A bovine parthenogenetic haploid stem cell and its acquisition method and culture medium

Abstract

The present invention relates to the field of biotechnology, and in particular to a bovine parthenogenetic haploid stem cell, a method for obtaining the same, and a culture medium. Parthenogenetic activation is performed on mature bovine oocytes to obtain parthenogenetic embryos, the parthenogenetic embryos are cultured in an in vitro culture medium to blastocysts, the parthenogenetic blastocysts are placed in a culture medium for culture, and bovine parthenogenetic haploid stem cells are obtained; wherein the culture medium is obtained by the following method: 10‑20 ng / mL FGF2, 1.5‑2.5 μM Activin A, 3‑5 μM CHIR99021, and 1.5‑3 μM XAV939 are added to each 500 mL mTeSR1 basal culture medium. The present invention provides a bovine parthenogenetic haploid stem cell culture medium, and bovine parthenogenetic haploid stem cells are obtained, whose genome is derived from oocytes, has self-replication ability and pluripotency, and can also be applied to spindle complex replacement, solves the problem of unclear genetic information of oocytes from slaughterhouses, obtains reconstructed oocytes with clear genetic information, and provides a new approach for biological breeding of cattle.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a bovine parthenogenetic haploid stem cell and an acquisition method and a culture medium thereof. Background Art

[0002] In the process of sexual reproduction, sperm and egg combine to form a fertilized egg, which develops into a complete individual under the regulation of the genetic material of both parents, that is, mammals are all diploid. Embryonic stem cells (ESCs) can be obtained from fertilized eggs. ESCs have the characteristics of self-renewal, multidirectional differentiation and unlimited proliferation in vitro, and play an important role in medical and biological research.

[0003] Without the participation of sperm, oocytes can develop into blastocysts through chemical activation or electrical activation, from which parthenogenetic haploid embryonic stem cells (PG-haESCs) can be obtained, which contain only one set of chromosomes. These PG-haESCs have many similar characteristics to ESCs and are undifferentiated pluripotent stem cells. Because of their advantage in producing homozygous genotypes, they have been used in gene screening at the mammalian cell level. Currently, PG-haESCs have been obtained in humans, monkeys, mice and other animals, and a certain level of research has been carried out, but the establishment of PG-haESCs cell lines in large livestock such as cattle is still blank.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a bovine PG-haESCs and a method for obtaining the same and a culture medium, aiming to solve the problem that the establishment of the existing bovine PG-haESCs cell line is still blank.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a culture medium suitable for growing bovine PG-haESCs is provided, wherein the culture medium comprises a basal culture medium and a FACX culture medium, the basal culture medium is mTeSR1 basal culture medium (purchased from Stemcell, 85850), the FACX culture medium comprises fibroblast growth factor 2 (FGF2), activin A (Activin A), CHIR99021 (CAS No.: 252917-06-9) and XAV939 (CAS No.: 284028-89-3),

[0008] The culture medium is prepared by the following method: 10-20 ng / mL FGF2, 1.5-2.5 μM Activin A, 3-5 μM CHIR99021, and 1.5-3 μM XAV939 are added to each 500 mL mTeSR1 basal culture medium.

[0009] The second aspect of the present invention provides a method for obtaining bovine PG-haESCs, which comprises:

[0010] Step 1, in vitro maturation of oocytes obtained from bovine ovaries;

[0011] Step 2, selecting mature oocytes for parthenogenetic activation to obtain parthenogenetic embryos;

[0012] Step 3, culturing the parthenogenetic embryo in vitro to obtain a parthenogenetic blastocyst;

[0013] Step 4: Cultivate the parthenogenetic blastocyst in the culture medium suitable for the growth of bovine PG-haESCs as described in the present invention to obtain bovine PG-haESCs.

[0014] Optionally, step 1 specifically includes: culturing oocytes obtained from bovine ovaries in in vitro maturation fluid for 20-22 hours, removing cumulus granulosa cells using hyaluronidase, and selecting mature oocytes that have expelled the first polar body.

[0015] Optionally, the step 2 specifically includes: activating mature oocytes by ionomycin to obtain parthenogenetic embryos.

[0016] Optionally, step 2 specifically includes: treating the selected mature oocytes in 5-10 μmol / L ionomycin (I) for 10 min, then adding 10-20 μg / mL actinomycete copper (Cycloheximide, CHX), and culturing for 4-6 h at 38.5°C and 5% CO2 saturated humidity to obtain parthenogenetic embryos.

[0017] Optionally, the step 3 specifically includes: culturing the parthenogenetic embryo in in vitro G1 culture medium (purchased from Vitrolife, a bicarbonate buffered culture medium containing hyaluronic acid) for 72 h, then transferring to G2 culture medium (purchased from Vitrolife, providing the embryo with a variety of amino acids and hyaluronic acid to optimize blastocyst development), and culturing for 7.5 days at 38.5°C and 5% CO2 saturated humidity conditions until the blastocyst is obtained.

[0018] Optionally, the step 4 specifically includes: culturing the parthenogenetic blastocyst in the culture medium suitable for the growth of bovine PG-haESCs as described in the present invention, and sorting out the bovine PG-haESCs by flow cytometry.

[0019] Optionally, step 4 specifically includes: culturing the parthenogenetic blastocyst in the culture medium suitable for the growth of bovine PG-haESCs described in the present invention; staining the cultured stem cells with Hoechst fluorescent dye 33342 (Hoechst 33342); then screening the cells to remove adherent cells, and then sorting out the bovine PG-haESCs using a flow cytometer.

[0020] The third aspect of the present invention provides bovine PG-haESCs, which are obtained by using the method for obtaining bovine parthenogenetic haploid stem cells described in the present invention.

[0021] Beneficial effects: The present invention provides a culture medium suitable for the growth of bovine PG-haESCs, obtains bovine parthenogenetic haploid stem cells, and establishes a bovine parthenogenetic haploid stem cell cell line. The entire genome of the bovine parthenogenetic haploid stem cells of the present invention is derived from oocytes, has the self-replication ability and pluripotency of stem cells, and the obtained cell line is a haploid cell line, which can be used for gene screening at the cell level. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a morphological picture of the parthenogenetic blastocyst obtained by in vitro culture in Example 1. The scale bar is 100 μm.

[0023] Figure 2 This is a morphological picture of the first generation (P1 generation) of bovine PG-haESCs established from parthenogenetic blastocysts in Example 1. The scale bar is 100 μm.

[0024] Figure 3 This is a diagram showing the purification results of bovine PG-haESCs flow sorting haploid stem cells in Example 1, removing spontaneously diploidized stem cells.

[0025] Figure 4 This is an immunofluorescence image of bovine PG-haESCs expressing pluripotency marker genes Octamer-binding transcription factor (OCT4), sex-determining region transcription factor 2 (SRY-boxtranscription factor 2, SOX2), and orthodenticle Homeobox 2 (OTX2) in Example 1. The scale bar is 50 μm.

[0026] Figure 5This is a diagram showing the results of the karyotype experiment of bovine PG-haESCs in Example 1, with a scale bar of 25 μm. DETAILED DESCRIPTION

[0027] The present invention provides bovine PG-haESCs and a method for obtaining the same and a culture medium. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] The embodiment of the present invention provides a culture medium suitable for the growth of bovine PG-haESCs, wherein the culture medium comprises a basal culture medium and a FACX culture medium, the basal culture medium is an mTeSR1 basal culture medium, and the FACX culture medium comprises FGF2, Activin A, CHIR99021 and XAV939;

[0029] The culture medium is prepared by the following method: 10-20 ng / mL FGF2, 1.5-2.5 μM Activin A, 3-5 μM CHIR99021, and 1.5-3 μM XAV939 are added to each 500 mL of basic culture medium.

[0030] The embodiment of the present invention obtains bovine PG-haESCs by providing a culture medium suitable for the growth of bovine PG-haESCs. By referring to the screening method of bovine haploid stem cell (AG-haESCs) culture medium, three culture mediums (LCDM culture medium, CTFR culture medium and bEPSC culture medium) that can culture bovine AG-haESCs were screened out from the nine culture mediums that have been disclosed, but they cannot form a stable bovine haploid stem cell cell line. On this basis, different combinations of 11 cytokines involved in the above three culture media, including CHIR99021, Dimethindene maleate, Minocycline Hydrochloride, Leukemia Inhibitory Factor (LIF), IWR-1-endo (CAS No.: 1127442-82-3), FGF2, XAV939, WH-4-023 (CAS No.: 837422-57-8), Transforming Growth Factor-β (TGFβ), Activin A and Vitamin C, were made and added to the mTeSR1 basal culture medium. It was found that adding FACE culture medium (FGF2, Activin A, CHIR99021, IWR1-1-endo) to the mTeSR1 basal culture medium can enable the solitary androgenic blastocysts to form stem cell colonies and form stable cell lines.

[0031] In the FACE culture medium, FGF2 plays an important role in regulating cell survival, division, differentiation and migration, and can regulate the self-renewal of various stem cells; Activin A plays an essential role in maintaining the self-renewal and pluripotency of stem cells; CHIR99021, as an inhibitor of glycogen synthase-3 (GSK3) inhibitor, can promote the differentiation of stem cells into specific cell types and promote the proliferation of stem cells; IWR1-1-endo inhibits the Wnt / β-catenin pathway by maintaining the stability of Axin, a destructor of the Wnt pathway, and can inhibit the differentiation of stem cells during the stem cell culture process. However, replacing IWR1-1-endo with AVX939 in the screening of bovine PG-haESCs culture medium has better stem cell morphology. XAV939 is a specific blocker of the Wnt / β-catenin signaling pathway, which directly binds to the Wnt receptor to prevent the interaction between the Wnt protein and the receptor, thereby inhibiting the conduction of the Wnt signal. As a direct inhibitor of the Wnt / β-catenin pathway, AVX939 was selected in the embodiment of the present invention and prepared into FACX culture medium for subsequent culture of bovine PG-haESCs.

[0032] The embodiment of the present invention provides a method for obtaining bovine PG-haESCs, which comprises:

[0033] Step 1, in vitro maturation of oocytes obtained from bovine ovaries;

[0034] Step 2, selecting mature oocytes for parthenogenetic activation to obtain parthenogenetic embryos;

[0035] Step 3, culturing the parthenogenetic embryo in vitro to obtain a parthenogenetic blastocyst;

[0036] Step 4: Cultivate the parthenogenetic blastocyst in the culture medium suitable for the growth of bovine parthenogenetic haploid stem cells as described in the embodiments of the present invention to obtain bovine PG-haESCs.

[0037] The bovine PG-haESCs of the embodiment of the present invention are obtained by in vitro culture. First, the oocytes obtained from the bovine ovary are matured in vitro, and then the mature oocytes are parthenogenetically activated by ionomycin to obtain parthenogenetic embryos, and then the parthenogenetic embryos are cultured in an in vitro culture medium to blastocysts to obtain parthenogenetic blastocysts. The parthenogenetic blastocysts are cultured in the culture medium described in the embodiment of the present invention to obtain bovine PG-haESCs.

[0038] In one embodiment, step 1 specifically includes: culturing oocytes obtained from bovine ovaries in in vitro maturation fluid for 20-22 hours, removing cumulus granulosa cells using hyaluronidase (0.001 g / mL), and selecting mature oocytes that have expelled the first polar body (PB1) and are in the second meiotic metaphase (M II) for parthenogenetic activation.

[0039] Each 10 mL of maturation medium includes 9 mL of a solution consisting of M199 culture medium, 10 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid, and 1 mL of a solution consisting of fetal bovine serum, 0.01 μg / mL luteinizing hormone, and 0.01 μg / mL estradiol. The M199 culture medium was purchased from Gibco and contains phenol red, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and L-glutamine.

[0040] In one embodiment, step 2 specifically includes: activating mature oocytes by ionomycin to obtain parthenogenetic embryos.

[0041] Mature oocytes were activated by ionomycin, and embryos that expelled the first polar body were selected to make it easier to obtain bovine PG-haESCs later.

[0042] In a specific embodiment, step 2 specifically includes: placing the selected mature oocytes in 5-10 μmol / L I for 10 min, placing them in 10-20 μg / mL CHX, and culturing them at 38.5°C and 5% CO2 saturated humidity for 4-6 h to obtain parthenogenetic embryos.

[0043] In one embodiment, step 3 specifically includes: culturing the parthenogenetic embryo in G1 culture medium in vitro for 72 hours, then transferring it to G2 culture medium, and culturing it for 7.5 days under 38.5°C, 5% CO2 saturated humidity conditions until it becomes a blastocyst, thereby obtaining a parthenogenetic blastocyst.

[0044] In one embodiment, the step 4 specifically includes: culturing the parthenogenetic blastocyst in the culture medium suitable for the growth of bovine PG-haESCs as described in the embodiment of the present invention, and sorting out the bovine PG-haESCs by flow cytometry.

[0045] Bovine PG-haESCs were obtained by culturing P1 generation bovine PG-haESCs in a culture medium suitable for the growth of bovine PG-haESCs and maintaining the proportion of haploid stem cells by flow cytometry.

[0046] In one embodiment, step 4 specifically includes: after removing the zona pellucida from the parthenogenetic blastocyst, culturing in the culture medium suitable for the growth of bovine parthenogenetic haploid stem cells as described in the embodiment of the present invention; incubating the cultured stem cells at 37°C for 30 min with 10 μg / mL Hoechst33342, shaking the cells every 10 min to ensure uniform staining; then sieving the cells to remove adherent cells, and then sorting out the bovine parthenogenetic haploid stem cells using a flow cytometer.

[0047] By sorting using a flow cytometer, stem cells that spontaneously diploidized during cell culture were removed, the proportion of haploid stem cells was maintained (the proportion could be maintained at around 65-70%), and a bovine PG-haESCs cell line was established.

[0048] The embodiment of the present invention provides a bovine PG-haESCs, wherein the bovine PG-haESCs are obtained by using the method for obtaining bovine PG-haESCs described in the embodiment of the present invention.

[0049] The embodiment of the present invention uses a culture medium suitable for the growth of bovine PG-haESCs to prepare bovine PG-haESCs. The obtained bovine PG-haESCs can be applied to spindle transfer (ST), which can solve the problem of unclear genetic information caused by the bovine oocytes coming from slaughterhouses. Through the ST technology, the genetic material of the donor bovine oocytes is replaced with bovine PG-haESCs with clear genetic information. The reconstructed oocytes can be used for in vitro fertilization to obtain offspring with clear genetic information of the parents.

[0050] The entire genome of the bovine PG-haESCs described in the embodiment of the present invention is derived from oocytes, and has the self-replication ability and pluripotency of stem cells. The obtained cell line is a haploid cell line and can be used for gene screening at the cellular level.

[0051] The present invention will be further described below through several specific embodiments.

[0052] Example 1

[0053] 1. In vitro culture of parthenogenetic blastocysts

[0054] Oocytes were obtained from bovine ovaries by aspiration, cultured in in vitro maturation medium for 21 h, cumulus granulosa cells were removed by hyaluronidase (0.001 g / mL), and mature oocytes at the M II stage with the first polar body expelled were selected. After the mature oocytes were treated in 8 μmol / LI for 10 min, they were placed in 15 μg / mL CHX and cultured for 5 h at 38.5°C and 5% CO2 saturated humidity to obtain parthenogenetic embryos. Subsequently, the parthenogenetic embryos were transferred to G1 culture medium and cultured for 72 h. At 48 h, the cleavage cells were picked out and transferred to G2 culture medium. They were cultured at 38.5°C and 5% CO2 saturated humidity until blastocysts (culture time was 7.5 d) to obtain parthenogenetic blastocysts (see Figure 1 as shown).

[0055] 2. Cultivation and acquisition of bovine PG-haESCs

[0056] A culture medium suitable for growing bovine PG-haESCs is provided, wherein the culture medium is prepared by the following method: 15 ng / mL FGF2, 2 μM Activin A, 4 μM CHIR99021, and 2 μM XAV939 are added to each 500 mL mTeSR1 basal culture medium.

[0057] The obtained parthenogenetic blastocysts were subjected to the action of pronase to remove the zona pellucida, transferred to feeder cells, and cultured in a culture medium suitable for the growth of bovine PG-haESCs. The inner cell mass can grow on the feeder cells, thereby obtaining P1 bovine PG-haESCs (see Figure 2 As shown). The feeder layer cells refer to primary cells of mouse fetuses that are cultured and treated with mitomycin after they have grown all over the culture dish. The culture medium in the cell culture dish is discarded, and a culture medium containing 10 ug / mL mitomycin is added, and the cells are incubated in an incubator for 2 hours. After the incubation, the culture medium containing mitomycin is discarded. The cells in the culture dish are washed with PBS, and the washed cells are digested, centrifuged, and frozen. The treatment process converts the primary cells of the mouse from fibroblasts into feeder layer cells that cannot proliferate, providing support and nourishment for embryonic stem cells.

[0058] 3. Flow cytometry sorting and purification of bovine PG-haESCs

[0059] Haploid stem cells will spontaneously diploidize during the culture process. In order to maintain the proportion of haploid stem cells, they need to be continuously sorted by flow cytometry during the culture process. Stem cells that have been cultured for 5 generations were incubated with 10 μg / mL Hoechst33342 at 37°C for 30 min. Every 10 min, the cells were shaken to ensure uniform staining. Before sorting, the cells were sieved to remove adherent cells, and then placed in the loading chamber of the BD FACS Ariall SORP flow cytometer for sorting. When using the BD FACS Ariall SORP flow cytometer to sort bovine parthenogenetic haploid stem cells, all sorting steps are performed aseptically. Haploid cell populations are selected for collection, spontaneously diploidized stem cells are removed, and the stem cells are purified, such as Figure 3 As shown in the figure, with the increase of sorting times, the proportion of haploids increased from 20% to 67%.

[0060] 4. Detection of pluripotency marker genes in bovine PG-haESCs

[0061] Immunofluorescence experiments were used to examine the expression of bovine PG-haESCs pluripotent stem cell marker genes OCT4 (Santa Cruz, antibody ratio of 1:200), SOX2 (Santa Cruz, antibody ratio of 1:200) and Orthodenticle Homeobox 2, OTX2 (R&D Systems, antibody ratio of 1:250). Bovine PG-haESCs were cultured for 4 days. The culture medium was first discarded, washed once with phosphate buffered saline (PBS), and 4% paraformaldehyde was added to fix the cells. The cells were then permeabilized and blocked, incubated with primary antibodies, secondary antibodies, and 4',6-diamidino-2-phenylindole (DAPI), and finally anti-fluorescence quenching agents were added to seal the slides. Bovine PG-haESCs were observed using a NIKON A1R laser confocal microscope, and the results are shown in the figure. Figure 4 As shown, it indicates that bovine PG-haESCs express pluripotent stem cell marker genes and have pluripotency, wherein Merged indicates an integrated image, and 4',6-diamidino-2-phenylindole (DAPI) indicates staining of cell nuclei.

[0062] 5. Karyotype Analysis of Bovine PG-haESCs

[0063] Cows have 60 chromosomes, of which PG-haESCs have 30 chromosomes. The number of chromosomes of sorted bovine PG-haESCs was verified by karyotype analysis. PG-haESCs with good growth status after sorting were selected, and fresh culture medium with 0.2 μg / mL colchicine was added. The cells were incubated at 37°C for 4 h to arrest the cells in metaphase. Then the culture medium was discarded, the cells were digested with trypsin, and a 0.56% (w / v) KCl solution preheated at 37°C was added, mixed evenly, and treated with hypotonicity for 30 min. The hypotonic solution was then discarded, and 8 mL of methanol-glacial acetic acid fixative precooled at -20°C was slowly added to it, and fixed twice, each time for 10 min. The cells were resuspended with an appropriate amount of fixative, and the cell suspension was dripped about 40 cm above the -20°C precooled slide. Finally, the cells were stained to observe the number of chromosomes. The results are as follows Figure 5 As shown, Merged indicates integrated images, DAPI indicates staining of cell nuclei, and DIC indicates bright field images. The results show that there are 30 chromosomes, proving that this stem cell is a haploid stem cell.

[0064] The above results all prove that the stem cells obtained by the culture medium provided by the present invention are pluripotent and have only one set of chromosomes, and are bovine parthenogenetic haploid stem cells.

[0065] In summary, the present invention provides a bovine parthenogenetic haploid stem cell and a method for obtaining the same and a culture medium. First, the oocyte obtained from the bovine ovary is matured in vitro, and then the mature oocyte is parthenogenetically activated by ionomycin to obtain a parthenogenetic embryo, and then the parthenogenetic embryo is cultured in an in vitro culture medium to a blastocyst to obtain a parthenogenetic blastocyst. The parthenogenetic blastocyst is cultured in the culture medium described in the embodiment of the present invention to obtain bovine PG-haESCs. The culture medium includes a basal culture medium and a FACX culture medium, and the basal culture medium is an mTeSR1 basal culture medium, and the FACX culture medium includes FGF2, Activin A, CHIR99021, and XAV939, and each 500 mL of the basal culture medium includes 10-20 ng / mL FGF2, 1.5-2.5 μM ActivinA, 3-5 μM CHIR99021, and 1.5-3 μM XAV939. During the culture process of bovine PG-haESCs, the proportion of bovine PG-haESCs can be maintained at around 65-70% through flow cytometric sorting.

[0066] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for obtaining bovine parthenogenetic haploid stem cells, characterized in that: include: Step 1, in vitro maturation of oocytes obtained from bovine ovaries; Step 2, selecting mature oocytes for parthenogenetic activation to obtain parthenogenetic embryos; Step 3, culturing the parthenogenetic embryo in vitro to obtain a parthenogenetic blastocyst; Step 4, culturing the parthenogenetic blastocyst in a culture medium suitable for the growth of bovine parthenogenetic haploid stem cells to obtain bovine parthenogenetic haploid stem cells; The culture medium is composed of a basal culture medium and a FACX culture medium, wherein the basal culture medium is an mTeSR1 basal culture medium, and the FACX culture medium is composed of FGF2, Activin A, CHIR99021 and XAV939. The culture medium was prepared by the following method: 15 ng / mL FGF2, 2 μM Activin A, 4 μM CHIR99021, and 2 μM XAV939 were added to each 500 mL mTeSR1 basal culture medium.

2. The method for obtaining bovine parthenogenetic haploid stem cells according to claim 1, characterized in that: The step 1 specifically comprises: culturing oocytes obtained from bovine ovaries in an in vitro maturation solution for 20-22 hours, removing cumulus granulosa cells using hyaluronidase, and selecting mature oocytes that have expelled the first polar body.

3. The method for obtaining bovine parthenogenetic haploid stem cells according to claim 1, characterized in that: The step 2 specifically includes: activating mature oocytes by ionomycin to obtain parthenogenetic embryos.

4. The method for obtaining bovine parthenogenetic haploid stem cells according to claim 3, characterized in that: The step 2 specifically includes: treating the selected mature oocytes in 5-10 μmol / L ionomycin for 10 min, then adding 10-20 μg / mL cycloheximide, and culturing for 4-6 h at 38.5°C and 5% CO2 saturated humidity to obtain parthenogenetic embryos.

5. The method for obtaining bovine parthenogenetic haploid stem cells according to claim 1, characterized in that: The step 3 specifically comprises: culturing the parthenogenetic embryo in a G1 culture medium in vitro for 72 hours, then transferring the parthenogenetic embryo to a G2 culture medium, and culturing the parthenogenetic embryo for 7.5 days at 38.5°C and 5% CO2 saturated humidity until the embryo becomes a blastocyst, thereby obtaining a parthenogenetic blastocyst; Among them, G1 culture medium was purchased from Vitrolife and is a bicarbonate buffered medium containing hyaluronic acid. G2 culture medium was purchased from Vitrolife and provides embryos with a variety of amino acids and hyaluronic acid to optimize blastocyst development.

6. The method for obtaining bovine parthenogenetic haploid stem cells according to claim 1, characterized in that: The step 4 specifically includes: culturing the parthenogenetic blastocyst in a culture medium suitable for the growth of bovine parthenogenetic haploid stem cells; staining the cultured stem cells with Hoechst33342; then screening the cells to remove adherent cells, and then sorting out the bovine parthenogenetic haploid stem cells using a flow cytometer.

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