Method for culturing excited stem cells of sheep as well as culture medium and application thereof

Abstract

The invention discloses a culture method of excited stem cells of sheep, a used culture medium and application. The invention belongs to the technical field of biology, and particularly relates to a culture method of excited stem cells of sheep, a used culture medium and application. The composition for culturing the excited state pluripotent stem cells of the sheep contains an N2B27 basic culture medium, cell factors and small molecules, the cell factors and the small molecules contain human Activin A, recovery human FGF2 and IWR1, the adding concentration of the human Activin A is 25 ng / mL, the adding concentration of the recovery human FGF2 is 25 ng / mL, and the adding concentration of the recovery human FGF2 is 25 ng / mL. The addition concentration of the recovery hub FGF2 is 12.5 ng / mL, and the addition concentration of the recovery hub FGF2 is 12.5 ng / mL; the adding concentration of the IWR1 is 5 mu M. The culture medium prepared from the composition can obtain stable sheep excited state pluripotent stem cells, and can be applied to construction of sheep gene editing model animals.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for culturing sheep stimulated stem cells and a culture medium and application thereof. Background Art

[0002] The pluripotency state of the embryonic epiblast undergoes a transition from Naïve to Formative and then to Primed states, and is also the source of pluripotent stem cells (PSCs). PSCs cultured in vitro can reflect the pluripotency state of the embryonic epiblast development in vivo, and different culture conditions can also help to establish PSCs with different pluripotency states in vitro. So far, stable PSCs with different pluripotency states, such as Naïve, Formative and Primed, have been successfully established in mice, humans and monkeys. They differ in signaling pathways, gene regulatory networks, epigenetics and metabolism. Primed PSCs have similar molecular characteristics and pluripotency states to those of the post-implantation epiblast. In addition to expressing core pluripotency marker genes, POU5F1 , SOX2 , NANOG In addition, it also activates Primed pluripotency-related transcription factors OTX2 , POU3F1 , FGF5 In the process of transition from Naïve to Primed pluripotency, methyltransferase upregulation is accompanied by an increase in CpG genome-wide DNA methylation levels, and one X chromosome in female cells is randomly inactivated. POU5F1 The expression of the primed PSCs is regulated by proximal enhancer elements, and the metabolic mode shifts to glycolytic metabolism. In terms of in vivo developmental capacity testing, primed PSCs do not have the ability to form germline chimeras. The establishment of livestock embryonic stem cells is expected to promote the development of biomedicine and accelerate the breeding process of livestock animals. Sheep are one of the important livestock animals in my country's animal husbandry production. The lack of understanding of the regulatory pathways for preimplantation embryonic development and epiblast pluripotency in sheep has limited the application of sheep pluripotent stem cells in agriculture and biomedicine. Stable primed stem cells with a clear pluripotency state have not yet been successfully established. Therefore, there is an urgent need to clarify the regulatory mechanism of epiblast pluripotency in early sheep embryos, optimize the culture system, and establish stable primed PSCs in sheep. Summary of the Invention

[0003] The main problem to be solved by the present invention is how to establish and culture sheep stimulated pluripotent stem cells.

[0004] In order to solve the above problems, the present invention provides a composition for culturing sheep stimulated pluripotent stem cells The composition for culturing sheep stimulated pluripotent stem cells provided by the present invention contains N2B27 basal culture medium and cytokines, wherein the cytokines contain human Activin A, recombinant human FGF2 and IWR1.

[0005] In the above composition, the added concentration of the human Activin A in the cytokine is 25 ng / mL; the added concentration of the recombinant human FGF2 is 12.5 ng / mL; and the added concentration of the IWR1 is 5 μM.

[0006] In the above composition, the N2B27 basal medium (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).

[0007] The present invention also provides a culture medium for culturing sheep stimulated pluripotent stem cells, wherein the active ingredient of the culture medium is the composition described above.

[0008] The present invention also provides a method for culturing sheep stimulated pluripotent stem cells, comprising the following steps: 1) selecting a sheep embryo at developmental day 12-14, mechanically removing the hypoblast and trophoblast cells, and digesting the epiblast cells of the embryo to obtain epiblast cell clusters; 2) Prepare a four-well plate with feeder cells 12 hours in advance and inoculate the digested epiblast cell clusters in the culture medium described above. After culturing for 4-6 days, obtain primary clones. 3) Digesting the primary clones obtained in step 2), re-seeding them in the culture medium described above, and performing subculture to obtain the sheep stimulated pluripotent stem cells.

[0009] In the above method, the digestion in step 1) is performed using TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes.

[0010] In the above method, the digestion in step 3) is performed by using Accutase (Gibco, A11105-01) at 37°C for 5 minutes; and the incubation temperature is 37°C.

[0011] The use of the above-mentioned composition and culture medium in the preparation of sheep stimulated pluripotent stem cell products, or in the preparation of products that increase the cell density of sheep stimulated pluripotent stem cells also falls within the scope of protection claimed by the present invention.

[0012] The present invention also provides the use of the method described above in preparing a sheep stimulated pluripotent stem cell product, or in preparing a product for increasing the cell density of sheep stimulated pluripotent stem cells.

[0013] The sheep primed pluripotent stem cells obtained by the culture medium developed by the present invention can be stably passaged for a long time for more than 100 generations and have clear Primed pluripotency characteristics. It can be used in the preparation of sheep primed pluripotent stem cells and has application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This figure illustrates the regulatory mechanism of pluripotency in the epiblast of early sheep embryos. A shows the changes in pluripotency in the epiblast of early sheep embryos; B shows the expression of genes that represent markers of pluripotency in the naive, formative, and primed states of the epiblast of early sheep embryos; and C shows the expression levels of key genes in the pluripotency regulatory signaling pathway of the epiblast of early sheep embryos.

[0015] Figure 2The establishment and pluripotency characterization of spPSCs. A shows the morphology and AP staining of spPSCs cultured in the AFIWR1 culture system. Scale bar, 50 μm. B shows immunofluorescence staining of the pluripotency marker genes POU5F1, SOX2, and NANOG in spPSCs cultured in the AFIWR1 culture system, with DAPI staining of nuclei. Scale bar, 50 μm. C shows the morphology and AP staining of spPSCs cultured in the AFXAV939 culture system. Scale bar, 50 μm. D shows immunofluorescence staining of the pluripotency marker genes POU5F1, SOX2, and NANOG in spPSCs cultured in the AFXAV939 culture system, with DAPI staining of nuclei. Scale bar, 50 μm. E shows the morphology and AP staining of spPSCs cultured in the AFIWP2 culture system. Scale bar, 50 μm. F: Immunofluorescence staining of the pluripotency marker genes POU5F1, SOX2, and NANOG in spPSCs cultured in the AFIWP2 system. DAPI staining of cell nuclei. Scale bar, 50 μm. G: Immunofluorescence staining of histone H3K27me3 in spPSCs. DAPI staining of cell nuclei. Scale bar, 100 μm. H: Immunofluorescence staining of β-catenin in spPSCs. DAPI staining of cell nuclei. Scale bar, 50 μm.

[0016] Figure 3 Functional analysis of cytokines and small molecules in the spPSC culture system. A shows the clonal morphology of spPSCs after depletion of IWR1, Activin A, or FGF2 from AFI medium. B shows representative images of AP staining after depletion of IWR1, Activin A, or FGF2 from AFI medium. Scale bars in A and B, 100 μm. C shows immunofluorescence staining of the pluripotency marker proteins POU5F1, SOX2, and NANOG after removal of individual components, with DAPI staining indicating cell nuclei. Scale bar, 100 μm. D shows quantitative analysis of pluripotency marker gene expression levels after removal of individual components. E shows quantitative analysis of differentiation marker gene expression levels after removal of individual components.

[0017] Figure 4 The differentiation capacity of spPSCs was assessed in vitro and in vivo. A shows immunofluorescence staining of embryoid body differentiation, with DAPI staining of cell nuclei. Scale bar, 50 μm. B shows H&E staining of teratoma tissue, demonstrating ectoderm, mesoderm, and endoderm lineages. Scale bar, 100 μm. C shows immunofluorescence staining of teratoma tissue. Scale bar, 100 μm. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0019] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0020] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0021] The sheep used in the following examples were purchased from Tianjin Academy of Agricultural Sciences.

[0022] Animal Handling and Ethics Statement: All animal experiments involving mice and sheep described in the following examples were approved in advance by the Institutional Animal Care and Ethics Committee (IACUC) of China Agricultural University and the IACUC of Beijing Agricultural College. CD-1® (ICR) IGS mice and BALB / c nude mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and used for 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 establishment of embryonic pluripotent stem cells.

[0023] All experiments in the following examples were performed with three biological and technical replicates. Graphical presentation and statistical analysis were performed using GraphPad Prism 8.0. The values ​​in the graphs are expressed as mean ± standard deviation, and statistical significance was calculated using Student's two-tailed t-test. P Values ​​less than 0.05 were considered statistically significant and are shown as * P <0.05,** P <0.01, and *** P <0.001.

[0024] Example 1: Study on the Culture Medium and Culture Method of Sheep Stimulated Stem Cells 1. Establishment of Sheep Stimulated Pluripotent Stem Cell (spPSCs) Culture Medium The primed PSCs are derived from the epiblast of the embryo before gastrulation. From the single-cell data analysis of early sheep embryos, we know that the classic primed pluripotency genes such as WNT5B , PITX2 , EOMES etc. begin to express highly from the epiblast of E12 embryos ( Figure 1Therefore, we attempted to establish excited PSCs from E12-E14 embryos.

[0025] Analysis of the pluripotency regulatory network of the early sheep embryonic epiblast showed that the FGF and TGF-β signaling pathways were activated in the epiblast at E12-E14, and the maintenance of stimulated pluripotency required the addition of FGF2 and Activin A. Further analysis showed that E12 embryos began to express gastrulation-related genes, which is at the critical point of embryonic gastrulation. Therefore, inhibition of the WNT signaling pathway is very important for the self-renewal of PSCs ( Figure 1 Based on this, the present invention established 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.

[0026] Sheep primed pluripotent stem cells (spPSCs) were cultured using N2B27 basal medium supplemented with the following small molecules and cytokines: 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 substituted with XAV939 (5 ​​μM, Selleckchem, S1180) or IWP2 (5 μM, Selleckchem, S7085).

[0027] 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).

[0028] The culture method of sheep primed pluripotent stem cells (sfPSCs) is as follows: 1) Epiblast cells were isolated from sheep embryos at E12-E14 (hypoblast and trophoblast cells were mechanically removed and digested with TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes to separate the epiblast into small cell clusters). Single cells were then inoculated into culture medium containing primed pluripotent stem cells (Table 1). Primary colonies formed 4-5 days after inoculation with 100% efficiency. 2) Primary clones were digested with Accutase to single cells and seeded onto feeder cells in Primed Pluripotent Stem Cell Medium for subculture. Subculture was performed every 3-4 days at a ratio of 1:4 to 1:5. Before subculturing, cells were washed once with DPBS and then digested with Accutase (Gibco, A11105-01) for 5 minutes at 37°C. After digestion, cells were dissociated into single cells by pipetting, and the cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, cells were resuspended in Primed Pluripotent Stem Cell Medium and seeded onto feeder cells. Cultured in a 37°C incubator with 20% O2 and 5% CO2.

[0029] The clone morphology of spPSCs is flat, similar to mouse EpiSCs and primed hESCs. spPSCs AP staining is positive ( Figure 2 Middle A), expressing pluripotency marker proteins POU5F1, SOX2, and NANOG ( Figure 2 B), where IWR1 can be replaced by XAV939 and IWP2 ( Figure 2 At the epigenetic level, immunofluorescence staining of histone H3K27me3 showed that one of the X chromosomes in female spPSCs was inactivated ( Figure 2 Middle G). The expression of β-catenin, a key protein in the WNT signaling pathway, was detected. The results showed that most of the β-catenin protein was stable in the cytoplasm ( Figure 2 Middle H).

[0030] 2. The requirement for each factor in the culture medium was tested through factor reduction experiments. The specific components to be examined are shown in Table 1. Table 1. Information on various culture medium components

[0031] The study found that: 1) After IWR1 removal, spPSCs clones showed obvious differentiation morphology, AP staining activity decreased, and the expression of pluripotency genes POU5F1, SOX2, NANOG, and SALL4 was significantly downregulated ( Figure 3 In AD, the expression of mesendoderm differentiation-related genes CER1, BMP4, and GATA6 was significantly upregulated ( Figure 3 Middle E); 2) After Activin A was removed, spPSCs could not proliferate well, clones showed differentiated morphology, AP staining activity decreased, and the expression of the pluripotency gene NANOG was significantly downregulated ( Figure 3 AC).

[0032] 3) After FGF2 withdrawal, 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 (Medium AE).

[0033] The above results indicate that the AFWNTi (Activin A, FGF2 and WNT inhibitor) culture system is the basic culture condition for maintaining spPSCs self-renewal.

[0034] 3. In vitro and in vivo differentiation ability testing of spPSCs 1) Detection of spPSCs differentiation ability in vitro The specific detection method is as follows: Disintegrate spPSCs into single cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in 2 mL of fibroblast culture medium (Table 2) and transfer the cells to a 3.5 cm dish. Incubate the cells in a 37°C cell culture incubator on a shaker at 70 rpm / min to allow the cells to aggregate. After approximately 3-4 days, the cells will aggregate and form embryoid bodies (EBs). Select well-preserved EBs and seed them into 12-well plates. Approximately 6-7 days after the EBs have adhered and differentiated, fix the cells and perform immunofluorescence staining.

[0035] Table 2. Fibroblast culture medium

[0036] The analysis found that spPSCs were able to form embryoid bodies in vitro. EBs spontaneously differentiated into three germ layers after adherent culture, and the expression of NESTIN (ectoderm marker), α-SMA (mesoderm marker), and GATA6 (endoderm marker) was detected by immunofluorescence. Figure 4 Middle A).

[0037] 2) Detection of spPSCs differentiation capacity in vivo The specific detection method is as follows: spPSCs were digested into single cells, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 100 µL of spPSC culture medium was added to resuspend the cells and placed on ice. The cell suspension was aspirated with an insulin injection needle, and the air in the syringe was expelled. The cells were then injected subcutaneously into the neck or foreleg of immunodeficient mice (BALB / c nude mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). The number of cells injected at each site was approximately 1 × 10 7 The mice were carefully raised and the growth of subcutaneous teratomas in the immunodeficient mice was examined after 4-6 weeks. The mice were killed by cervical dislocation and the teratoma tissue was removed and sampled for testing.

[0038] The results showed that spPSCs injected into nude mice could form teratomas. Immunohistochemical staining showed that teratomas contained tissues of the inner, middle and outer germ layers ( Figure 4 (B, C).

[0039] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A composition for culturing sheep stimulated pluripotent stem cells, characterized in that: The composition contains N2B27 basal culture medium and cytokine small molecules, wherein the cytokine contains human Activin A, recombinant human FGF2 and IWR1.

2. The composition according to claim 1, wherein: The added concentration of the human ActivinA in the cytokines is 25 ng / mL; the added concentration of the recombinant human FGF2 is 12.5 ng / mL; and the added concentration of the IWR1 is 5 μM.

3. The composition according to claim 1 or 2, characterized in that: The N2B27 basal medium (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), 5.0 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), and 5% knockout serum. replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544) and 2.5 μM ROCK inhibitor Y27632 (Selleckchem, S1049).

4. A culture medium for culturing sheep stimulated pluripotent stem cells, characterized in that: The active ingredient of the culture medium is the composition according to any one of claims 1 to 3.

5. A method for culturing sheep stimulated pluripotent stem cells, characterized in that: The following steps are involved: 1) selecting a sheep embryo at developmental day 12-14, mechanically removing the hypoblast and trophoblast cells, and digesting the epiblast cells of the embryo to obtain epiblast cell clusters; 2) Prepare a four-well plate with feeder cells 12 hours in advance, inoculate the digested epiblast cell clusters onto the feeder cells containing the culture medium of claim 4, and culture for 4-6 days to obtain primary clones; 3) Digesting the primary clones obtained in step 2), re-seeding them into feeder layer cells containing the culture medium according to claim 4, and performing subculture to obtain the sheep stimulated pluripotent stem cells.

6. The method according to claim 5, characterized in that: In step 1), the digestion was performed using TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes.

7. The method according to claim 5 or 6, characterized in that: Step 3) The digestion was performed using Accutase (Gibco, A11105-01) at 37°C for 5 minutes; the incubation temperature was 37°C.

8. Use of the composition according to any one of claims 1 to 3 or the culture medium according to claim 4 in preparing a product of sheep-primed pluripotent stem cells, or in preparing a product for increasing the cell density of sheep-primed pluripotent stem cells.

9. Use of the method according to claims 5-7 in preparing a product of sheep-primed pluripotent stem cells, or in preparing a product for increasing the cell density of sheep-primed pluripotent stem cells.

Images