Construction method and application of bovine originating embryonic stem cell line

Abstract

The invention relates to the technical field of biotechnology, and discloses a construction method of a bovine originating embryonic stem cell line, which comprises the following steps: (1) obtaining in vitro fertilized bovine early blastocyst; (2) trophoblast cells of the blastocyst are removed, an inner cell cluster is inoculated into a culture system containing a feeder layer, and the culture system is a basic culture medium added with FGF2 and WNT pathway inhibitors; and (3) culturing until an inner cell cluster forms a cell set drop, and carrying out digestion passage to obtain the stably proliferated bovine originated embryonic stem cell line. According to the construction method and the application of the bovine embryonic stem cell line, a specific initial induction combination of 'FGF2 + WNT inhibitor IWR1' is adopted, and a cell mass in an early blastocyst is used for establishing the line, so that the bovine embryonic stem cell line which is uniform in form, can be stably passaged for more than 26 generations and still keeps typical embryonic characteristics is successfully obtained; the problems of unstable state and easy differentiation of the bovine stem cell line in the prior art are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a method for constructing a bovine naive embryonic stem cell line and application thereof. BACKGROUND

[0002] Blastocysts are used to establish bovine stem cell lines representing different pluripotent states, such as "naive" cells with stronger somatic cell differentiation tendency. However, the existing technology focuses more on the preliminary establishment and basic characterization of cell lines, and the obtained cell lines often face problems such as unstable state, slow proliferation, unclear pluripotency maintenance mechanism, etc.

[0003] The main reason for the above problems is that the existing culture system is mostly based on the research experience of mouse or human stem cells, without fully considering the species specificity of cattle as a ruminant in terms of embryonic development timing, signal pathway dependence and epigenetic regulation. For example, how the core signal pathways (such as WNT, TGF-β / Activin) and epigenetic factors that drive the stable maintenance of bovine naive pluripotency work together is still a "black box" that has not been systematically analyzed, which restricts the establishment of stable and efficient bovine stem cell culture system and its large-scale application in breeding practice. SUMMARY

[0004] To solve the problems raised in the background art, the present application provides the following technical solution: a method for constructing a bovine naive embryonic stem cell line, comprising the following steps:

[0005] (1) obtaining an in vitro fertilized bovine early blastocyst;

[0006] (2) removing the trophoblast cells of the blastocyst and inoculating the inner cell mass into a culture system containing a feeder layer, wherein the culture system is a basic culture medium supplemented with FGF2 and a WNT pathway inhibitor;

[0007] (3) after the inner cell mass forms cell colonies, digestion and subculture are performed to obtain a stable proliferating bovine naive embryonic stem cell line.

[0008] Preferably, in step (2), the basic culture medium is N2B27 medium; and the WNT pathway inhibitor is IWR1.

[0009] Preferably, during the subculture process, IWR1 in the culture system is replaced with Activin A to form NBAF culture conditions, so as to maintain and optimize the pluripotency state and proliferation ability of the stem cells.

[0010] Preferably, in step (1), the bovine early blastocyst is a blastocyst cultured in vitro for 7 days.

[0011] Preferably, in step (3), the cell line can maintain the original morphology and pluripotency when subcultured to the 26th generation.

[0012] Preferably, the constructed bovine naive embryonic stem cell line expresses pluripotency marker genes OCT4, NANOG and SOX2, and is positive for alkaline phosphatase staining.

[0013] Preferably, the constructed bovine naive embryonic stem cell line has the ability to differentiate into ectoderm, mesoderm and endoderm in vitro.

[0014] Preferably, the constructed bovine naive embryonic stem cell line can integrate and form a chimeric blastocyst after injection into a bovine 8-cell embryo.

[0015] The bovine naive embryonic stem cell line constructed by the method has the application in preparing biological agents for beef cattle genetic breeding or studying bovine reproductive development and pluripotency regulation pathway models.

[0016] A bovine naive embryonic stem cell line cultured and maintained under NBAF culture conditions.

[0017] Compared with the prior art, the present application provides a construction method of a bovine naive embryonic stem cell line and its application, which has the following beneficial effects:

[0018] 1. The construction method of the bovine naive embryonic stem cell line and its application successfully obtains a bovine embryonic stem cell line with uniform morphology, which can be stably subcultured for more than 26 generations and still maintain typical naive characteristics, by using the specific initial induction combination of “FGF2 + WNT inhibitor IWR1” and using early blastocyst inner cell mass for line construction, solving the problem of unstable state and easy differentiation of bovine stem cell lines in the prior art.

[0019] 2. The construction method of the bovine naive embryonic stem cell line and its application discovers and uses Activin A to replace IWR1 to form NBAF culture conditions, which not only significantly speeds up the proliferation rate of stem cells, but also further improves the expression level of core pluripotency genes (such as NANOG), providing technical support for obtaining a large amount of high-quality bovine stem cells.

[0020] 3. The construction method of the bovine naive embryonic stem cell line and its application proves by removing that Activin A is a key factor for maintaining the morphology and state of the cell line constructed by the present application, and FGF2 is not essential, which deepens the understanding of bovine specific pluripotency regulation network and has important theoretical value.

[0021] 4、The method for constructing the bovine primed embryonic stem cell line and the application thereof, after strict verification, the cell line constructed by the application not only expresses core pluripotency markers and has the ability to differentiate into three germ layers in vitro, but also exhibits high embryonic chimeric ability in in vivo experiments, so that it becomes an ideal model for studying the early development mechanism of bovine and provides a core germplasm resource for genetic improvement of beef cattle through stem cell-mediated gene editing.

[0022] 5、The method for constructing the bovine primed embryonic stem cell line and the application thereof, by using the cell line and culture system provided by the application, the influence of different signal pathways and epigenetic factors (such as WNT, LIF, TSA, etc.) on the state of bovine stem cells can be efficiently and accurately screened and evaluated, which provides an irreplaceable platform tool for finally clarifying the unique regulation network. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A diagram for establishing a bovine primed embryonic stem cell by the application from an IVF blastocyst;

[0024] Figure 2 A diagram for detecting the expression of pluripotency marker genes of embryonic stem cells by immunofluorescence in the application;

[0025] Figure 3 A diagram for detecting the expression of pluripotency marker genes of embryonic stem cells by qPCR in the application;

[0026] Figure 4 A diagram for AP staining and cell proliferation rate detection in the application;

[0027] Figure 5 A diagram for cell proliferation characteristics under different conditions and qPCR detection of pluripotent gene changes in the application;

[0028] Figure 6 A diagram for the influence of removing FGF2 and Activin A on pluripotency in the application;

[0029] Figure 7 A diagram for the chimeric ability of bovine primed embryonic stem cells in the application;

[0030] Figure 8 A diagram for the influence of epigenetic factors and signal pathways on SOX2 expression in the application;

[0031] Figure 9 A diagram for the influence of epigenetic factors and signal pathways on cell proliferation in the application;

[0032] Figure 10 A diagram for in vitro formation of EB spheres in the application;

[0033] Figure 11 A diagram for directional three-germ layer differentiation of bovine primed embryonic stem cells in the application;

[0034] Figure 12 Figure 1. Chimeric ability of bovine primed embryonic stem cells. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0036] Referring to Figures 1-12 The present application provides a technical solution: a construction method of a bovine naive embryonic stem cell line and application thereof.

[0037] Embodiment 1: Basic construction of bovine naive embryonic stem cell line

[0038] 1. Embryo preparation: bovine early embryos were obtained by in vitro fertilization technology, and cultured in vitro to the 7th day, and expanded blastocysts with good morphology were selected.

[0039] 2. Inner cell mass separation: the trophoblast cells of the blastocyst were removed by mechanical method to obtain the complete inner cell mass.

[0040] 3. Initial inoculation and culture: the inner cell mass was transferred in advance to a culture plate supplemented with mouse fetal fibroblasts as a feeder layer. The culture system was N2B27 basic culture medium, and 20 ng / mL of human basic fibroblast growth factor and 5 μM of WNT pathway inhibitor IWR1 were added.

[0041] 4. Acquisition and passage of primary clones: 5-7 days after inoculation, the inner cell mass adhered and formed outgrowth. Continue to culture to form cell colonies with clear boundaries, use trypsin-EDTA solution for digestion, and use mechanical method to pick good morphology clones, transfer to new feeder layer for passage, recorded as the first generation. After passage, the cells gradually formed flat clones with uniform morphology, clear boundaries and high nuclear-cytoplasmic ratio, which is the typical morphology of bovine naive embryonic stem cells.

[0042] 5. Stable maintenance of stem cells: through regular passage once every 5-7 days, the cell line can stably proliferate for more than 26 generations, and still maintain its typical naive state morphology after multiple passages.

[0043] Embodiment 2: Optimization of stem cell culture system

[0044] After the cell line constructed in Example 1 was passaged to the 5th generation, the culture system was adjusted to optimize the proliferation rate and pluripotency state of the cell line:

[0045] IWR1 in the culture system was replaced with 20 ng / mL Activin A while FGF2 was retained, and this condition was recorded as NBAF culture condition.

[0046] It was found that, compared with the original IWR1 system, the proliferation rate of the cells cultured under the NBAF condition was significantly improved, and the colony morphology was more compact and better raised.

[0047] Example 3: Verification of pluripotency of bovine naive embryonic stem cells

[0048] The bovine naive embryonic stem cell line constructed and maintained by the methods of Examples 1 and 2 (selected at the 15th generation) was subjected to pluripotency identification:

[0049] 1. Immunofluorescence staining: After fixation and permeabilization of the cells, staining was performed using anti-OCT4, anti-NANOG and anti-SOX2 primary antibodies and corresponding fluorescent secondary antibodies. The results showed that more than 95% of the cell nuclei highly expressed SOX2 protein, and OCT4 and NANOG proteins were also positively expressed.

[0050] 2. qPCR detection: After extraction of total RNA and reverse transcription into cDNA, quantitative PCR was used to detect the expression of pluripotency genes. The results showed that, compared with the bovine fibroblast control group, the mRNA expression levels of endogenous OCT4, NANOG and SOX2 genes in the stem cell line were significantly up-regulated.

[0051] 3. Alkaline phosphatase staining: The cells were stained using an alkaline phosphatase detection kit. The results showed that the cell colonies showed strong positive staining (dark purple), indicating that the cell line had high levels of alkaline phosphatase activity.

[0052] The above results collectively indicate that the cell line constructed in the present application has typical pluripotent stem cell characteristics.

[0053] Example 4: Verification of in vitro differentiation potential of stem cells

[0054] To verify the multi-directional differentiation ability of the constructed cell line, a pseudo-embryoid body formation experiment was performed:

[0055] 1. The stem cells were digested into a single cell suspension and transferred to a low-adhesion culture dish for suspension culture in N2B27 medium containing 5% FBS.

[0056] 2. After 3-5 days, clear edge and compact structure pseudo-embryoid bodies were observed.

[0057] 3. Transfer the well-formed embryoid bodies to gelatin-coated culture plates for adherent culture, and continue differentiation for 1-2 weeks.

[0058] 4. Perform immunofluorescence staining on the differentiated cells, and use three embryonic layer-specific marker antibodies: β-III-Tublin (ectoderm), α-SMA (mesoderm), and Vimentin (endoderm). The results show that positive signals of the three markers can be detected simultaneously in the differentiation product, proving that the stem cell line has the potential to differentiate into the three germ layers.

[0059] Example 5: Verification of the in-vivo chimeric potential of the stem cells

[0060] To test the ability of the established stem cell line to participate in in-vivo development, a chimeric body formation experiment was performed:

[0061] 1. Construct a bovine primed embryonic stem cell line stably expressing H2B-mcherry fluorescent protein through lentiviral transduction.

[0062] 2. Use microinjection technology to inject 10-15 positive nucleus-positioned mCherry bovine stem cells into the perivitelline space of a bovine 8-cell stage embryo.

[0063] 3. Continue to culture the injected embryo in vitro to the blastocyst stage.

[0064] 4. Under a fluorescence microscope, obvious GFP-positive cells can be observed in the inner cell mass and trophoblast layer of the blastocyst, and these cells integrate well with the host embryo cells. This result shows that the stem cell line constructed by the present application has good chimeric ability and can participate in normal embryonic development.

[0065] Example 6: Application of stem cells in signal pathway research

[0066] 1. Use the bovine primed embryonic stem cell line constructed by the present application to study the effects of different signal pathways and epigenetic factors on the state of stem cells:

[0067] Under the NBAF basic culture conditions, the following factors are added respectively: histone deacetylase inhibitor TSA, histone methylation inhibitor DZnep, IWR1+LIF.

[0068] 2. After 72 hours of culture, it is found that the addition of TSA causes the cell morphology to deteriorate and differentiation signs to appear; the addition of DZnep makes the cell morphology deteriorate but does not cause obvious differentiation; the addition of IWR1 and LIF makes the clone smaller but still maintains SOX2 positivity.

[0069] 3. Through cell counting method to detect the proliferation speed, it is found that the addition of TSA promotes proliferation but is accompanied by differentiation, while the NBAF basic condition maintains pluripotency while maintaining a relatively optimal proliferation level.

[0070] This example demonstrates that this cell line is an excellent model to study the bovine specific pluripotency regulatory network and identifies a key role of Activin A signaling in maintaining the bovine naive state.

[0071] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are only examples of the principles and application of the application. Many variations and modifications, as well as alternatives, can be made to the embodiments without departing from the spirit and scope of the application, and all such variations and modifications and alternatives are to be included within the scope of the application as defined by the following claims and their equivalents.

Claims

1. A method for constructing a bovine naive embryonic stem cell line, characterized by, The method comprises the following steps: (1) obtaining bovine early blastocysts cultured in vitro; (2) removing the trophoblast cells of the blastocysts, inoculating the inner cell mass into a culture system containing a feeder layer, and adding FGF2 and a WNT pathway inhibitor to a basic culture medium; (3) after the inner cell mass forms cell colonies, performing digestion and subculture to obtain a stable bovine primed embryonic stem cell line.

2. The construction method of claim 1, wherein, In step (2), the basic culture medium is N2B27 culture medium, and the WNT pathway inhibitor is IWR1.

3. The construction method according to claim 1 or 2, characterized in that, During the subculture process, IWR1 in the culture system is replaced with Activin A to form NBAF culture conditions, so as to maintain and optimize the pluripotency state and proliferation ability of the stem cells.

4. The construction method of claim 1, wherein, In step (1), the bovine early blastocysts are blastocysts cultured in vitro for 7 days.

5. The construction method of claim 1, wherein, In step (3), the cell line can still maintain the primed state morphology and pluripotency when subcultured to the 26th generation.

6. The construction method of claim 1, wherein, The constructed bovine primed embryonic stem cell line expresses pluripotency marker genes OCT4, NANOG and SOX2, and is positive for alkaline phosphatase staining.

7. The construction method of claim 1, wherein, The constructed bovine primed embryonic stem cell line has the ability to differentiate into ectoderm, mesoderm and endoderm in vitro.

8. The construction method of claim 1, wherein, The constructed bovine primed embryonic stem cell line can integrate and form a chimeric blastocyst after being injected into a bovine 8-cell embryo.

9. The application of a bovine primed embryonic stem cell line constructed by the method of any one of claims 1-8 in preparing biological agents for beef cattle genetic breeding or in researching bovine reproductive development and pluripotency regulation pathway models.

10. A bovine primed embryonic stem cell line cultured and maintained by the NBAF culture conditions of claim 3.

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