Construction method of rat embryonic stem cells

Abstract

The invention belongs to the technical field of biology, and provides a method for constructing rat embryonic stem cells, which is characterized in that the rat embryonic stem cells are constructed by using an rLC3DM culture medium through two ways: 1, the rat embryonic stem cells are directly constructed from an SD strain rat blastocyst; secondly, the rat embryonic stem cells cultured under the 2i condition are converted into novel stem cells; the two methods effectively avoid the inherent defects of a traditional 2i culture system, including the technical problems that the cell genome stability is poor, the polyploidy proportion is abnormally increased, the germline transmission capacity is sharply reduced along with the increase of the passage times, and cells are spontaneously differentiated in the culture process. The novel rat embryonic stem cell established by the invention shows excellent genome stability and continuous totipotency characteristics in long-term culture, and is suitable for establishing a rat embryonic stem cell line with higher genome stability and long-term germline transmission capability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and relates to mammalian embryonic stem cell technology, specifically to a method for constructing rat embryonic stem cells. Background Technology

[0002] Rats are widely used laboratory animals, offering several advantages over mice. However, the application rate of transgenic mice far exceeds that of rats, a significant factor being the relatively underdeveloped research on rat embryonic stem cells. Transgenic rats require the generation of corresponding rat embryonic stem cells, and the first successful generation of rat embryonic stem cells was not achieved until 2008. Currently, the widely used rat embryonic stem cell culture conditions are based on the 2i (PD0325901, CHIR99021) culture conditions used for mice. However, rat embryonic stem cells cultured under 2i conditions have some drawbacks. First, rat embryonic stem cells cultured under 2i conditions are prone to genomic instability issues such as polyploidy during the culture process. One research team injected successfully gene-targeted rat embryonic stem cells into embryos but failed to obtain gene-targeted rats. A key reason for this was the presence of over 65% polyploid cells in the cells, leading to low germline transfer efficiency. Studies have shown that rat embryonic stem cells established using 2i conditions have germline transfer capability up to 7 generations, but beyond 7 generations, they cannot produce complete rat individuals. Therefore, the chromosomal abnormalities caused by these conditions will affect the further application of rat embryonic stem cells. In addition, rat embryonic stem cells cultured under 2i conditions will show differentiation after long-term culture.

[0003] Currently, the most widely used culture condition for rat embryonic stem cells is still 2i, but cells under this condition suffer from several drawbacks, including genomic instability and easy differentiation. In recent years, several research teams have been optimizing the culture conditions for rat embryonic stem cells. Studies have shown that using the small molecule combination YPAC (Y27632, PD0325901, A-83-01, CHIR99021) to establish rat embryonic stem cells under these conditions exhibits stem cell gene expression and germline transferability; however, the proportion of abnormal chromosomes increases with each generation. Furthermore, some studies have found that PKC inhibitors and PD0325901 support the establishment of multi-strain rat embryonic stem cells, but the germline transferability of cells under these conditions requires further verification. Optimization of rat embryonic stem cell culture conditions continues, but rat embryonic stem cells under these conditions still suffer from several drawbacks, including easy differentiation and genomic instability. The small molecule PD0325901 can cause chromosomal abnormalities in mouse embryonic stem cells. Since current rat embryonic stem cell culture media contain this small molecule, it is necessary to obtain rat embryonic stem cell culture conditions that are independent of PD0325901.

[0004] In 2017, a study established extended pluripotent stem cells (EPSCs) in humans and mice using the small molecule combination LCDM (LIF, CHIR99021, dimethindene maleate, and minocycline hydrochloride). These cells possess the molecular characteristics and developmental potential of embryonic stem cells. Compared to traditional embryonic stem cells, EPSCs have a higher germline transfer capacity and can be rapidly used to obtain gene-edited mice through tetraploid compensation experiments, making them better seed cells. In the past two years, researchers have established porcine EPSCs using the same small molecule combination LCDM. The LCDM small molecule combination does not contain PD0325901, supporting the establishment of embryonic stem cells with a more stable genome. However, to date, rat embryonic stem cells have not been established using this small molecule combination. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing rat embryonic stem cells, which obtains rat embryonic stem cells by culturing in rLC3DM medium, in order to solve the problems of genomic instability, high polyploidy ratio, rapid loss of germline transferability and easy differentiation of rat embryonic stem cells under existing technologies (such as 2i culture system).

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a method for constructing rat embryonic stem cells, characterized in that: the novel rat embryonic stem cells are LCDM-rES, the construction method includes method one and method two, both of which use rLC3DM culture medium, method one is a novel rat embryonic stem cell established from SD strain rat blastocysts, and method two is a novel rat embryonic stem cell obtained from 2i-rES rat embryonic stem cells under 2i conditions.

[0007] Preferably, the method includes the following step:

[0008] S1. Embryo Acquisition and Pretreatment: Blastocysts were flushed from the uterus of SD strain rats at 3-6 days of gestation and the zona pellucida of the blastocysts was removed after treatment with a tert-type solution.

[0009] S2. Initial culture: The blastocysts after the zona pellucida was removed in step S1 were seeded onto pre-prepared feeder cells and cultured in an incubator, with the culture medium being replaced with fresh medium every other day.

[0010] S3. Clonal proliferation and passage: After the first culture, pick the raised cell clusters formed on the feeder layer, cut them into small pieces with a glass needle, and re-inoculate them onto new feeder layer cells;

[0011] S4. Obtaining embryonic stem cell lines: After the second culture, the obtained cell clones are rat embryonic stem cells.

[0012] Preferably, the benchtop liquid treatment time in step S1 is 1-5 min, the first culture time in step S3 is 4-8 days, and the second culture time in step S4 is 2-5 days.

[0013] Preferably, the steps of method two are as follows:

[0014] A. Cell conversion: Rat embryonic stem cells cultured for 2 hours were digested into single-cell suspensions using an enzyme dissociation solution (Accutase), seeded onto feeder cells, and cultured in rLC3DM medium, with the medium being replaced with fresh medium every other day;

[0015] B. Observation of morphological transformation: During the culture process, the morphology of cell clones gradually changed from heterogeneous to uniform raised clone morphology;

[0016] C. Stable passage culture: Passage is performed by Accutase digestion. During the culture process, the suspended cells in the supernatant need to be collected, centrifuged and resuspended before continuing the culture.

[0017] D. Cell line acquisition: The finally obtained stable passaged cells are the novel rat embryonic stem cells under the new rLC3DM culture system.

[0018] Preferably, the rLC3DM medium is a mixture of N2B27 basal medium and rLIF, CHIR99021, dimethindene maleate and minocycline hydrochloride, and the mixture is stored at low temperature.

[0019] Preferably, the rLIF content is 10 ng / ml, the CHIR99021 content is 3 μM, the Dimethindenemaleate content is 2 μM, the Minocycline hydrochloride content is 2 μM, and the storage temperature is 0~8℃.

[0020] Preferably, the N2B27 basal culture medium comprises DF12, Neurobasal, N2, B27, Glutamax, and NEAA.

[0021] Preferably, the volume ratio of the N2B27 basal culture medium components is DF12:Neurobasal:N2:B27:Glutamax:NEAA=96.5:96.5:1:2:2:2.

[0022] The beneficial effects of this invention are as follows: The key innovation of the rLC3DM medium used in this invention lies in its absence of PD0325901, which is used in existing mainstream culture systems. This fundamentally avoids the chromosomal abnormalities and polyploidy problems that this small molecule may induce, resulting in rat embryonic stem cells with higher genomic stability. Compared to cells cultured for 2 years that lose their germline transmission ability after 7 generations, the stem cells obtained by this invention can maintain their developmental pluripotency after long-term culture, exhibiting stable developmental capacity and providing a reliable cell source for preparing gene knockout or transgenic rat models. At the same time, this culture system can effectively maintain the undifferentiated state of cells, significantly reducing spontaneous differentiation during culture and ensuring the long-term stability of cell population homogeneity and stemness characteristics. Furthermore, this invention is the first to successfully apply the LCDM small molecule combination to a rat system, establishing a novel type of extended pluripotent rat stem cell. This is not only a significant breakthrough in existing rat embryonic stem cell technology but also lays a solid foundation for using rats, a model animal with greater advantages in physiological and neuroscience fields, to conduct more in-depth developmental biology research and precise gene editing. Attached Figure Description

[0023] Figure 1 This is an immunofluorescence staining map of stem gene expression detected in this invention, with a scale bar of 100 μm;

[0024] Figure 2 This is a teratoma staining detection diagram from the present invention. Teratoma staining detects the differentiation potential of novel rat embryonic stem cells with three germ layers. Scale bar: 100 μm.

[0025] Figure 3 In this invention, the proportion of polyploids is analyzed by flow cytometry, and those with a DNA content exceeding 4n are considered polyploids.

[0026] Figure 4 This refers to karyotype analysis in this invention;

[0027] Figure 5 These are morphological images of the 2i-rES and rL3CDM-rES clones established from rat blastocysts in this invention, with a scale bar of 100 μm. Detailed Implementation

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] It should be noted that rat LIF, CHIR99021, dimethindene maleate, and minocycline hydrochloride used in this invention are all commercially available.

[0032] Comparative Example 1: Preparation of 2i-rES

[0033] The uterus of 4.5-day-old pregnant SD rats was harvested, and blastocysts were flushed out. The blastocysts were placed in a Thyroid strain solution, and the zona pellucida surrounding them was removed. The zona-pellucida-removed blastocysts were then seeded onto feeder cells using 2 IU of medium. The raised structures were picked out with a glass needle and cut into small pieces. These pieces were then placed onto new feeder cells. After 3-4 days, most of the raised cell clusters would have grown clones, which are the first-generation cells. Subsequently, the cells can be digested with Accutase into single cells and seeded onto feeder cells using 2 IU of medium.

[0034] Example 1: Novel rat embryonic stem cells established from SD strain rat blastocysts

[0035] S1. Embryo Acquisition and Pretreatment: The uterus of 4.5-day-old pregnant SD rats was removed, and the blastocysts were flushed out using M2. The flushed blastocysts were then placed in a Teflon solution for 1 minute to remove the zona pellucida.

[0036] S2. Initial Culture: Blastocysts with the transparent bag removed are seeded onto feeder cells and placed in an incubator. All reagents used in embryo manipulation must be placed in the incubator in advance to equilibrate the temperature, and the culture medium should be replaced with fresh medium every other day.

[0037] S3. Clonal proliferation and passage: After 5-7 days, raised cell clusters will grow on the feeder cells. Use a glass needle to pick up the raised structures, cut them into small pieces, and place them on new feeder cells.

[0038] S4.3-4 days later, clones grow out, which are novel rat embryonic stem cells established from blastocysts.

[0039] The culture medium was rLC3DM, composed of N2B27 basal medium supplemented with rat LIF (10 ng / ml, Millipore, LIF3010), CHIR99021 (3 μM, Selleck, S2924), dimethindene maleate (2 μM, Tocris, 1425), and minocycline hydrochloride (2 μM, Santa Curz, 13614-98-7). The prepared medium was stored at 4°C for no more than one week.

[0040] The components of the N2B27 basal culture medium are shown in Table 1.

[0041] Table 1. Composition of N2B27 basal culture medium

[0042]

[0043] Example 2: Novel rat embryonic stem cells obtained from 2i-rES rat embryonic stem cells under 2i conditions

[0044] A. Cell conversion: After the 2i-rES cells reached confluence, they were digested into single cells with Accutase, resuspended in rLC3DM medium, and seeded onto feeder cells; the rLC3DM medium used was the same as in Example 1;

[0045] B. Observation of morphological transformation: Under rLC3DM conditions, the clonal morphology will gradually change from multiple cell morphologies to a uniform, raised clonal morphology.

[0046] C. Stable passage culture: Due to poor cell adhesion, the supernatant of the suspended clones needs to be collected during medium change and passage. Change the medium every other day. When changing the medium, aspirate the supernatant into a centrifuge tube, centrifuge, resuspend in fresh medium, and transfer to the original well. After about 3-4 days, the cells will reach confluence. Then passage the cells. When passaged, digest them into single cells with Accutase, centrifuge, and resuspend in fresh medium.

[0047] D. After approximately three generations or more, the cells reach a stable state, thus forming a novel type of rat embryonic stem cell.

[0048] Example 3 Detection

[0049] 1. Immunofluorescence staining detection

[0050] 1.1 Method

[0051] (a) Wash the cells three times with PBS;

[0052] (b) Fixation: Add an appropriate amount of 4% paraformaldehyde and treat at room temperature for 25 minutes;

[0053] (c) Wash three times with PBS, each time for 5 minutes;

[0054] (d) Permeability sealing: Add permeability sealing solution and treat at room temperature for 1 hour;

[0055] (e) Primary antibody incubation: Prepare primary antibody incubation solution with permeable blocking solution according to the ratio, add appropriate amount of primary antibody incubation solution, and incubate overnight at 4°C;

[0056] (f) Wash 3 times with PBS, 5 minutes each time;

[0057] (g) Secondary antibody incubation: Prepare secondary antibody incubation solution with secondary antibody dilution solution according to the ratio, add an appropriate amount of secondary antibody incubation solution, and treat at 37°C for 1 hour. From this step onwards, pay attention to avoiding light.

[0058] (h) Wash 3 times with PBS, 5 minutes each time;

[0059] (i) DAPI staining: Add DAPI to ensure that the cells are covered, and stain for 1-3 minutes;

[0060] (j) Wash 3 times with PBS, 5 minutes each time; observe and take pictures.

[0061] 1.2 Results

[0062] The novel rat embryonic stem cells (LCDM-rES) established using rLC3DM possess the basic characteristics of traditional pluripotent stem cells. Firstly, the expression levels of stem cell genes were analyzed using immunofluorescence staining. Similar to the rat embryonic stem cells established using rLC3DM, the novel rat embryonic stem cells expressed key pluripotency marker genes OCT4, SOX2, and NANOG. Figure 1 ).

[0063] 2. HE staining detection of teratomas

[0064] 2.1 Method

[0065] Cells were injected subcutaneously into the back of immunodeficient mice. After approximately 3-6 weeks, the mice were sacrificed, and the tumor tissue (teratoma, no more than 1.5 cm in diameter) was removed. The removed teratoma was fixed, first embedded in paraffin, then sectioned in paraffin, and finally stained with hematoxylin and eosin (HE).

[0066] The steps for HE staining are as follows:

[0067] (a) Sample preparation, including fixing the sample;

[0068] (b) Dewaxing: The sections are placed in xylene for dewaxing, and xylene is removed by gradient ethanol treatment;

[0069] (c) Hematoxylin staining: Immerse the slides in hematoxylin staining solution and rinse with tap water to remove the hematoxylin;

[0070] (d) Eosin staining: Immerse the slide in eosin staining solution;

[0071] (e) Dehydration and mounting: Dehydration is performed by gradient ethanol, followed by mounting with neutral resin;

[0072] (f) Take a photo.

[0073] 2.2 Results

[0074] Further analysis of cell differentiation potential using teratoma formation experiments showed that typical three germ layer tissues could be observed in teratomas formed from novel rat embryonic stem cells established by rLC3DM. Figure 2 Therefore, the novel rat embryonic stem cells established possess the molecular characteristics and differentiation potential of traditional embryonic stem cells.

[0075] 3. PI staining detection

[0076] 3.1 Method

[0077] The Cell Cycle and Apoptosis Detection Kit C1052 (Beyotime) was used. The experimental procedure is as follows:

[0078] (a) Preparation of cell samples: Prepare a cell sample of more than 1 x 10⁶. Digest the cells into single cells with Accutase and carefully aspirate the supernatant.

[0079] (b) Cell fixation: Resuspend cells in 1 ml of pre-chilled PBS. Slowly add the cell suspension to pre-chilled ethanol at -20°C, until the final ethanol concentration is 70-75%. Fix for 12-24 hours. After fixation, centrifuge for 5 minutes. Aspirate the supernatant. Wash cells with 1 ml of pre-chilled PBS, centrifuge again, and discard the supernatant.

[0080] (c) PI staining: Add 0.5 ml of PI staining solution to each sample, mix gently, and incubate at 37°C in the dark for 30 minutes.

[0081] Flow cytometry detection and analysis: Red fluorescence was detected using a BD FACSVerse flow cytometer. Finally, FlowJO software was used for cellular DNA content analysis and light scattering analysis.

[0082] 3.2 Conclusion

[0083] Rat embryonic stem cells established in 2i were seeded under rLC3DM conditions and cultured for 15 days or more. Analysis by PI staining revealed that the proportion of polyploid cells cultured in rLC3DM was significantly lower than that in cells cultured under 2i conditions. Figure 3Therefore, novel rat embryonic stem cells under rLC3DM conditions have a more stable genome.

[0084] 4. Karyotype analysis

[0085] 4.1 Methods

[0086] Cells were expanded into T25 culture flasks, with a cell confluence of 60-80%. Samples were then sent to Hangzhou Polar Gene Technology Co., Ltd. for karyotype analysis.

[0087] 4.2 Results

[0088] Further passage of novel rat embryonic stem cells established under rLC3DM to the 22nd generation revealed no polyploidy in karyotype analysis. Simultaneously, rat embryonic stem cells established under 2i conditions were passaged to the 21st generation, and karyotype analysis showed that 25% of the cells had 80-84 chromosomes, indicating polyploidy. Figure 4 Therefore, the novel rat embryonic stem cells established by rLC3DM in this study have a more stable genome, providing better seed cells for obtaining genetically modified rats.

[0089] 5. Clonal morphology analysis

[0090] 5.1 Method

[0091] On days 2-3 of cell culture, the clonal morphology was photographed using a regular microscope.

[0092] 5.2 Results

[0093] Unlike the diverse clonal morphologies observed under the 2i condition, cells in rLC3DM maintained a uniform, undifferentiated, raised clonal morphology even after long-term passage. Therefore, this novel rat embryonic stem cell line exhibits a more stable clonal morphology and is more stable in undifferentiated behavior after long-term passage. Figure 5 ).

[0094] In summary, this invention establishes a novel rat embryonic stem cell based on culture conditions independent of PD0325901, and this cell possesses the characteristics of traditional rat embryonic stem cells, has a more stable genome, and does not differentiate after long-term passage.

[0095] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for constructing rat embryonic stem cells, characterized in that: The novel rat embryonic stem cells are LCDM-rES. The construction method includes method one and method two. Both method one and method two use rLC3DM medium. Method one is a novel rat embryonic stem cell established from SD strain rat blastocysts. Method two is a novel rat embryonic stem cell obtained from 2i-rES rat embryonic stem cells under 2i conditions.

2. The method for constructing rat embryonic stem cells according to claim 1, characterized in that: The method consists of the following steps: S1. Embryo Acquisition and Pretreatment: Blastocysts were flushed from the uterus of SD strain rats at 3-6 days of gestation and the zona pellucida of the blastocysts was removed after treatment with a tert-type solution. S2. Initial culture: The blastocysts after the zona pellucida was removed in step S1 were seeded onto pre-prepared feeder cells and cultured in an incubator, with the culture medium being replaced with fresh medium every other day. S3. Clonal proliferation and passage: After the first culture, pick the raised cell clusters formed on the feeder layer, cut them into small pieces with a glass needle, and re-inoculate them onto new feeder layer cells; S4. Obtaining embryonic stem cell lines: After the second culture, the obtained cell clones are rat embryonic stem cells.

3. The method for constructing rat embryonic stem cells according to claim 2, characterized in that: The benchtop liquid treatment time in step S1 is 1-5 min, the first culture time in step S3 is 4-8 days, and the second culture time in step S4 is 2-5 days.

4. The method for constructing rat embryonic stem cells according to claim 1, characterized in that: The steps of method two are as follows: A. Cell conversion: 2i cultured rat embryonic stem cells were digested into single-cell suspensions using an enzymatic dissociation solution, seeded onto feeder cells, and cultured in rLC3DM medium, with fresh medium replaced every other day; B. Observation of morphological transformation: During the culture process, the morphology of cell clones gradually changed from heterogeneous to uniform raised clone morphology; C. Stable passage culture: Passage is performed by digestion with enzyme dissociation solution. During the culture process, the suspended cells in the supernatant need to be collected, centrifuged and resuspended before continuing the culture. D. Cell line acquisition: The finally obtained stable passaged cells are the novel rat embryonic stem cells under the new rLC3DM culture system.

5. The method for constructing rat embryonic stem cells according to claim 1, characterized in that: The rLC3DM medium is a mixture of N2B27 basal medium and rat LIF, CHIR99021, dimethylindole maleate and minocycline hydrochloride, and the mixture is stored at low temperature.

6. The method for constructing rat embryonic stem cells according to claim 5, characterized in that: The ra t The LIF content is 10 ng / ml, the CHIR99021 content is 3 μM, the dimethylindidine maleate content is 2 μM, the minocycline hydrochloride content is 2 μM, and the storage temperature is 0~8℃.

7. The method for constructing rat embryonic stem cells according to claim 6, characterized in that: The N2B27 basal culture medium consists of DF12, Neurobasal, N2, B27, glutamine, and NEAA.

8. The method for constructing rat embryonic stem cells according to claim 7, characterized in that: The volume ratio of the N2B27 basal culture medium components is DF12:Neurobasal:N2:B27:glutamine:NEAA = 96.5:96.5:1:2:2:2.

Images