A method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts

Abstract

The present invention discloses a method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts. The method combines frozen blastocysts with a culture medium specific for the embryonic stem cell derivation stage to establish a stable bovine embryonic stem cell derivation system, achieving an 80% or greater probability of blastocyst adhesion and outgrowth, and an embryonic stem cell line establishment efficiency of 50% or greater. This method addresses the time-consuming issue of deriving bovine embryonic stem cells from fresh embryos, improves the efficiency and stability of bovine embryonic stem cell line establishment, facilitates the efficient establishment of in vitro breeding models, accelerates the breeding process, and reduces breeding costs, possessing significant practical value for the development of new dairy cattle breeds (lines).

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for deriving Holstein bovine embryonic stem cells from vitrified frozen blastocysts. Background Art

[0002] Embryonic stem cells are pluripotent cells isolated from the inner cell mass of mammalian blastocysts. They possess the ability to proliferate indefinitely, self-renew, and differentiate into all major cell lineages. Embryonic stem cells can be used for research in embryogenesis, tissue differentiation, drug screening, disease model development, transplantation therapy, animal breeding, and transgenic animal engineering. Therefore, embryonic stem cells have enormous potential for application in agriculture, biology, and medicine, and are a hot topic and a cutting-edge area in life science research.

[0003] The first animal embryonic stem cells to be isolated and established were mouse embryonic stem cells, with their results published in Nature and PNAS in 1981, respectively, by M.J. Evans and M.H. Kaufman of the University of Cambridge and Gail.R. Martin of the University of California. Subsequently, embryonic stem cells from macaques, humans, and rats were successfully isolated and established. In 2018, Y.S. Bogliotti isolated and established bovine embryonic stem cells from fresh bovine embryos, successfully establishing a bovine embryonic stem cell culture system similar to EpiSCs. This demonstrated the feasibility of using bovine embryonic stem cells in combination with genome-wide selection to construct in vitro breeding models, thus possessing significant commercial potential. Despite the ability to isolate and derive bovine embryonic stem cells in vitro, the efficiency of establishing bovine embryonic stem cell lines remains low, at around 30%. Therefore, addressing this low efficiency is crucial for achieving in vitro breeding of dairy cows.

[0004] Current research focuses on isolating embryonic stem cells from fresh bovine embryos. Fresh embryo development is continuous and time-sensitive, and the efficiency and pluripotency of embryonic stem cell lines isolated from bovine embryos at different developmental stages vary. Therefore, the efficiency and pluripotency of embryonic stem cell lines are closely related to the stage of the donor embryo. Establishing embryonic stem cell lines for in vitro breeding requires a large number of embryos. Using fresh blastocysts for in vitro breeding of embryonic stem cell lines requires rapid isolation of embryonic stem cells within a specific timeframe of embryonic development. Using large numbers of fresh embryos for embryonic stem cell line establishment not only requires significant manpower and material resources, but also creates tight timelines and a significant workload, inevitably impacting the efficiency of embryonic stem cell line establishment. Using frozen embryos for bovine embryonic stem cell line establishment allows for the rapid cryopreservation of blastocyst-stage bovine embryos, enabling the mass isolation and establishment of embryonic stem cells, which could significantly address many of the challenges associated with fresh embryonic stem cell line establishment. However, current research on technologies for establishing bovine embryonic stem cell lines using frozen embryos remains lacking. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for deriving Holstein bovine embryonic stem cells from vitrified frozen blastocysts, comprising culturing the revived Holstein bovine vitrified frozen blastocysts in three stages of derivation using specific cell culture fluids; stage I: isolating the inner cell mass of the blastocyst to the attachment stage, using type I cell culture fluid: modified mTeSR medium + 8-10% FBS + 10-15uMY27632; stage II: from the inner cell mass attachment to the cell passaging stage, using type II cell culture fluid: modified mTeSR medium + 8-10% FBS; stage III: the cell culture stage 24 hours after passaging. Specific cell culture medium was formulated for each stage, and type III cell culture medium was used: modified mTeSR medium + 3-8% KSR; the modified mTeSR medium was prepared by adding a final concentration of 0.979mM GABA, 0.984uM Pipecolic Acid, 0.98mM LiCl, 20ng / mL Activin A, 20ng / mL human FGF2, 0.1mM β-mercaptoethanol and 2.5μM IWR1 to mTeSR1 medium.

[0008] As a preferred embodiment of the present invention, the steps of stage I are: using acidic benchtop liquid to treat the revived and expanded frozen blastocysts, removing the zona pellucida, cleaning the remaining blastocyst cells with type I cell culture medium, placing the blastocyst cells in a cell culture plate with a feeder layer, adding type I cell culture medium and culturing in a 37°C, 5% CO2 incubator for 6 days.

[0009] As a further preferred embodiment of the present invention, the type I cell culture medium is a modified mTeSR medium + 10% FBS + 10uM Y27632.

[0010] As a preferred embodiment of the present invention, the stage II step is as follows: after the cells are attached, the cell culture medium is replaced with type II cell culture medium, and the culture is continued for 3-5 days to allow cell clusters to form outgrowth, the outgrowth is digested with TrypLE for 2-3 minutes, DPBS is added to terminate the digestion, and the cells are gently blown into small cell clusters with a pipette, the supernatant is removed by centrifugation, the stage I culture medium is added, and the cells are evenly blown and inoculated on the MEF feeder layer and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0011] As a further preferred embodiment of the present invention, the type II cell culture medium is: modified mTeSR medium + 10% FBS.

[0012] As a preferred embodiment of the present invention, the stage III step is: replacing the cell culture medium used in the previous step of passage for 24 hours with the stage III culture medium, changing the medium every day, and continuing to culture for 3-5 days. When the cell density reaches 70%, digestion is carried out with TrypLE and passage is carried out at a ratio of 1:6.

[0013] As a further preferred embodiment of the present invention, the type III cell culture medium is: modified mTeSR medium + 5% KSR.

[0014] As a further preferred embodiment of the present invention, the steps are:

[0015] Stage I: Treat the expanded frozen blastocysts with acidic benchtop solution, remove the zona pellucida, and clean the remaining blastocyst cells with type I cell culture medium. Place the blastocyst cells in a 24-well cell culture plate with a feeder layer. Add 500 μL of type I cell culture medium and culture in a 37°C, 5% CO2 incubator for 6 days. After 24 hours, observe the cell attachment. For cell clusters that have not attached within 48 hours, press them with a 30g sterile needle to assist attachment. On the third day, add 250 μL of type I cell culture medium without changing the medium to prevent cells from being sucked away due to poor attachment.

[0016] Phase II: After cells attach, the cell culture medium is replaced with type II cell culture medium. Culture is continued for 3-5 days to allow cell clusters to form outgrowths. The outgrowths are digested with TrypLE for 2-3 minutes, and DPBS is added to terminate the digestion. The cells are gently pipetted into small cell clusters. The supernatant is removed by centrifugation, and the phase I culture medium is added. After pipetting evenly, the cells are inoculated onto the MEF feeder layer and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0017] Phase III: Replace the cell culture medium used in the previous step for 24 hours with the phase III culture medium. Change the medium daily and continue culturing for 3-5 days. When the cell density reaches 70%, digest with TrypLE and subculture at a ratio of 1:6.

[0018] As a preferred embodiment of the present invention, in vitro cultured Holstein bovine embryos are vitrified when they develop from the morula to the blastocyst stage. When establishing embryonic stem cell lines, frozen blastocysts are thawed in batches according to the required number, and the thawed blastocysts are cultured in a 38°C, 6% CO2 incubator. The thawed and expanded frozen blastocysts are used for stage I processing.

[0019] Beneficial effects:

[0020] Establishing a bovine embryonic stem cell line is a key step in establishing an in vitro breeding model for dairy cows. The efficiency of establishing a line and the stability of its pluripotency determine the efficiency and cost of the in vitro breeding model. Existing studies have isolated and derived bovine embryonic stem cells from fresh embryos. However, when the number of donor embryos is large, the time required is tight and the efficiency of establishing a line is low (30%), which cannot meet the needs of in vitro breeding. The present invention combines frozen blastocysts with a culture medium specific for the embryonic stem cell derivation stage to establish a stable bovine embryonic stem cell derivation system. The probability of blastocyst adhesion and outgrowth is ≥80%, and the efficiency of embryonic stem cell line establishment is ≥50%. The present invention solves the time-consuming issue of deriving bovine embryonic stem cells from fresh embryos, improves the efficiency and stability of bovine embryonic stem cell line establishment, facilitates the efficient establishment of in vitro breeding models, accelerates the breeding process, reduces breeding costs, and has important practical value for the development of new dairy cow breeds (lines). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Expanded blastocyst after 10 hours of vitrified blastocyst thawing.

[0022] Figure 2 :After the zona pellucida is removed, the blastocyst attaches and forms outgrowth;

[0023] Figure 3 :Bovine embryonic stem cells derived after passage

[0024] Figure 4 :Alkaline phosphatase staining of bovine embryonic stem cells

[0025] Figure 5 :Immunofluorescence staining to detect pluripotency factor expression DETAILED DESCRIPTION

[0026] The experimental methods described in the following examples are all conventional methods unless otherwise specified. The main reagents mTeSR (stem cell, 05896) and Y-27632 (stem cell, 72304) were purchased from Jiangsu Ruijie Biological Co., Ltd.; GABA (A5835), LiCl (746460), and Pipecolic Acid (P2519) were purchased from Sigma Biological Co., Ltd.; TRYPLE (Gibco, 12563011) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Activin A was purchased from Beijing Sino Biological Technology Co., Ltd.; and embryo vitrification thawing solution was purchased from Jinan Yuanqi Technology Co., Ltd. (ORIGIO, 12295002F).

[0027] (1) Preparation of cell culture medium

[0028] 1. Preparation of modified mTeSR medium

[0029] Modified mTeSR medium was prepared by adding 0.979 mM GABA, 0.984 μM Pipecolic Acid, 0.98 mM LiCl, 20 ng / mL Activin A, 20 ng / mL human FGF2, 0.1 mM β-mercaptoethanol, and 2.5 μM IWR1 to mTeSR1 (Cat. No. 05896).

[0030] 2. Type I cell culture medium: modified mTeSR medium + 10% FBS + 10uM Y27632;

[0031] 3. Type II cell culture medium: modified mTeSR medium + 10% FBS;

[0032] 4. Type III cell culture medium: modified mTeSR medium + 5% KSR.

[0033] Example 1

[0034] (1) Frozen blastocyst recovery

[0035] The thawing and recovery of vitrified blastocysts adopts a four-step method, and the embryo vitrification thawing solution includes: 1, 2, 3, and 4 solutions.

[0036] 1. Prepare thawing solution (preheat the reagents in advance: solution 1: 37°C, solutions 2 to 4: room temperature) and Pasteur pipettes;

[0037] 2. Remove the hot stage from the microscope, place it on the side of the microscope, and turn on the hot stage;

[0038] 3. Make 200 μl droplets on each of the two large dishes (make liquid 1 on one dish and place it on a hot plate, and make liquids 2, 3, and 4 on another dish and place it at room temperature);

[0039] 4. When using the small droplet made from liquid 1, take it from the hot stage to the microscope and put it back on the hot stage to keep warm after use;

[0040] 5. Quickly immerse the frozen carrier into Liquid 1, time for 1 minute, and transfer the embryos from Liquid 1 to the bottom of Liquid 2, time for 3 minutes;

[0041] 6. Transfer the embryos from liquid 2 to the bottom of liquid 3 and time for 5 minutes;

[0042] 7. Transfer the embryos from liquid 3 to the bottom of liquid 4 and rinse the embryos in different positions;

[0043] 8. Transfer the embryos to embryo culture medium and culture them in a 38°C, 6% CO2 incubator for 8 hours. Figure 1 shown.

[0044] (II) Establishment and passage of embryonic stem cell lines

[0045] Stage I: After the blastocyst expands (such as Figure 1 Treat the expanded blastocysts with acidic benchtop solution for 5 minutes to remove the zona pellucida. Rinse the remaining blastocyst cells with Type I cell culture medium. Place the blastocysts in a 24-well cell culture plate covered with a feeder layer. Add 500 μL of Type I cell culture medium and culture in a 37°C, 5% CO2 incubator for 6 days. After 24 hours, observe cell attachment. For cell clusters that have not attached within 48 hours, press with a 30g sterile needle to assist attachment. On the third day, add 250 μL of Type I cell culture medium without changing the medium to prevent loose attachment and cell aspiration.

[0046] Phase II: After the cells have attached, the cell culture medium is replaced with type II cell culture medium and culture is continued for 5 days until the cell clusters form outgrowth (e.g. Figure 2 The outgrowth was digested with TrypLE for 3 min, and then digested with DPBS. The cells were gently pipetted into small cell clusters and centrifuged at 1000 g for 5 min. The supernatant was removed, and the culture medium from stage I was added. After being pipetted evenly, the cells were inoculated onto the MEF feeder layer and cultured in a 37°C, 5% CO2 incubator.

[0047] Phase III: After 24 hours of cell passage, the cell culture medium was replaced with Phase III culture medium. The cells were cultured for 4 days after changing the medium every day. Figure 3 as shown), digested with TrypLE, and passaged 1:10.

[0048] (3) Pluripotency Verification

[0049] 1. Alkaline phosphatase detection

[0050] Discard the cell culture medium and add DPBS to wash the cells. Add 4% paraformaldehyde and fix at room temperature for 15 minutes; discard the paraformaldehyde and wash twice with DPBS. Add 500μL of BCIP / NBT working solution to each well and stain at room temperature in the dark for 15 minutes to 2 hours. Discard the staining solution; add DPBS to wash, stop staining with distilled water, and observe and photograph under an inverted microscope. Figure 4 .

[0051] 2. Immunofluorescence staining

[0052] Wash the cells slowly with 500uL DPBS 3 times, 5 minutes each time. Add 200uL of 4% paraformaldehyde to fix the cells and place them at room temperature for 15-30 minutes; wash the cells with DPBS 3 times, 5 minutes each time; permeabilize with 0.5% Triton X-100 for 20 minutes, wash 3 times with PBS, 5 minutes each time; block the cells with blocking solution for 1 hour; add primary antibody (diluted according to the primary antibody dilution ratio) and incubate at 4°C in the dark overnight; then, wash with PBS 3 times, 5 minutes each time; add secondary antibody (diluted according to the secondary antibody dilution ratio) and incubate at room temperature in the dark for 1 hour; wash the cells with PBS 3 times, 5 minutes each time; wash the cells once with DPBS, 5 minutes each time; add 200uL Hoechst33342 to stain in the dark for 5 minutes, wash the cells with PBS, and observe and photograph under a fluorescence microscope. Results are shown in Figure 5 .

[0053] (IV) Statistics on system building efficiency

[0054] Nine frozen blastocysts were thawed using the method of the present invention, of which eight outgrowths were generated by attachment, with a probability of 88% for blastocyst attachment and outgrowth. Six lines were established using outgrowth separation, and the efficiency of embryonic stem cell line establishment was 66.7%, which is significantly higher than the reported efficiency of fresh embryo line establishment (as shown in Table 1).

[0055] Table 1 Comparison of the efficiency of establishing bovine embryonic stem cell lines using different recovery methods

[0056] outgrowth generation rate bESCs lineage establishment efficiency Method of the present invention 88％ 66.7％ <![CDATA[CTFR [1] ]]> - 52％

[0057] References

[0058] [1]Bogliotti,YS,Wu,J.,Vilarino,M.,Okamura,D.,Soto,DA,Zhong,C.,Sakurai,M.,Sampaio,RV,Suzuki,K.,Izpisua Belmonte,JC,and Ross,PJ(2018)Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts.Proceedings of the National Academy of Sciences of the United States of America 115,2090-2095.

Claims

1. A method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts, characterized in that Specific cell culture media were used for the three stages of derivation of revived Holstein bovine vitrified frozen blastocysts; Stage I: Isolation of the inner cell mass of the blastocyst to the attachment stage, cultured in Type I cell culture media, the formula of Type I cell culture media is: modified mTeSR medium + 8-10% FBS + 10-15 μM Y27632; Stage II: From the attachment of the inner cell mass to the cell passaging stage, after the cells attach, the cell culture media is replaced with Type II cell culture media, and culture is continued for 3-5 days to form in vitro derivatives (outgrowth) of the cell mass, which are digested with TrypLE for 2-3 minutes, and DPBS is added to terminate the digestion. The cells are gently blown into small cell clusters with a pipette, and the supernatant is removed by centrifugation. The Stage I culture media is added, and after being blown evenly, they are inoculated on the MEF feeder layer and placed in a 37°C, 5% The cells were cultured in a CO2 incubator for 24 hours, and the type II cell culture medium was modified mTeSR medium + 8-10% FBS; stage III: the cell culture stage 24 hours after passaging, using type III cell culture medium: modified mTeSR medium + 3-8% KSR; the modified mTeSR medium was prepared by adding a final concentration of 0.979 mM GABA, 0.984 μM Pipecolic Acid, 0.98 mM LiCl, 20 ng / mL Activin A, 20 ng / mL human FGF2, 0.1 mM β-mercaptoethanol and 2.5 μM IWR1 to mTeSR1 medium.

2. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to claim 1, characterized in that The steps of the described stage I are: using acidic benchtop liquid to treat the thawed and expanded frozen blastocysts, removing the zona pellucida, washing the remaining blastocyst cells with type I cell culture medium, placing the blastocyst cells in a cell culture plate with a feeder layer, adding type I cell culture medium and culturing in a 37°C, 5% CO2 incubator for 4-7 days.

3. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to claim 2, characterized in that The type I cell culture medium is modified mTeSR medium + 10% FBS + 10 uM Y27632.

4. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to claim 1, characterized in that The type II cell culture medium: modified mTeSR medium + 10% FBS.

5. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to claim 1, characterized in that The stage III step is as follows: the cell culture medium used in the previous step for passage for 24 hours is replaced with the stage III culture medium, the cells are cultured for 3-5 days after the medium is changed every day, and when the cell density reaches 70%, they are digested with TrypLE and passaged at a ratio of 1:

6.

6. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to claim 5, characterized in that The type III cell culture medium: modified mTeSR medium + 5% KSR.

7. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to any one of claims 1 to 6, characterized in that Contains steps: Stage I: Treat the thawed and expanded frozen blastocysts with acidic benchtop solution, remove the zona pellucida, and wash the remaining blastocyst cells with type I cell culture medium. Place the blastocyst cells in a 24-well cell culture plate with a feeder layer. Add 500 μl of type I cell culture medium and culture in a 37°C, 5% CO2 incubator for 6 days. After 24 hours, observe the cell attachment. For cell clusters that have not attached within 48 hours, press them with a 30g sterile needle to assist attachment. On the third day, add 250 μl of type I cell culture medium without changing the medium to prevent cells from being sucked away due to poor attachment. Phase II: After cells attach, the cell culture medium is replaced with type II cell culture medium. Culture is continued for 3-5 days to allow cell clusters to form in vitro derivatives. The in vitro derivatives are digested with TrypLE for 2-3 minutes, and then digested with DPBS. The cells are gently pipetted into small cell clusters. The supernatant is removed by centrifugation, and the phase I culture medium is added. After pipetting evenly, the cells are inoculated onto the MEF feeder layer and cultured in a 37°C, 5% CO2 incubator for 24 hours. Phase III: Replace the cell culture medium used in the previous step for 24 hours with the phase III culture medium. Change the medium daily and continue culturing for 3-5 days. When the cell density reaches 70%, digest with TrypLE and subculture at a ratio of 1:

6.

8. The method for deriving Holstein bovine embryonic stem cells from vitrified blastocysts according to claim 7, characterized in that Holstein bovine embryos cultured in vitro were vitrified when they developed from the morula to the blastocyst stage. When establishing embryonic stem cell lines, frozen blastocysts were thawed in batches according to the required number and cultured in a 37°C, 5% CO2 incubator. The thawed and expanded frozen blastocysts were used for stage I processing.

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