CN115197901A - Method for in vitro differentiation of novel human hyper-potential stem cells into oocyte-like cells - Google Patents
Method for in vitro differentiation of novel human hyper-potential stem cells into oocyte-like cells Download PDFInfo
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- 210000004027 cell Anatomy 0.000 title claims abstract description 59
- 230000004069 differentiation Effects 0.000 title claims abstract description 27
- 238000000338 in vitro Methods 0.000 title claims abstract description 23
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 20
- 210000001778 pluripotent stem cell Anatomy 0.000 claims abstract description 28
- 230000021121 meiosis Effects 0.000 claims abstract description 10
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000003822 preparative gas chromatography Methods 0.000 claims description 34
- 210000004681 ovum Anatomy 0.000 claims description 14
- 210000000287 oocyte Anatomy 0.000 claims description 12
- 108010000912 Egg Proteins Proteins 0.000 claims description 8
- 102000002322 Egg Proteins Human genes 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000010410 layer Substances 0.000 claims description 6
- 210000004602 germ cell Anatomy 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 230000035800 maturation Effects 0.000 claims description 4
- 230000005012 migration Effects 0.000 claims description 4
- 238000013508 migration Methods 0.000 claims description 4
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 claims description 3
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 3
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 3
- 102000006771 Gonadotropins Human genes 0.000 claims description 3
- 108010086677 Gonadotropins Proteins 0.000 claims description 3
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 claims description 3
- 101100407657 Mus musculus Pgc gene Proteins 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 210000002459 blastocyst Anatomy 0.000 claims description 3
- 239000002622 gonadotropin Substances 0.000 claims description 3
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims description 3
- 238000001727 in vivo Methods 0.000 claims description 3
- 230000001776 parthenogenetic effect Effects 0.000 claims description 3
- 210000004508 polar body Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000002356 single layer Substances 0.000 claims description 3
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- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims 1
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- 230000008901 benefit Effects 0.000 abstract description 5
- 210000002894 multi-fate stem cell Anatomy 0.000 abstract description 5
- 230000002457 bidirectional effect Effects 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 101100310648 Mus musculus Sox17 gene Proteins 0.000 abstract 1
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- 201000004535 ovarian dysfunction Diseases 0.000 description 5
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- 101000652324 Homo sapiens Transcription factor SOX-17 Proteins 0.000 description 2
- 102100030243 Transcription factor SOX-17 Human genes 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- 208000015124 ovarian disease Diseases 0.000 description 2
- 231100000543 ovarian dysfunction Toxicity 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 229960003237 betaine Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2501/30—Hormones
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- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
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Abstract
The invention discloses a method for differentiating a novel human pluripotent stem cell into an oocyte-like cell in vitro, which comprises the following steps: s1, obtaining EPS: firstly, obtaining human EPS, starting meiosis by PGC when reaching a second stage, and then developing the meiosis into gametes; s2, specialization of PGC: under BMP 4-induced differentiation conditions, EPS will gradually differentiate and give rise to SOX 17-expressing cells using a corresponding cell extraction method. The invention obtains a kind of multipotential stem cells with embryo and extraembryonic tissue bidirectional development potential by using chemical micromolecule combination, the multipotential stem cells and classical human multipotential stem cells and
compared with the multipotent stem cells, the pluripotent stem cells have multiple advantages: 1. the homogeneity of each cell line is good, and the super potential stem cell of the inventionThe culture conditions are suitable for culturing multiple strains of pluripotent stem cells, and the cells have strong homogeneity, so that the differentiation process caused by cell heterogeneity is prevented from being interfered.
Description
Technical Field
The invention relates to the technical field of cells, in particular to a method for differentiating a novel human hyper-potent stem cell into an oocyte-like cell in vitro.
Background
With the development of socioeconomic development and the change of fertility concept, the women have a delayed birth age, more and more elderly women requiring fertility are required, but with the increasing age, the female ovarian reserve function is reduced, the ovarian response capability to ovulation-promoting drugs is reduced, and survey data indicates that the successful delivery rate of the embryo transplantation cycle of in vitro fertilization of non-donated ova is reduced with the increasing age, the successful delivery rate of the cycle for obtaining donated ova is stable, the cycle for obtaining high-quality ova of young women is possible to be pregnant even for menopausal women, except for the elderly women, patients with ovarian dysfunction also need to supply ova, the incidence rate of ovarian dysfunction is about 3% in women with the reproductive age at present, and the incidence rate shows a trend of increasing year by year with the increasing work and life rhythm and the increasing environmental pollution in recent years. Although the treatment of patients with ovarian insufficiency can cause intermittent ovulation, only 5% -10% of patients with ovarian insufficiency can spontaneously ovulate and successfully conceive, relatively speaking, the treatment of supply of eggs is an effective means for helping pregnancy of patients with ovarian insufficiency, so that the patients have the hope of fertility, and stem cells are cells with two characteristics of self-renewal and differentiation potential.
The existing stem cells have low efficiency in culture and differentiation, a single evaluation system and greater disputes, which are related to the fact that the classical human pluripotent stem cells are in a late stage pluripotent state which is difficult to participate in embryonic development, so that the development potential is lowered, the differentiation efficiency is lowered, the effect of providing lacked ova for the female reproductive process cannot be achieved, and certain limitations exist in application.
Disclosure of Invention
The invention provides a method for differentiating a novel human hyper-potential stem cell into an oocyte-like cell in vitro aiming at the defects in the background technology.
In order to solve the phenomenon, the invention adopts the following technical scheme that a method for in vitro differentiation of novel human hyper-potential stem cells into oocyte-like cells comprises the following steps:
s1, firstly, obtaining human EPS, starting meiosis by PGC when reaching a second stage, and then developing the meiosis into gametes;
s2, under the condition of BMP4 induced differentiation, EPS is gradually differentiated and cells expressing SOX17 are generated by adopting a corresponding cell extraction method;
s3, carrying part of non-germ cells by PGC (PGC) specified from EPS, separating from the bottom of the culture dish and suspending in a culture medium;
s4, collecting the lumps separated from the bottom of the dish, and culturing the lumps into primordial follicles in a follicle maturation culture medium;
s5, wherein the developing follicle can continue to grow and finally the ovum is discharged, thus obtaining the oocyte-like cell.
As a further preferred embodiment of the present invention, in step S2, upon obtaining PGC, human embryo develops to 5-10 weeks to produce PGC, mouse PGC is produced at embryonic stage 10.5-13.5 days, and in vivo, PGC undergoes two developmental stages, the first stage: PGCs appear in the extra-embryonic layer and accumulate outside the urogenital spine, and at this stage, PGCs have stem cell properties.
As a further preferred mode of the invention, in step S2, the transition of the pluripotent stem cells-PGCs and PGC-germ cells in vitro can be taken as two common stages in the development of gametes.
In a further preferred embodiment of the present invention, the cell extraction method used in step S2 is a monolayer adherence method.
In a further preferred embodiment of the present invention, in step S3, a total time of 16.5 hours is consumed during the migration of PGCs, so that the effect of differentiation of the upper and lower layers of cells is achieved.
As a further preferred mode of the invention, in step S4, these oocytes have zona pellucida and express ZP2 and ZP3 membrane proteins.
In a further preferred embodiment of the invention, the oocytes are then induced to activate using pregnant mare serum gonadotropin and human chorionic gonadotropin to allow expulsion from the polar body in step S4, revealing that these cells can complete the first meiosis and can develop to the blastocyst stage by parthenogenetic activation.
In a more preferred embodiment of the present invention, the largest diameter of the discharged ovum in step S5 is 75 μm.
The invention obtains a kind of multipotential stem cell with embryo and extraembryonic tissue bidirectional development potential by utilizing chemical micromolecule combination, the multipotential stem cell and classical human multipotential stem cell and
compared with the multipotential stem cells, the pluripotent stem cells have multiple advantages: 1. the cell lines have good homogeneity, the hyper-potential stem cell is suitable for culturing a plurality of lines of multi-potential stem cells, and the homogeneity of the cell is very strong, so that the differentiation process caused by the heterogeneity of the cell is prevented from being interfered; 2. the PGC cell has totipotent development potential, different from traditional multipotent stem cell, the PGC cell is meiotically divided into ovum and sperm, the two are combined to develop into a complete individual, the EPS cell has development potential similar to that of the EPS cell, can participate in the development of embryo and extraembryonic tissue, and its development potential is obviously superior to that of classical multipotent stem cell and conventional multipotent stem cell
Stem cells, which allow the use of pluripotent stem cells for transformation of oocytes in vitro at a higher starting point.
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FIG. 1 is a scheme of differentiation scheme of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a method for in vitro differentiation of novel human pluripotent stem cells into oocyte-like cells comprises the following steps:
s1, firstly, obtaining human EPS, starting meiosis by PGC when reaching a second stage, and then developing the meiosis into gametes;
s2, under the condition of BMP4 induced differentiation, EPS is gradually differentiated and cells expressing SOX17 are generated by adopting a corresponding cell extraction method;
s3, carrying part of non-germ cells to separate from the bottom of a culture dish and suspending the non-germ cells in a culture medium by PGC (poly glycine betaine) specially obtained from EPS;
s4, collecting the lumps separated from the bottom of the dish, and culturing the lumps into primordial follicles in a follicle maturation culture medium;
s5, wherein the developing follicle can continue to grow and finally the ovum is discharged, thus obtaining the oocyte-like cell.
In step S2, upon obtaining PGCs, human embryos develop to produce PGCs by week 5-10, mouse PGCs are produced at embryonic stage 10.5-13.5 days, and PGCs undergo two developmental stages in vivo, the first stage: PGCs appear in the extra-embryonic layer and accumulate outside the urogenital spine, and at this stage, PGCs have stem cell properties.
In step S2, the transition of pluripotent stem cells-PGCs and PGC-germ cells in vitro can be seen as two common stages in the development of gametes.
In step S2, the cell extraction method is a monolayer adherence method.
In the step S3, 16.5 hours are consumed in the process of PGC migration, and the effect of cell upper and lower layer differentiation is achieved.
In step S4, these oocytes have zona pellucida and express ZP2 and ZP3 membrane proteins.
In step S4, the oocytes are then induced and activated using pregnant mare serum gonadotropin and human chorionic gonadotropin to allow for expulsion of the polar bodies, revealing that these cells can complete the first meiosis and can develop to the blastocyst stage by parthenogenetic activation.
In step S5, the largest diameter of the discharged ovum can reach 75 μm.
In conclusion, the invention obtains a type of pluripotent stem cells with embryo and extraembryonic tissue bidirectional development potential by utilizing chemical micromolecule combination, the pluripotent stem cells and the classical human pluripotent stem cells and
compared with the multipotential stem cells, the pluripotent stem cells have multiple advantages: 1. the culture conditions of the hyper-potential stem cells are suitable for culturing a plurality of lines of the hyper-potential stem cells, and the homogeneity of the cells is very strong, thereby avoiding the differentiation process caused by the heterogeneity of the cells from being interfered; 2. the PGC cell has totipotent development potential, different from traditional multipotent stem cell, the PGC cell is meiotically divided into ovum and sperm, the two are combined to develop into a complete individual, the EPS cell has development potential similar to that of the EPS cell, can participate in the development of embryo and extraembryonic tissue, and its development potential is obviously superior to that of classical multipotent stem cell and conventional multipotent stem cell
Stem cells, which allow the use of pluripotent stem cells for transformation of oocytes in vitro at a higher starting point; 3. the cell line is more stable, the traditional pluripotent stem cell has the defects of poor stability of in vitro long-term passage genome and variable pluripotent state, and limits the subsequent clinical application of the pluripotent stem cell, the genome of the pluripotent stem cell can be kept stable after long-term subculture, the development potential and the development efficiency of the pluripotent stem cell are not obviously changed, the chimeric rate of the pluripotent stem cell can reach more than 10% no matter 10 generations or 50 generations, in addition, the pluripotent stem cell also has the advantages of strong single cell survival capability, high proliferation speed and the like, the human and mouse pluripotent stem cells have consistent development potential and the culture conditions are the same, the fact shows that the two pluripotent stem cells are in similar pluripotent state, namely in similar starting point, and the subject is to be a novel human pluripotent stem cellThe cell is used as a starting cell, is differentiated into PGC cells in vitro, and is induced to be transformed into oocyte-like cells, so that a new solution can be provided for solving the clinical problem that the available ovum is lacked in the process of female assisted reproduction.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (8)
1. A method for in vitro differentiation of novel human pluripotent stem cells into oocyte-like cells is characterized in that the differentiation steps comprise the following steps:
s1, obtaining EPS: firstly, obtaining human EPS, starting meiosis by PGC when reaching a second stage, and then developing the meiosis into gametes;
s2, specialization of PGC: under the condition of BMP4 induced differentiation, ES gradually differentiates and generates cells expressing EGFP by adopting a corresponding cell extraction method;
s3, PGC migration: PGC specialized from EPS, carry some non-germ cells to break away from the bottom of the culture dish, and suspend in culture medium;
s4, follicle assembly: collecting the lumps separated from the bottom of the dish, and culturing the lumps into primordial follicles in a follicle maturation culture medium;
s5, follicular maturation: wherein the follicle can continue to grow and develop, and the ovum can be discharged finally, so that the oocyte-like cell can be obtained.
2. The method for the in vitro differentiation of the human novel pluripotent stem cell into an oocyte-like cell according to claim 1, wherein in step S2, the human embryo develops to 5-10 weeks to produce PGCs, the mouse PGCs are produced at embryonic stage 10.5-13.5 days, and the PGCs undergo two developmental stages in vivo, the first stage: PGCs appear in the extra-embryonic layer and accumulate outside the urogenital spine, and at this stage, PGCs have stem cell properties.
3. The method for in vitro differentiation of human novel pluripotent stem cells into oocytes according to claim 1, wherein in step S2, the transformation of pluripotent stem cells-PGC and PGC-germ cells in vitro occurs as two common stages during gamete development.
4. The method for in vitro differentiation of human novel pluripotent stem cells into oocyte-like cells according to claim 1, wherein the cell extraction method adopted in step S2 is a monolayer adherence method.
5. The method for in vitro differentiation of human novel pluripotent stem cells into oocytes according to claim 1, wherein the step S3 is performed within 16.5 hours of the migration of PGCs, so as to achieve the effect of differentiation of upper and lower layers of cells.
6. The method for in vitro differentiation of human novel pluripotent stem cells into primordial-like germ cells according to claim 1, wherein the oocytes have zona pellucida and express ZP2 and ZP3 membrane proteins in step S4.
7. The method for in vitro differentiation of human pluripotent stem cells into oocytes according to claim 1, wherein in step S4, the oocytes are induced to be activated and then expelled from the polar body by using pregnant mare serum gonadotropin and human chorionic gonadotropin, which reveals that the cells can complete the first meiotic division and can develop to the blastocyst stage by parthenogenetic activation.
8. The method for in vitro differentiation of human pluripotent stem cells into oocytes according to claim 1, wherein the largest diameter of the expulsed ova in step S5 is up to 75 μm.
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