Construction method of human haploid blastocyst
By inducing human haploid embryonic stem cells to form blastocysts in vitro, a genetic screening problem in early human embryonic development research has been solved, providing a research model with similar structure and cellular composition.
Patent Information
- Application Number
- CN202510957925.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-11-21
AI Technical Summary
Currently, there is a lack of methods to construct human haploid blastocysts in vitro, which prevents their use for genetic screening studies of early human embryonic development.
Human haploid embryonic stem cells (hahESCs) were placed in a specific blastocyst induction system and induced to form human haploid blastocysts through a combination of aggregation and expansion culture media.
The constructed human haploid blastocyst has a similar structure and cellular composition to the human blastocyst, providing a genetic screening platform and research model for early human embryonic development.
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Figure CN120988978A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell culture technology, and in particular relates to a method for constructing human haploid blastocysts. Background Technology
[0002] Due to ethical and other limitations, our understanding of early human embryonic development is currently limited. Human embryonic stem cells (HECs) are pluripotent stem cells with self-renewal and multi-lineage differentiation potential, widely used in developmental biology and other research fields. They are ideal in vitro research models for early human embryonic development and have enormous application prospects in regenerative medicine. Although various structures simulating the post-implantation developmental stages of early human embryos have been generated using HECs, models that reproduce the embryogenetic events of human blastocysts are currently lacking. Human haploid embryonic stem cells (hahESCs) are derived from activated oocytes or semi-enucleated fertilized eggs. Due to their advantages such as single genome and stem cell characteristics, they are widely used in genetic screening research. However, it remains unclear whether it is possible to construct human haploid blastocyst-like cells in vitro for genetic screening research on the developmental process of early human embryos. Summary of the Invention
[0003] To address the aforementioned technical problems, this invention provides a method for constructing human haploid blastocysts.
[0004] The technical solution adopted in this invention is: a method for constructing human haploid blastocysts, wherein human haploid embryonic stem cells (hahESCs) are placed in a blastocyst induction system to culture human haploid blastocysts.
[0005] Preferably, the blastocyst induction system includes an aggregation culture medium, a first expansion culture medium, and a second expansion culture medium;
[0006] The aggregation culture medium consisted of Ndiff227 culture medium supplemented with 0.3% BSA and 10 μM Y-27632;
[0007] The first extended culture medium was Ndiff227 culture medium supplemented with 1 μM PD0325901, 1 μM A83-01, 1 μM 1-Oleoyl lysophosphatidic acid, 10 ng / ml recombinant human LIF and 10 μM Y-27632.
[0008] The second extended culture medium consisted of Ndiff227 culture medium supplemented with 0.5 μM 1-Oleoyl lysophosphatidicacid and 10 μM Y-27632.
[0009] Preferably, the specific steps are as follows:
[0010] Step 1: Human haploid embryonic stem cells were digested and passaged, and haploid cells in human haploid embryonic stem cells were enriched by flow cytometry.
[0011] Step 2: Place the isolated haploid cells in aggregate culture medium for 1 day, then transfer them to the first expansion culture medium for 3-4 days of induction, and then transfer them to the second expansion culture medium for 2 days of induction to obtain human haploid blastocysts.
[0012] Preferably, in step one, hahESCs are cultured in conventional human embryonic stem cell culture medium until the confluence reaches 70-80%, and then dissociated into single cells using TrypLE enzyme; the cell suspension is incubated in a tissue culture dish coated with 0.2% gelatin at 37°C for 45-60 min to remove feeder cells, and then filtered through a 40μm cell sieve.
[0013] Preferably, in step two, the culture conditions are a humidified incubator with 37°C, 5% CO2, and 5% O2.
[0014] Preferably, 1×10 5 One hahESC was resuspended in 1 mL of aggregated culture medium and seeded into the wells of a 24-well Aggrewell 400. The first and second expansion cultures were then replaced sequentially.
[0015] Methods for constructing human haploid blastocysts and the resulting human haploid blastocysts.
[0016] Preferably, the human haploid blastocyst has a human blastocyst-like structure, including: a human blastocyst-like structure containing a cell mass-like structure, a trophectoderm-like structure, and a visible cavity, while also containing haploid cells.
[0017] Preferably, during in vitro culture, the human haploid blastocyst is first placed in the first embryo culture medium and cultured until it adheres to the wall, then the second embryo culture medium is replaced.
[0018] The first embryo culture medium was advanced DMEM / F12 supplemented with 20% fetal bovine serum, 2 mM glutamine, 1% ITS-X supplement, 8 nM β-estradiol, 200 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1 mM sodium pyruvate and 10 μM Y-27632.
[0019] The second embryo culture medium consisted of advanced DMEM / F12 supplemented with 30% serum substitute, 2 mM glutamine, 1% ITS-X supplement, 8 nM β-estradiol, 200 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1 mM sodium pyruvate, and 10 μM Y-27632.
[0020] Application of human haploid blastocysts in cell models or screening platforms for human embryonic development research.
[0021] The advantages and positive effects of this invention are: by placing human haploid embryonic stem cells in a blastocyst induction system, human haploid blastocysts are constructed; these human haploid blastocysts have similar structures and cellular compositions to human blastocysts and have the same developmental potential; they can provide a good genetic screening platform and in vitro cell research model for the study of early human embryonic development. Attached Figure Description
[0022] Figure 1 White light image of hahESCs cells, scale bar 100μm;
[0023] Figure 2 The results of flow cytometry analysis of DNA content in hahESCs;
[0024] Figure 3 White light field image of a human haploid blastocyst, scale bar 100μm;
[0025] Figure 4 The results of flow cytometry analysis of DNA content in diploid control H9 and human haploid blastocysts are shown.
[0026] Figure 5 Immunofluorescence images for identifying human haploid blastocysts, scale bar 100 μm;
[0027] Figure 6 A composite image of single-cell sequencing of human haploid blastocysts and human blastocysts;
[0028] Figure 7 White light field images and immunofluorescence images of human embryonic stem cells, human trophoblast stem cells, and human extraembryonic mesoderm established for human haploid blastocysts, scale bar 100 μm;
[0029] Figure 8 Immunofluorescence images of in vitro cultures of human haploid blastocysts, scale bar 100 μm. Detailed Implementation
[0030] The embodiments of the present invention will now be described with reference to the accompanying drawings.
[0031] This invention relates to a method for constructing human haploid blastocysts. First, haploid cells are sorted and placed in a human blastocyst induction system to produce human haploid blastocyst-like structures with haploid properties, referred to as human haploid blastocysts. The constructed human haploid blastocysts have a structure and cellular composition similar to human blastocysts, and can establish human embryonic stem cells (HECs) in different cell induction culture media. Human haploid blastocysts, in addition to having a similar structure and function to human blastocysts, also possess the advantages of haploid cells, making them suitable for research such as generating human haploid embryo-related cell lines and genetic screening for early human embryonic development.
[0032] Human haploid embryonic stem cells (hahESCs) were selected and constructed into human haploid blastocysts through specific culture and induction. The specific steps are as follows:
[0033] Step 1: Culture and sorting of human haploid embryonic stem cells; hahESCs were cultured in conventional human embryonic stem cell culture medium; routine digestion and passage were performed using 1 mg / mL collagenase IV; haploid cells in hahESCs were enriched by flow cytometry sorting.
[0034] Step 2: Construction of human haploid blastocysts; 1×10 5 One hahESC was resuspended in 1 mL of aggregation culture medium and inoculated into one well of a prepared 24-well Aggrewell 400 plate; the plate was incubated in a humidified incubator at 37°C, 5% CO2, and 5% O2; after 1 day, the culture medium was replaced with the first expansion culture medium, and the culture medium was replaced every other day; after 2-3 days, once the cavity structure appeared, the culture medium was replaced with the second expansion culture medium; after 2 days, human haploid blastocysts could be observed to form under a microscope.
[0035] The traditional culture medium for human embryonic stem cells includes: 20% Knockout Serum Replacement, 2mM Glutamax, 0.5% 100× penicillin antibody, 0.1mM β-mercaptoethanol and 10ng / mL bFGF supplemented in Knockout DMEM.
[0036] The aggregation culture medium consisted of Ndiff227 culture medium supplemented with 0.3% BSA and 10 μM Y-27632.
[0037] The formulation of the first extended culture medium is as follows: Ndiff227 culture medium supplemented with 1 μM PD0325901, 1 μM A83-01, 1 μM 1-Oleoyl lysophosphatidic acid, 10 ng / ml recombinant human LIF and 10 μM Y-27632.
[0038] The second extended culture medium was formulated as follows: 0.5 μM 1-Oleoyllysophosphatidic acid and 10 μM Y-27632 were added to Ndiff227 culture medium.
[0039] The cellular composition of the constructed human haploid blastocysts was determined using single-cell sequencing and immunofluorescence techniques, and they exhibited structures and functions similar to human blastocysts. These human haploid blastocysts were then cultured in vitro. After adhering to the culture medium in a first embryo culture medium, the medium was replaced with a second embryo culture medium.
[0040] The first embryo in vitro culture medium formula is advanced DMEM / F12 supplemented with 20% fetal bovine serum, 2mM glutamine, 1% ITS-X supplement, 8nM β-estradiol, 200ng / ml progesterone, 25μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1mM sodium pyruvate and 10μM Y-27632.
[0041] The second embryo in vitro culture medium formulation is advanced DMEM / F12 supplemented with 30% serum substitute, 2mM glutamine, 1% ITS-X supplement, 8nM β-estradiol, 200ng / ml progesterone, 25μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1mM sodium pyruvate and 10μM Y-27632.
[0042] Experiments revealed that the constructed human haploid blastocysts possessed similar developmental potential to human blastocysts. They were able to be established and produced under different types of induction culture media. Human haploid blastocysts, including those in a primed state, human embryonic stem cells, human trophoblast stem cells, and human extraembryonic endoderm stem cell lines, were used to construct an easily accessible, multifunctional, standardized platform for human haploid genetic screening. This platform can serve to elucidate the mechanisms of early human embryonic development and peri-implantation development, as well as to develop derivative biomedical research methods.
[0043] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.
[0044] Example 1: Construction of human haploid blastocysts
[0045] 1.1 Culture and sorting of human haploid embryonic stem cells
[0046] Culture of hahESCs: HahESCs were derived from Cell Biology, Chinese Academy of Sciences, catalog number SCSP-311. Cells were resuscitated on feeder cells and cultured in a humidified incubator at 37°C and 5% CO2 using conventional human embryonic stem cell culture medium. Cells were passaged every 3-4 days using 1 mg / mL collagenase IV. Cell growth status is as follows. Figure 1 As shown.
[0047] Traditional human embryonic stem cell culture medium formulation consists of Knockout DMEM (Gibco, 10829018) supplemented with 20% Knockout Serum Replacement (Gibco, 10828028), 2mM Glutamax (Thermo, 35050061), 0.5% 100× penicillin antibiotic (Gibco, 15140122), 0.1mM β-mercaptoethanol (Thermo, 21985023), and 10ng / mL bFGF (Peprotec, 100-18B).
[0048] Haploid cell enrichment of hahESCs: hahESCs with 70%-90% confluence were digested with TrypLE enzyme (Thermo, 12604021), stained with Hoechst 33342 (Invitrogen, H3570, 5 μg / ml) at 37°C for 20 min, and filtered through a 40 μm cell sieve (CORNING, 352340). Human embryonic stem cells H9 were used as a diploid control. Cells were sorted and analyzed using flow cytometry. Flow cytometry results are shown below. Figure 2 As shown, the 1n peak is selected as a haploid cell.
[0049] 1.2 Construction of human haploid blastocysts
[0050] Preparation of 24-well Aggrewell 400 plates: Add 500 μL of anti-adhesion treatment solution (STEMCELL, 07010) to each well of a 24-well Aggrewell 400 (STEMCELL Technologies, 34411) plate and incubate overnight at 37°C. Before use, wash the wells once with 2 mL of Ndiff 227, then add 500 μL of aggregation culture medium.
[0051] HahESCs obtained through routine sorting were cultured in conventional human embryonic stem cell culture medium until approximately 70% confluence. Then, the hahESCs were dissociated into single cells using TrypLE enzyme. Subsequently, the cell suspension was incubated at 37°C for 45–60 minutes in tissue culture dishes coated with 0.2% gelatin (Sigma, V900863) to remove feeder cells, and the cells were filtered through a 40 μm cell sieve. Staining with 0.4% trypan blue (Thermo, 15250061) was performed, and cell counts were performed using an automated cell counter (LOGOSBIOSYSTEMS, LUNA-II™ Automated Cell Counter).
[0052] 1×10 5 One hahESC was resuspended in 1 mL of aggregation culture medium and inoculated into one well of a prepared 24-well Aggrewell 400 plate. The plate was incubated in a humidified incubator (Thermo Scientific 311) at 37°C, 5% CO2, and 5% O2. After one day of incubation, the culture medium was replaced with the first expansion medium, and then changed every other day. After 2-3 days of incubation, once cavity structures appeared, the culture medium was replaced with the second expansion medium. After 2 days of incubation, human haploid blastocysts could be observed forming under a microscope, with morphology as shown in the image. Figure 3 As shown.
[0053] Aggregation culture medium formulation: Ndiff227 (Takara, Y40002) culture medium supplemented with 0.3% BSA (Sigma, A1933) and 10 μM Y-27632 (Selleck, S1049).
[0054] The formulation of the first expansion culture medium was as follows: Ndiff227 culture medium supplemented with 1 μM PD0325901 (MCE, HY-10254), 1 μM A83-01 (MCE, HY-10432), 1 μM 1-Oleoyl lysophosphatidic acid (MCE, HY-137862), 10 ng / ml recombinant human LIF (SinoBiological, 14890-HNAH) and 10 μM Y-27632.
[0055] The second extended culture medium was formulated by supplementing Ndiff227 culture medium with 0.5 μM 1-Oleoyllysophosphatidic acid and 10 μM Y-27632.
[0056] Example 2: Characterization of human haploid blastocysts
[0057] 2.1 DNA content analysis of human haploid blastocysts
[0058] Haploid blastocysts were digested with TrypLE enzyme, stained with Hoechst 33342 (5 μg / ml) at 37°C for 20 minutes, and filtered through a 40 μm cell sieve. Human embryonic stem cell line H9 was used as a diploid control. Cells were sorted and analyzed using flow cytometry. Flow cytometry results are shown below. Figure 4 As shown, the blastocysts formed by the self-assembly of human haploid embryonic stem cells have a high haploid ratio, with the 1n peak ratio being 24.4%.
[0059] 2.2 Immunofluorescence staining and single-cell sequencing
[0060] Human haploid blastocysts were manually isolated using a pipette under a stereomicroscope. They were fixed for 30 minutes at room temperature with 4% paraformaldehyde (Sigma, 158127). Then, they were blocked and punched in 0.1% Triton X-10 (Sigma, T8787) prepared with 5% BSA for 2 hours at room temperature. The samples were then incubated overnight at 4°C with a primary antibody diluted with 5% BSA. After overnight incubation, the samples were washed three times with PBS containing 0.1% Tween-20 (Solarbio, T8220) and then incubated with a secondary antibody for 2 hours at room temperature in the dark. Finally, the nuclei were stained with Hoechst 33342 for 15 minutes at room temperature. Immunofluorescence images were produced under a laser confocal microscope (Leica, TCS SP8), and the staining results are shown below. Figure 5As shown. Immunofluorescence results indicate that human haploid blastocysts contain three lineages of human blastocysts: epiblast (OCT4 / SOX2), hypoblast (GATA6 / GATA4), and TE (GATA3 / CDX2). Single-cell sequencing results and comparisons with known human blastocyst single-cell sequencing results are shown below. Figure 6 As shown, the results indicate that human haploid blastocysts contain a cellular composition similar to that of early human blastocysts. These results demonstrate that human haploid blastocysts possess a structure and cellular composition similar to that of human blastocysts.
[0061] Example 3: Establishment of three lineage-specific stem cell lines through in vitro blastocyst formation
[0062] The resulting human haploid blastocysts were placed in traditional human embryonic stem cell culture medium, human trophoblast stem cell culture medium, and human extraembryonic endoderm stem cell culture medium for line establishment, respectively, to establish human embryonic stem cell lines. (and primed state), human trophoblast stem cells and human extraembryonic endoderm stem cell lines.
[0063] Traditional human embryonic stem cell culture medium: The formula is Knockout DMEM supplemented with 20% Knockout Serum Replacement, 2mM Glutamax, 0.5% 100× penicillin antibody, 0.1mM β-mercaptoethanol and 10ng / mL bFGF.
[0064] Human trophoblast stem cell culture medium: The formula is DMEM / F12 (Gibco, 11320033) culture medium supplemented with 0.1 mM β-mercaptoethanol, 0.2% fetal bovine serum (HyClone, SH30406.05), 0.5% 100× penicillin antibody, 0.3% BSA, 1% ITS-X supplement (Thermo, 51500056), 1.5 μg / mL VC (Sigma, 1043003), 50 ng / mL human EGF (Peprotec, AF-100-15-1MG), 2 μM CHIR99021 (MCE, HY-10182), 0.5 μM A83-01, 1 μM MSB431542 (MCE, HY10431), 0.8 mM VPA (Sigma, P4543), and 5 μM Y-27632.
[0065] Human extraembryonic endoderm stem cell culture medium: The formula is Ndiff227 culture medium supplemented with 100 ng / ml Activin A (MCE, HY-P700159AF), 3 μM CHIR99021 and 10 ng / ml recombinant human LIF.
[0066] Morphological observation and immunofluorescence staining results are as follows Figure 7 As shown. The results indicate that human haploid blastocysts can be used to establish human embryonic stem cells with typical cell morphology and expression of corresponding lineage-specific proteins. (and primed state), human trophoblast stem cells and human extraembryonic endoderm stem cell lines. The human haploid blastocysts prepared in Example 1 have blastocyst differentiation capacity.
[0067] Example 4: In vitro culture of human haploid blastocysts
[0068] Human haploid blastocysts were washed twice with a drop of first embryo culture medium using an oral pipette, and then transferred to 8-well μ-Slide (ibidi, 80826) plates containing the first embryo culture medium; half of the culture medium was replaced after 24 hours. Human haploid blastocysts typically attach to the plate within two days; after attachment, the culture medium was replaced with the second embryo culture medium and cultured for another two days for immunofluorescence staining.
[0069] The first embryo in vitro culture medium formulation consisted of advanced DMEM / F12 (Gibco, 12634010) supplemented with 20% fetal bovine serum, 2 mM glutamine (Sigma, G8540), 1% ITS-X supplement, 8 nM β-estradiol (MCE, HYB0141), 200 ng / ml progesterone (MCE, HYN0437), 25 μM N-acetyl-L-cysteine (MCE, HY-B0215), 0.22% sodium lactate (Sigma, L4263), 1 mM sodium pyruvate (Sigma, P4562) and 10 μM Y-27632.
[0070] The second embryo in vitro culture medium formulation was advanced DMEM / F12 (Gibco, 12634010) supplemented with 30% Knockout Serum Replacement, 2 mM glutamine, 1% ITS-X supplement, 8 nM β-estradiol, 200 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1 mM sodium pyruvate and 10 μM Y-27632.
[0071] Immunofluorescence staining results as follows Figure 8 As shown, human haploid blastocysts can further develop into structures similar to those of human embryos around implantation, with yolk sac and amnion cavities and expressing corresponding lineage-specific proteins (amnion: SOX2, yolk sac: GATA6).
[0072] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.
Claims
1. A method for constructing a human haploid blastocyst, characterized in that: Human haploid embryonic stem cells (hahESCs) were cultured in a blastocyst induction system to obtain human haploid blastocysts.
2. The method for constructing human haploid blastocysts according to claim 1, characterized in that: The blastocyst induction system includes an aggregation culture medium, a first expansion culture medium, and a second expansion culture medium. The aggregation culture medium was supplemented with 0.3% BSA and 10 μM Y-27632 to Ndiff227 culture medium; The first extended culture medium was Ndiff227 culture medium supplemented with 1 μM PD0325901, 1 μM A83-01, 1 μM 1-Oleoyl lysophosphatidic acid, 10 ng / ml recombinant human LIF and 10 μM Y-27632. The second extended culture medium consisted of Ndiff227 culture medium supplemented with 0.5 μM 1-Oleoyl lysophosphatidicacid and 10 μM Y-27632.
3. The method for constructing human haploid blastocysts according to claim 1 or 2, characterized in that: The specific steps are as follows: Step 1: Human haploid embryonic stem cells were digested and passaged, and haploid cells in human haploid embryonic stem cells were enriched by flow cytometry. Step 2: Place the isolated haploid cells in aggregate culture medium for 1 day, then transfer them to the first expansion culture medium for 3-4 days of induction, and then transfer them to the second expansion culture medium for 2 days of induction to obtain human haploid blastocysts.
4. The method for constructing human haploid blastocysts according to claim 3, characterized in that: In step one, hahESCs are cultured in conventional human embryonic stem cell culture medium until the confluence reaches 70-80%, and then the hahESCs are dissociated into single cells using TrypLE enzyme. The cell suspension is incubated in a tissue culture dish coated with 0.2% gelatin at 37°C for 45-60 min to remove feeder cells, and then filtered through a 40μm cell sieve.
5. The method for constructing human haploid blastocysts according to claim 3, characterized in that: In step two, the culture conditions are a humidified incubator at 37°C, 5% CO2, and 5% O2.
6. The method for constructing human haploid blastocysts according to claim 3 or 5, characterized in that: 1×10 5 One hahESC was resuspended in 1 mL of aggregated culture medium and seeded into the wells of a 24-well Aggrewell 400. The first and second expansion cultures were then replaced sequentially.
7. Human haploid blastocysts constructed by the method for constructing human haploid blastocysts according to any one of claims 1-6.
8. The human haploid blastocyst according to claim 7, characterized in that: It includes cell cluster-like structures, trophectoderm-like structures and visible cavities, and blastocyst-like structures containing haploid cells.
9. The human haploid blastocyst according to claim 7 or 8, characterized in that: During in vitro culture, human haploid blastocysts are first placed in the first embryo culture medium and cultured until they adhere to the wall. Then, the second embryo culture medium is replaced. The first embryo culture medium was advanced DMEM / F12 supplemented with 20% fetal bovine serum, 2 mM glutamine, 1% ITS-X supplement, 8 nM β-estradiol, 200 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1 mM sodium pyruvate and 10 μM Y-27632. The second embryo culture medium consisted of advanced DMEM / F12 supplemented with 30% serum substitute, 2 mM glutamine, 1% ITS-X supplement, 8 nM β-estradiol, 200 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, 0.22% sodium lactate, 1 mM sodium pyruvate, and 10 μM Y-27632.
10. The use of the human haploid blastocyst as described in any one of claims 7-9 in a cell model or screening platform for human embryonic development research.