the above composition, the concentration of human Activin A in the cytokines is 25 ng / mL; the concentration of recombinant human FGF2 is 12.5 ng / mL; and the concentration of IWR1 is 5 μM.

[0007] In the above composition, the N2B27 basal culture medium, calculated in 500 mL, contains 227 mL DMEM / F12 (Thermo Fisher Scientific, 10565-018), 227 mL Neurobasal (Gibco, 21103-049), 2.5 mL N2 supplement (Gibco, 17502-048), 5 mL B27 supplement (Gibco, 12587-010), 2.5 mL 100× GlutaMAX (Gibco, 35050-061), 5.0 mL 100× nonessential amino acids (Gibco, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5.0 mL 100× penicillin-streptomycin (Gibco, 15140-122), and 5% knockout serum. replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544) and 2.5 μM ROCKinhibitor Y27632 (Selleckchem, S1049).

[0008] The present invention also provides a culture medium for culturing sheep excited-state pluripotent stem cells, wherein the active ingredient of the culture medium is the composition described above.

[0009] This invention also provides a method for culturing sheep excited-state pluripotent stem cells, comprising the following steps:

[0010] 1) Select sheep embryos in the 12th-14th day of development, mechanically remove the hypoblast and trophoblast cells, and digest the epiblast cells of the embryo to obtain epiblast cell clusters;

[0011] 2) Prepare a four-well plate with feeder cells 12 hours in advance, and inoculate the digested epiblast cell clusters into the culture medium described above. After culturing for 4-6 days, the primary clones will be obtained.

[0012] 3) Digest the primary clone obtained in step 2), re-inoculate it in the culture medium containing the above-mentioned culture medium, and perform passage culture to obtain the sheep excited-state pluripotent stem cells.

[0013] In the above method, the digestion in step 1) is performed by TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes.

[0014] In the above method, step 3) involves digestion with Accutase (Gibco, A11105-01) at 37°C for 5 minutes; the culture temperature is 37°C.

[0015] The application of the composition and culture medium described above in the preparation of sheep excited-state pluripotent stem cell products, or in the preparation of products that increase the cell density of sheep excited-state pluripotent stem cells, also falls within the scope of protection claimed by this invention.

[0016] The present invention also provides the application of the method described above in the preparation of sheep excited-state pluripotent stem cell products, or in the preparation of products that increase the cell density of sheep excited-state pluripotent stem cells.

[0017] The sheep excited-state pluripotent stem cells obtained from the culture medium developed in this invention can be stably passaged for more than 100 generations and have clear Primed pluripotency characteristics. They can be used for the preparation of sheep excited-state pluripotent stem cells and have application prospects. Attached Figure Description

[0018] Figure 1 This study elucidates the regulatory mechanism of epiblast cell pluripotency in early sheep embryos. A represents the changes in epiblast cell pluripotency status in early sheep embryos; B represents the expression of marker genes indicating different pluripotency statuses in the Naïve, Formative, and Primed epiblast cells of early sheep embryos; and C represents the analysis of the expression levels of key genes in the epiblast cell pluripotency regulatory signaling pathway in early sheep embryos.

[0019] Figure 2This section describes the establishment and pluripotency identification of spPSCs. A shows the morphology and AP staining of spPSCs in the AFIWR1 culture system. Scale bar, 50 μm. B shows the immunofluorescence staining of spPSCs in the AFIWR1 culture system with pluripotency marker genes POU5F1, SOX2, and NANOG, and DAPI staining of cell nuclei. Scale bar, 50 μm. C shows the morphology and AP staining of spPSCs in the AFXAV939 culture system. Scale bar, 50 μm. D shows the immunofluorescence staining of spPSCs in the AFXAV939 culture system with pluripotency marker genes POU5F1, SOX2, and NANOG, and DAPI staining of cell nuclei. Scale bar, 50 μm. E shows the morphology and AP staining of spPSCs in the AFIWP2 culture system. Scale bar, 50 μm. F shows immunofluorescence staining of spPSC pluripotency marker genes POU5F1, SOX2, and NANOG in the AFIWP2 culture system, with DAPI staining of cell nuclei, scale bar, 50 μm. G shows immunofluorescence staining of histone H3K27me3 in spPSCs, with DAPI staining of cell nuclei, scale bar, 100 μm. H shows immunofluorescence staining of β-catenin protein in spPSCs, with DAPI staining of cell nuclei, scale bar, 50 μm.

[0020] Figure 3 This study describes the detection of cytokine and small molecule functions in spPSC culture systems. A shows the clonal morphology of spPSCs after removing IWR1, Activin A, or FGF2 from AFI medium; B shows representative images of AP staining after removing IWR1, Activin A, or FGF2 from AFI medium. Scale bars for A and B are 100 μm. C shows immunofluorescence staining of pluripotency marker proteins POU5F1, SOX2, and NANOG after removing individual components; DAPI staining indicates cell nuclei. Scale bar is 100 μm. D shows the quantitative analysis of pluripotency marker gene expression levels after removing individual components. E shows the quantitative analysis of differentiation marker gene expression levels after removing individual components.

[0021] Figure 4 This section describes the in vitro and in vivo differentiation capacity of spPSCs. A shows immunofluorescence staining for embryoid differentiation, with DAPI staining of cell nuclei (scale bar, 50 μm). B shows H&E staining of teratoma tissue, displaying the ectoderm, mesoderm, and endoderm lineages (scale bar, 100 μm). C shows immunofluorescence staining of teratoma tissue (scale bar, 100 μm). Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. 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 invention in any way.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0025] The sheep used in the following examples were purchased from the Tianjin Academy of Agricultural Sciences.

[0026] Animal Handling and Ethical Statement: All animal experiments involving mice and sheep described in the following examples were approved in advance by the Animal Care and Ethics Committee (IACUC) of China Agricultural University and the IACUC of Beijing College of Agriculture. CD-1® (ICR) IGS mice and BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. for the isolation of mouse embryonic fibroblasts and teratoma formation experiments. All sheep were in natural estrus and mated for the collection of early embryonic single cells and the establishment of embryonic pluripotent stem cells.

[0027] All experiments in the following examples were performed with three biological and technical replicates. Graphical representations and statistical analyses were performed using GraphPad Prism 8.0. Values ​​in the graphs are expressed as mean ± standard deviation, and statistical significance was calculated using Student's two-tailed t-test. P A value less than 0.05 is considered statistically significant and is shown as *. P <0.05,** P <0.01, and *** P <0.001.

[0028] Example 1: Study on culture medium and culture method of sheep excited-state stem cells

[0029] 1. Establishment of culture medium for excited-state pluripotent stem cells (spPSCs) in sheep

[0030] Excited-state pluripotency cells (PSCs) originate from the epiblast of the pre-gastrulation embryo. Analysis of single-cell data from early sheep embryos reveals that classic Primed pluripotency genes, such as... WNT5B , PITX2 , EOMES High expression begins in the epiblast of E12 embryos ( Figure 1(A, B). Therefore, an attempt was made to establish excited-state PSCs from E12-E14 embryos.

[0031] Analysis of the epiblast pluripotency regulatory network in early sheep embryos revealed that the FGF and TGF-β signaling pathways are activated in the E12-E14 epiblasts, and the maintenance of excited-state pluripotency requires the addition of FGF2 and Activin A. Further analysis showed that the E12 embryo begins to express genes related to gastrulation, representing a critical point in embryonic gastrulation; therefore, inhibition of the WNT signaling pathway is crucial for PSC self-renewal. Figure 1 (C). Based on this, the present invention establishes a culture system containing Activin A, FGF2 and IWR1 (AFI), which can efficiently isolate and establish stable spPSCs from the epiblast of E12-E14 embryos.

[0032] The culture system for excited-state pluripotent stem cells (spPSCs) was based on N2B27 medium, and the following small molecules and cytokines were investigated for addition: human Activin A (25 ng / mL, PeproTech, 120-14E), recombinant human FGF2 (12.5 ng / mL, PeproTech, 100-18B), and IWR1 (5 μM, Selleckchem, S7086). IWR1 could be replaced with XAV939 (5 ​​μM, Selleckchem, S1180) or IWP2 (5 μM, Selleckchem, S7085).

[0033] Among them, N2B27 basal medium (500 mL): 227 mL DMEM / F12 (Thermo Fisher Scientific, 10565-018), 227 mL Neurobasal (Gibco, 21103-049), 2.5 mL N2supplement (Gibco, 17502-048), 5 mL B27 supplement (Gibco, 12587-010), 2.5 mL 100× GlutaMAX (Gibco, 35050-061), 5.0 mL 100× nonessential amino acids (Gibco, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5.0 mL 100×penicillin-streptomycin (Gibco, 15140-122), 5% knockout serum replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544) and 2.5 μM ROCKinhibitor Y27632 (Selleckchem, S1049).

[0034] The culture method for excited-state pluripotent stem cells (sfPSCs) is as follows:

[0035] 1) Epiblast cells were isolated from sheep embryos that had developed to E12-E14 (hypoblast and trophoblast cells were mechanically removed, and the epiblast was digested in TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes to separate the epiblast into small cell clusters). The cells were digested into single cells and seeded into a culture medium containing excited pluripotent stem cells (Table 1). Primary clones were formed 4-5 days after seeding, with an efficiency of 100%.

[0036] 2) Primary clones were digested with Accutase to form single cells, which were then seeded onto feeder cells containing excited-state pluripotent stem cell culture medium for passage. Passage was performed every 3-4 days at a ratio of 1:4 to 1:5. Before passage, the cells were washed once with DPBS, then digested with Accutase (Gibco, A11105-01) at 37°C for 5 minutes. After digestion, the cells were pipetted into single cells, the cell suspension was collected, and centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, the cells were resuspended in excited-state pluripotent stem cell culture medium and seeded onto feeder cells, then cultured in an incubator containing 20% ​​O2 and 5% CO2 at 37°C.

[0037] spPSCs exhibit a flattened clonal morphology, similar to mouse EpiSCs and primed hESCs. spPSCs are positive for AP staining. Figure 2 (A), expressing pluripotency marker proteins POU5F1, SOX2, and NANOG ( Figure 2 (B), where IWR1 can be replaced by XAV939 and IWP2 ( Figure 2 In the epigenetic level, immunofluorescence staining results for histone H3K27me3 indicated that one of the X chromosomes in female spPSCs was inactive. Figure 2 (G). The expression of β-catenin, a key protein in the WNT signaling pathway, was detected, and the results showed that the vast majority of β-catenin protein was stably located in the cytoplasm. Figure 2 (H).

[0038] 2. The requirements for each factor in the culture medium were determined by factor reduction experiments. The specific components examined are shown in Table 1.

[0039] Table 1. Information on various culture medium components

[0040]

[0041] Research has found that:

[0042] 1) After IWR1 withdrawal, spPSC clones showed obvious differentiation morphology, decreased AP staining activity, and significantly downregulated expression of pluripotency genes POU5F1, SOX2, NANOG, and SALL4. Figure 3 In mesoderm differentiation-related genes CER1, BMP4, and GATA6, expression was significantly upregulated (AD). Figure 3 (E);

[0043] 2) After Activin A was removed, spPSCs could not proliferate well, the clones showed differentiation morphology, AP staining activity decreased, and the expression of the pluripotency gene NANOG was significantly downregulated. Figure 3 AC (Chinese)

[0044] 3) After FGF2 removal, cell proliferation was severely impaired, the expression of pluripotency genes POU5F1 and NANOG was significantly downregulated, and the expression of ectoderm differentiation gene PAX6 was significantly upregulated. Figure 3 (AE).

[0045] The above results indicate that the AFWNTi (Activin A, FGF2 and WNT inhibitor) culture system is the basic culture condition for maintaining the self-renewal of spPSCs.

[0046] 3. In vivo and in vitro differentiation capacity detection of spPSCs

[0047] 1) Detecting the in vitro differentiation capacity of spPSCs

[0048] The specific detection method is as follows: spPSCs were digested into single cells, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 2 mL of fibroblast culture medium (Table 2) was added to resuspend the cells, and the cells were transferred to 3.5 cm dishes. The dishes were placed on a shaker in a 37℃ cell culture incubator at 70 rpm / min to allow the cells to aggregate. After approximately 3-4 days, the cells aggregated to form embryoid bodies (EBs). Well-formed EBs were picked out and seeded in 12-well plates. After approximately 6-7 days of EB adhesion and differentiation, the cells were fixed and subjected to immunofluorescence staining.

[0049] Table 2. Fibroblast Culture Medium

[0050]

[0051] Analysis revealed that spPSCs could form embryonic bodies in vitro. After adherent culture, EBs spontaneously differentiated into three germ layers, and the expression of NESTIN (ectoderm marker), α-SMA (mesoderm marker), and GATA6 (endoderm marker) was detected by immunofluorescence. Figure 4 (A)

[0052] 2) Detect the in vivo differentiation capacity of spPSCs

[0053] The specific detection method is as follows: spPSCs were digested into single cells, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 100 µL of spPSC culture medium and placed on ice. The cell suspension was aspirated using an insulin injection needle, air was expelled from the syringe, and the cells were injected subcutaneously into the neck or foreleg of immunodeficient mice (BALB / c nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The number of cells injected at each site was approximately 1 × 10-1. 7 Mice were carefully fed and, after 4-6 weeks, the growth of subcutaneous teratomas in immunodeficient mice was examined. Mice were then euthanized by cervical dislocation, and the teratoma tissue was extracted and collected for testing.

[0054] The results showed that spPSCs injected into nude mice could form teratomas, and immunohistochemical staining revealed that the teratomas contained tissues from the endoderm, mesoderm, and ectoderm. Figure 4 (B, C)

[0055] 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 composition for culturing ovine primed pluripotent stem cells, characterized by: The composition contains N2B27 basal medium, cytokine small molecules, the cytokine contains human Activin A, recombinant human FGF2 and IWR1; The addition concentration of the human Activin A in the cytokine is 25 ng / mL; the addition concentration of the recombinant human FGF2 is 12.5 ng / mL; and the addition concentration of the IWR1 is 5 μM.

2. The composition of claim 1, wherein: The N2B27 basal medium contains, in 500 mL, 227 mL DMEM / F12 (Thermo Fisher Scientific, 10565-018), 227 mL Neurobasal (Gibco, 21103-049), 2.5 mL N2 supplement (Gibco, 17502-048), 5.0 mL B27 supplement (Gibco, 12587-010), 2.5 mL 100× GlutaMAX (Gibco, 35050-061), 5.0 mL 100× nonessential amino acids (Gibco, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5.0 mL 100× penicillin-streptomycin (Gibco, 15140-122), 5% knockout serum replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544), and 2.5 μM ROCK inhibitor Y27632 (Selleckchem, S1049).

3. A culture medium for culturing ovine primed pluripotent stem cells, characterized in that: The active ingredient of the culture medium is the composition of claim 1 or 2.

4. A method of culturing ovine primed pluripotent stem cells, characterized by: Comprising the following steps: 1) selecting sheep in vivo embryos at the development stage of 12-14 days, mechanically removing hypoblast and trophoblast cells, and digesting the epiblast cells of the embryo to obtain epiblast small cell clusters; 2) preparing a four-well plate coated with feeder cells 12 h in advance, inoculating the digested epiblast small cell clusters in the feeder cells containing the culture medium of claim 3, and culturing for 4-6 days to obtain primary clones; 3) digesting the primary clones obtained in step 2) and reseeding in feeder cells containing the culture medium of claim 3 for subculture to obtain the sheep primed pluripotent stem cells.

5. The method of claim 4, wherein: The digestion in step 1) is TrypLE™ Express (Gibco, 12605010) digestion at 37°C for 3 minutes.

6. The method according to claim 4 or 5, characterized in that: Step 3) the digestion is Accutase (Gibco, A11105-01) digestion for 5 minutes at 37°C; and the culture temperature is 37°C.

7. Use of the composition of claim 1 or 2 or the medium of claim 3 in the manufacture of a product of ovine primed pluripotent stem cells.

8. Use of the composition of claim 1 or 2 or the medium of claim 3 in the manufacture of a product for increasing the cell density of ovine primed pluripotent stem cells.

9. Use of the method of any one of claims 4 to 6 in the manufacture of ovine primed pluripotent stem cells.

10. Use of the method of any one of claims 4 to 6 in increasing the cell density of ovine primed pluripotent stem cells.

Images