CN113881623A - Method for forming ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic embryo - Google Patents
Method for forming ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic embryo Download PDFInfo
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Abstract
The invention discloses a method for forming an ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic, which comprises the steps of treating strontium ions and cytochalasin D to obtain parthenogenetic activated embryos, establishing parthenogenetic embryonic stem cell lines, further differentiating to form primordial germ cell-like cells, and transplanting the primordial germ cell-like cells and mouse female embryonic gland cells into a combined immunodeficient mouse in a polymerization manner to obtain the ovum; the technology can produce infinite normal functional ova, and has important significance for clinically treating infertility or premature ovarian failure and obtaining enough high-quality ova in the scientific research process.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for forming an ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenesis.
Background
Germ cells are the only cells in an organism that are capable of transmitting totipotency and genetic material of the organism by generational inheritance, including mature germ cells, i.e., sperm and eggs, that differentiate from Primordial Germ Cells (PGCs) to terminally differentiated. With the change of life style of human, the number of infertility patients increases year by year worldwide. It is important to understand human germ cells, including eggs and sperm. But is limited by the number of human ova and the difficulty in sampling, as well as ethical constraints, which seriously hinder the research on the maturation mechanism of the ova and the improvement of the treatment level of clinical reproductive related diseases. Embryonic Stem Cells (ESCs) are plastic cells capable of self-renewal and differentiation potential, can be differentiated into two hundred or more cells and dozens of tissues and organs in animals, and have immeasurable significance in studying the basic laws of mammalian ontogeny, the principles of disease development and regenerative medicine application. The use of the pluripotency of embryonic stem cells to induce human embryonic stem cells (hESCs) into human ova in vitro has become a focus of the present research.
At present, there has been a report of using pluripotent stem cells to differentiate to form specific normal functional ova, which uses female Embryonic Stem Cells (ESCs) or female Induced Pluripotent Stem Cells (iPSCs) as starting cells to form primordial germ cell-like cells (PGCLCs) through a two-step differentiation method under the action of recombinant proteins and small molecular compounds. Differentiation of pluripotent stem cells into epiblast-like cells (EpiLCs) was performed in the first step by adding FGF2 and Activin a, and further by adding BMP4, LIF, SCF, and EGF, and differentiation of epiblast-like cells (EpiLCs) into primordial germ-like cells (PGCLCs) was performed under 3D culture conditions. The primordial germ-like cells (PGCLCs) are polymerized with mouse embryonic stage E12.5 female glandular cells and transplanted into combined immunodeficient mouse kidney cysts to form ova with normal fertilization capability.
However, this technique has the following major drawbacks in application:
1. embryonic stem cells and induced pluripotent stem cells have limitations in human applications.
The production of Embryonic Stem Cells (ESCs) necessarily destroys normal embryonic architecture and therefore has a strong ethical problem. And embryonic stem cells cannot be produced from the same individual, there is a risk of immunological rejection in future disease treatments and applications.
The formation process of most Induced Pluripotent Stem Cells (iPSCs) stimulates the endogenous gene expression of somatic cells by introducing exogenous genes, thereby reprogramming somatic cells into induced pluripotent stem cells. And the existence of the exogenous gene has certain tumorigenicity. In addition, induced pluripotent stem cells cannot be widely used because they accumulate genetic mutations during their formation, thereby causing genomic instability.
2. The process of pluripotent stem cell differentiation to form ova is often accompanied by incomplete erasure/reconstruction of genomic imprints, thereby reducing the efficiency of functional ova production.
The human genome is 2-fold, with half of the genome from the female parent and the other half from the male parent. In addition to carrying genetic information on DNA, maternal and paternal genomes also present genomic imprinting information dominated by DNA methylation. These genomic imprinting messages are different in maternal and paternal genomes, which can regulate normal gene expression. Embryonic stem cells are derived from fertilized embryos, while induced pluripotent stem cells are derived from reprogramming of individual somatic cells, and thus these cells all contain the paternal genome. During the process of forming primordial germ cells, genomic imprints of paternal and maternal origin are erased, followed by reconstitution of genomic imprints of maternal origin during differentiation of primordial germ cells to form an egg.
3. In the process of forming the ovum by the human pluripotent stem cell, two X chromosomes of the formed human primordial germ cell-like cells are incompletely activated, so that the two X chromosomes cannot fully enter the meiosis process, and the ovum with normal function cannot be generated. In general, the activity of X-staining from a parent source is lower than that of chromosomes from a parent source.
Parthenogenetic activation is the in vitro stimulation of oocytes in MII stage by physical and chemical means, resulting in the formation of pronuclei and development of embryos. The blastocyst obtained after Parthenogenetic activation of the oocyte is called Parthenogenetic embryo, and the isolated ICM establishes an embryonic stem cell line called Parthenogenetic embryonic stem cell (pESC) line. At present, no report on the differentiation of parthenogenetic embryonic stem cells into fully functional ova is available.
Disclosure of Invention
The invention aims to provide a method for forming an ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenesis, which aims to solve the problems in the prior art. The invention relates to a method for activating parthenogenesis of an ovum by using strontium ions and cytochalasin D, which is used for forming parthenogenetic embryonic stem cells (pESCs) with good developmental capacity and further differentiating the parthenogenetic embryonic stem cells to form the ovum. Parthenogenetic embryonic stem cells (pESCs) can be expanded indefinitely in vitro by cell culture methods, thus allowing unlimited cell sources. Further, an unlimited number of functional ova can be produced by cell differentiation techniques. The technology has important significance for clinically treating infertility or premature ovarian failure and obtaining sufficient high-quality ova in the scientific research process.
More importantly, the parthenogenetic embryonic stem cells (pESCs) can be obtained from the patient per se, and no exogenous gene is introduced in the parthenogenetic activation process, so that the restriction of ethical problems can be avoided, and the application risk is reduced to the minimum. Furthermore, because parthenogenetic embryonic stem cells (pESCs) are derived from an ovum, they do not have a genomic imprint of paternal origin, making it easier to complete erasure of the imprint during formation of primordial germ cell-like cells (PGCLCs) and to establish a genomic imprint of maternal origin suitable for an ovum. And parthenogenetic embryonic stem cell (pESCs) -derived primordial germ-like cells (PGCLCs) have two X chromosomes from maternal origin and should therefore more readily enter the meiotic process and form structurally and functionally normal ova.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for forming an ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic embryo, which comprises the following steps: treating the ovum with strontium ions and cytochalasin D to obtain parthenogenetic activated embryo, establishing parthenogenetic embryonic stem cell line, further differentiating to form primordial germ cell-like cells, and transplanting the primordial germ cell-like cells and female embryonic glandular cells of the mouse into a combined immune defective mouse in a polymerization manner to obtain the ovum.
Further, the method specifically comprises the following steps:
(1) establishment of parthenogenetic embryonic stem cell line
In-vitro mouse MII stage ovum is adopted, granular cells are digested in HKSOM solution containing hyaluronidase, then the cleaned granular cells are washed in pure potassium ion optimized culture solution containing HEPES and sodium pyruvate, and the cleaned granular cells are transferred into parthenogenetic activation culture solution to perform parthenogenetic activation;
cleaning the parthenogenetic activated embryos in the balanced KSOM and culturing until the embryos develop to blastula;
collecting blastocysts on a feeding layer treated by mitomycin C, adding a culture solution of a system building to culture until obvious outward growth can be seen;
collecting the outgrowth, digesting the outgrowth in trypsin, stopping digestion by using a stem cell culture solution, inoculating the outgrowth on a feeding layer treated by mitomycin C, and culturing and amplifying to obtain the parthenogenetic embryo stem cell line;
(2) directed differentiation and sorting of primordial germ-like cells
Culturing the female embryonic stem cells and the parthenogenetic embryonic stem cells for 3-5 generations, and then starting induction; inoculating the female embryonic stem cells or parthenogenetic embryonic stem cells into a cell culture plate treated by the recombinant human fibronectin package, and culturing by using an EpiLC induction culture solution;
after induction is finished, digesting cells and counting, adopting PGCLCs to induce a differentiation culture solution to differentiate, and then adopting SSEA1-APC and CD61-PE direct standard antibodies to sort to obtain primordial germ cell sample cells;
(3) magnetic bead sorting of E12.5 female glandular cells
Digesting E12.5 day female gonads in TE, screening by using a cell sieve and centrifuging; discarding the supernatant, using a magnetic bead antibody prepared by a magnetic bead sorting buffer solution to resuspend cells, and incubating on ice in a dark place; then, continuously washing, resuspending and separating by using a magnetic bead sorting buffer solution to obtain E12.5-day female gonad somatic cells with PGCs removed;
(4) in vitro repolymerization of repolymerized ovaries
Polymerizing the primordial germ cell-like cells obtained in the step (2) and the gonad somatic cells obtained in the step (3) according to the quantity ratio of 1:5-1:10 to form a reunion ovary;
(5) and (3) obtaining a combined immunodeficiency mouse, injecting the refocus ovary obtained in the step (4) into a kidney capsule of the combined immunodeficiency mouse, and culturing to obtain a normally functional ovum.
Further, the mass percent of HEPES in the pure potassium ion optimization culture solution containing HEPES and sodium pyruvate is 1%, and the concentration of the sodium pyruvate is 0.22 g/mL.
Further, the parthenogenetic activation culture solution is Ca2+-free potassium ion-only optimized culture solution, 10mM SrCl2And 20. mu.g/ml cytochalasin D.
Further, in the step (2), after the female embryonic stem cells and parthenogenetic embryonic stem cells are cultured for 3 to 5 passages in a culture system containing N2B27+ t2iL, induction is started when the cell state is good.
The invention discloses the following technical effects:
the method comprises the steps of differentiating parthenogenetic embryonic stem cells activated by strontium ions to form ova, namely treating the parthenogenetic activated embryos by the strontium ions and cytochalasin D to obtain parthenogenetic activated embryos, establishing parthenogenetic embryonic stem cell lines, further differentiating to form primordial germ cell-like cells, and transplanting the primordial germ cell-like cells and mouse E12.5 female embryo E12.5 sexual gland cells into a combined immunodeficient mouse in a polymerization manner to obtain the ova.
According to the parthenogenetic activation method, the parthenogenetic embryonic stem cells with high quality can be generated by combining strontium ions with cytochalasin D. Strontium ions can simulate the continuous calcium oscillation process in the fertilization process of the MII ovum, and cytochalasin D can inhibit the discharge of the second polar body in the fertilization process of the MII ovum, so that the diploid embryo with complete parthenogenetic activation can be efficiently formed. The parthenogenetic activated diploid embryo has the capacity of developing into a blastula similar to that of a normal fertilized embryo, the blastula can form parthenogenetic embryonic stem cells (pESCs) through embryo establishment, and the parthenogenetic embryonic stem cells (pESCs) have the same quality and development potential as those of normal mouse Embryonic Stem Cells (ESCs).
Parthenogenetic embryonic stem cells (pESCs) differentiate to form primordial germ cell-like cells (PGCLCs) that have similar levels of gene expression and methylation to normal Primordial Germ Cells (PGCs) in vivo, and can also be fertilized in vitro to obtain healthy, fertile mouse offspring.
By using the method, a high-quality parthenogenetic embryonic stem cell (pESCs) cell line can be generated by 16 MII ova on average. When parthenogenetic embryonic stem cells (pESCs) are expanded in vitro for 10 generations, 2.4X 10 can be obtained by differentiation6One ovum (on average each MII ovum can be amplified to obtain 1.5X 105Individual eggs).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of the process of in vitro differentiation of an ovum from a parthenogenetic embryonic stem cell line.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
(1) Establishment of parthenogenetic embryonic stem cell (pESCs) line
a. Injecting 5IU of Pregnant Mare Serum Gonadotropin (PMSG) into the abdominal cavity of each female mouse with 6-8 weeks old Actin-GFP, and injecting 5IU of Human chorionic gonadotropin (hCG) into the abdominal cavity of each mouse for superovulation 46-48h after injection;
b. 14h after hCG injection, MII ova were collected from the oviduct, and granular cells were digested in a preheated HKSOM solution containing 0.3% hyaluronidase, then washed 3 times in a preheated Potassium-only conditioning (Potassium single-plex-optimized HEPES and sodium bicarbonate, HKSOM) solution containing 1% HEPES and 0.22g/ml sodium pyruvate, transferred to 50. mu.l of estrus soliquous activation medium, and activated for 4h in an incubator, and 2 pronucleus soliquous (2 clear pronucleus were visible after estrus soliquous activation)The formula of the live culture solution is as follows: ca2+-free pure Potassium ion-optimized medium (KSOM) +10mM SrCl2+20μg/ml cytochalasin D)；
KSOM Stock 10X composition is as follows:
KSOM 50ml fraction as follows:
HKSOM 50ml has the following composition:
c. cleaning the parthenogenetic activated embryos in the balanced KSOM for 3 times, culturing in the KSOM, observing 2-cell embryos the next day, taking out and discarding the embryos which do not grow to 2 cells, and allowing the remaining embryos to develop to blastula;
d. the blastocysts are inoculated on a feeding layer (4-hole plate) treated by 20 mu g/ml mitomycin C, and a system establishing culture solution (knock-out DMEM culture medium + 20% KSR +1mM L-glutamine + 1% non-essential amino acid +50units/ml penicillin +50mg/ml streptomycin +0.1mM 2-mercaptoethanol +1mM PD0325901+1000IU/ml LIF) is added for culture, the culture solution is changed every other day, and obvious outgrowth (outgrowth) production can be seen after 7 days of culture;
e. the growth was picked up with a 10. mu.l pipette tip, digested for 5min in 0.25% trypsin, then digested with stem cell culture medium (knockout DMEM medium + 15% FBS +1mM L-glutamine + 1% non-essential amino acids +50units/ml penicillin +50mg/ml streptomycin +0.1mM 2-mercaptoethanol +1000IU/ml LIF), and inoculated on mitomycin C treated feeder layers for culture and amplification, using pronuclear typing.
(2) Directed differentiation and sorting of primordial germ-like cells (PGCLCs)
a. ESCs and pESCs were cultured for 3-5 passages in a culture system of N2B27+ t2iL (1000IU/ml LIF, 1mM PD0325901 and 3mM CHIR99021), and PGCLCs were induced when the cells were in good condition;
b. ESCs or pESCs were inoculated into recombinant human Fibronectin (human Fibronectin, hFN) -coated cell culture plates, and cultured for 2d using EpiLC induction medium (N2B27 medium +20ng/ml activin A + bFGF + 1% KSR), with fresh EpiLC medium being replaced daily;
c. after 2d induction by EpiLC, digesting the cells, counting the cells, inducing differentiation medium (GMEM medium + 15% KSR +1mM sodium pyruvate +1mM L-glutamine + 1% non-essential amino acid +50units/ml penicillin +50mg/ml streptomycin +0.1mM 2-mercaptoethanol +500ng/ml BMP4+1000IU/ml mLIF +100ng/ml SCF +50ng/ml EGF) using PGCLCs, differentiating, inoculating the cells into low-adhesion 96-well culture plates according to 3000 cells per Embryoid body (EB, EBs), and culturing;
d. after differentiation of PGCLCs for 4d, sorting was performed by Flow cytometric sorting (FACS) using SSEA1-APC and CD61-PE direct labeled antibody.
(3) Magnetic bead sorting of E12.5 female glandular cells
a. E12.5 day gonad was isolated and digested in 0.25% TE buffer for 10min at 37 ℃;
b. after digestion is stopped, the mixture is sieved by a 40-micron cell sieve and centrifuged for 5min at 300 g;
c. discarding the supernatant, resuspending the cells with magnetic bead antibody prepared with 100. mu.l of magnetic bead sorting buffer, incubating for 20min on ice in the dark, installing a magnetic frame during the incubation, and balancing the adsorption column with 1ml of magnetic bead sorting buffer;
d. after washing the cells with 4ml of magnetic bead sorting buffer, resuspending the cells with 1ml of magnetic bead sorting buffer, and gently adding the cell suspension to an adsorption column, taking care not to generate air bubbles, and collecting the remaining liquid with a 1.5ml EP tube;
e. centrifuging the liquid, removing supernatant, precipitating to remove PGCs, and resuspending with appropriate amount of culture solution.
(4) In vitro repolymerization and meiosis induction of repolymerized ovaries
a. Polymerizing gonadal somatic cells separated from magnetic beads and PGCLCs sorted by a flow cytometer in a low-adhesion 96-well culture plate according to the ratio of 1:5-1:10, and gradually aggregating the two to form a refocused ovary (rOvary);
b. culturing the refocus ovary in vitro for 24h, injecting into NOD-SCID mouse kidney cyst, collecting refocus ovary on 25-28 days of injection, and performing tissue section to observe mature follicle wrapped by multiple layers of granular cells, wherein the follicle has follicle cavity, and the ovum diameter is 50-100 μm;
c. for the relevant samples for meiosis detection, 3 μ M all-trans Retinoic Acid (RA) was added to the refolding culture solution for refolding, and the subsequent operations were the same as those of the normal samples.
(5) Renal cyst transplantation
a. Using NOD-SCID mice of 4-6 weeks old and anaesthetizing before operation, wherein general anaesthesia can be maintained for 20-30min for enough time to transplant;
b. the mice are placed on the side, and the refocus ovary is injected into the kidney cyst of the mice, and the whole process does not exceed 10 min.
(6) In vitro maturation and in vitro fertilization
a. In vitro maturation solution (a-MEM Medium + 5% FBS +0.24mM sodium pyruvate +1IU/ml PMSG +1.5IU/ml hCG) 5% CO at 37 deg.C in advance2Equilibrating in a warm bath incubator for 30min (lid unscrewing), and preparing 35mm dish in vitro maturation solution (using 35mm Petri-dish, spot 7 drops of 50. mu.l in vitro maturation solution, cover it with 3ml mineral oil, CO2Culturing for at least 2 h);
b. taking out ovum with normal function from mouse kidney cyst, selecting ovum with normal shape, placing in IVM culture drop, and adding CO2Culturing in incubator for 6-7h until the first time of number reductionMetaphase MI can be reached if the medium is cultured for 10h (16-17 h in total);
c. preparing a pseudopregnant mouse one day in advance, and detecting thrombus on the day of fertilization, and marking as E0.5; one day in advance 1 fertilization drop was made (90. mu.l/drop, 5 drops/dish in 35mm Pteri-dish, covered with 3ml mineral oil, drops not well over round) and 2 KSOM embryo culture drops or G1-Plus embryo culture drops (7 drops of 50. mu.l) were made, equilibrated overnight;
d. killing male mouse 2h before ovum collection, painlessly according to standard ethical operation, collecting epididymis tail in 35mm Petri-dish with 2ml HTF receiving semen, obtaining sperm from epididymis, microscopic examination, if sperm swim rapidly, indicating quality is good, controlling sperm concentration at about 5 × 106Per ml;
e. putting MII ovum obtained by in vitro maturation into fertilization drop (about 20 ovum/drop), adding 20 μ l of supernatant fluid, and fertilizing in incubator for 6 h;
f. washing with preheated semen for 2 times, then washing ovum with preheated KSOM or G1-Plus embryo culture solution for 3 times and culturing in KSOM or G1-Plus embryo culture drop, and washing to clean as much as possible to prevent residual sperm from influencing the development of fertilized embryo;
g. the next day, embryos that entered the 2-cell stage were picked and placed into KSOM culture drops for culture or direct transfer, and if unfertilized or dead embryos were present in the culture drops, the overall quality of the embryo development would be affected.
As shown in FIG. 1 and the following table, a high quality parthenogenetic embryonic stem cell (pESCs) cell line can be generated by 16 MII eggs on average using the above method. When parthenogenetic embryonic stem cells (pESCs) are expanded in vitro for 10 generations, 2.4X 10 can be obtained by differentiation6One ovum (on average each MII ovum can be amplified to obtain 1.5X 105Individual eggs).
Second step amplification of pESC
Third step, PGCLCs induction
Fourth step reoccumulation of ovarian formation (each polymer contained 2X 10)4PGCLCs and 10x104Female gland cell)
The fifth step is the GV ovum number of each reunion ovary
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (5)
1. A method for forming an ovum by in vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenesis comprises the following steps: treating the ovum with strontium ions and cytochalasin D to obtain parthenogenetic activated embryo, establishing parthenogenetic embryonic stem cell line, further differentiating to form primordial germ cell-like cells, and transplanting the primordial germ cell-like cells and female embryonic glandular cells of the mouse into a combined immune defective mouse in a polymerization manner to obtain the ovum.
2. The method according to claim 1, characterized in that it comprises in particular the steps of:
(1) establishment of parthenogenetic embryonic stem cell line
In-vitro mouse MII stage ovum is digested granular cells in HKSOM solution containing hyaluronidase, then washed in pure potassium ion optimized culture solution containing HEPES and sodium pyruvate, transferred into parthenogenetic activation culture solution, and parthenogenetic activation is carried out;
cleaning the parthenogenetic activated embryos in the balanced KSOM and culturing until the embryos develop to blastula;
collecting blastocysts on a feeding layer treated by mitomycin C, adding a culture solution of a system building to culture until obvious outward growth can be seen;
collecting the outgrowth, digesting the outgrowth in trypsin, stopping digestion by using a stem cell culture solution, inoculating the outgrowth on a feeding layer treated by mitomycin C, and culturing and amplifying to obtain the parthenogenetic embryo stem cell line;
(2) directed differentiation and sorting of primordial germ-like cells
Culturing the female embryonic stem cells and the parthenogenetic embryonic stem cells for 3-5 generations, and then starting induction; inoculating the female embryonic stem cells or parthenogenetic embryonic stem cells into a cell culture plate treated by the recombinant human fibronectin package, and culturing by using an EpiLC induction culture solution;
after induction is finished, digesting cells and counting, adopting PGCLCs to induce a differentiation culture solution to differentiate, and then adopting SSEA1-APC and CD61-PE direct standard antibodies to sort to obtain primordial germ cell sample cells;
(3) magnetic bead sorting of E12.5 female glandular cells
Digesting E12.5 day female gonads in TE, screening by using a cell sieve and centrifuging; discarding the supernatant, using a magnetic bead antibody prepared by a magnetic bead sorting buffer solution to resuspend cells, and incubating on ice in a dark place; then, continuously washing, resuspending and separating by using a magnetic bead sorting buffer solution to obtain E12.5-day female gonad somatic cells with PGCs removed;
(4) in vitro repolymerization of repolymerized ovaries
Polymerizing the primordial germ cell-like cells obtained in the step (2) and the gonad somatic cells obtained in the step (3) according to the quantity ratio of 1:5-1:10 to form a reunion ovary;
(5) and (3) obtaining a combined immunodeficiency mouse, injecting the refocus ovary obtained in the step (4) into a kidney capsule of the combined immunodeficiency mouse, and culturing to obtain a normally functional ovum.
3. The method according to claim 2, wherein the mass percentage of HEPES in the potassium ion-only optimized culture solution containing HEPES and sodium pyruvate is 1%, and the concentration of the sodium pyruvate is 0.22 g/mL.
4. The method according to claim 2, wherein the parthenogenetic activation medium is formulated with Ca2+-free potassium ion-only optimized culture solution, 10mM SrCl2And 20. mu.g/ml cytochalasin D.
5. The method according to claim 2, wherein in the step (2), the induction is started when the cell condition is good after the female embryonic stem cell and the parthenogenetic embryonic stem cell are cultured in the culture system containing N2B27+ t2iL for 3 to 5 passages.
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