CN106754654A - Mammalian artificial embryo based on stem cell builds method and its model that animal cloning is bred - Google Patents
Mammalian artificial embryo based on stem cell builds method and its model that animal cloning is bred Download PDFInfo
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- CN106754654A CN106754654A CN201611093084.3A CN201611093084A CN106754654A CN 106754654 A CN106754654 A CN 106754654A CN 201611093084 A CN201611093084 A CN 201611093084A CN 106754654 A CN106754654 A CN 106754654A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0273—Cloned animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/106—Primate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
Abstract
Method and its model that animal cloning is bred are built the invention discloses a kind of mammalian artificial embryo based on stem cell, using mammalian egg oolemma or equivalent to oolemma artificial synthesized structure as the stem cell acceptor of artificial embryology, it is induced differentiation into mammal preimplantation embryos structure under in vitro conditions after inserting stem cell.Two Key Technologies Designs are induced to the differentiation capability application of initial stage embryo, the early-stage differentiation of embryos of the stem cell ovum oolemma complex of structure with stem cell versatility.Main four technical steps be the preparation of stem cell, the preparation of oocyte or body early embryo oolemma, artificial embryology build induced with cell differentiation, animal cloning is bred.This method is possible in important technical of the future as animal cloning raising technology commercial application, while having important medical application to be worth.
Description
Technical field
The invention belongs to animals and humans reproduction bioengineering new technology, a kind of food in one's mouth based on stem cell is related generally to
Method and its model that newborn animal artificial embryology structure-animal cloning is bred.
Background technology
Many since Britain is born from clone sheep in 1997, studies on Animal Cloning in Forty is fast-developing, most of experimental animal
It is already successful with cloning domestic animal, serve huge with group expanding for life science basic research and individual the breeding of particular animals
Big impetus, and start to be advanced to commercial application with the cloning domestic animal technology of Niu Weizhu.But, due to cloning at present
Technical merit it is limited, the clone embryos complex manufacturing process based on nuclear transplantation, the cloned animal after embryo transfer
Production efficiency is still very low, and the production cost of every cloned animal is high, it is difficult to carry out large-scale promotion application.
On the other hand, the perfect and its versatility or totipotency of stem cells technology are characterized as animal breeding, human medical
Clinical practice provides possibility, and this is also the basis of artificial embryology structure and animal cloning raising technology model.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of mammalian artificial's embryo's structure based on stem cell
Build-method bred of animal cloning and its model.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：A kind of lactation based on stem cell
The method that animal artificial embryology structure-animal cloning is bred, comprises the following steps：
(1) preparation of mammalian stem cell：The induction that mammalian stem cell is used for artificial embryology builds, and passes through respectively
Self-control or commercial sources are obtained；
(2) preparation of oocyte or body early embryo oolemma：Carrying out every time, artificial embryology structure is first 24 hours, according to
The quantity of manufacturing artificial embryo, preparation needs the ovum or body early embryo of quantity 120%；The ovum or body early embryo of collection with
Micromanipulation mode is cut oolemma and suctions out ooplasm or cell division ball, is made the oolemma of ghost；
(3) artificial embryology builds and cell differentiation induction：
A. stem cell injects ovum oolemma：Various stem cells are directly injected into the oolemma of ghost and induce people
Work embryo, the stem cell group cultivated with the trypsin treatment of mass percent concentration 0.5%, then with micromanipulation mode to
40-60 stem cell of injection, is combined stem cell-oolemma in the oolemma of the ghost of each oocyte or body early embryo
Body is concentrated, and is carried out in stem cell medium in vitro culture 12-24 hours according to different plant species, makes stem cell continue to rise in value
60-80 cell mass is formed, the condition of culture of stem cell-oolemma complex is 5%CO2, 5%O2And 90%N2；
B. the external evoked culture of artificial embryology：The stem cell that step A is obtained-ovum oolemma complex, in 5%CO2
In the condition of culture of 95% air, Fiber differentiation 12-24 hours in induction broth 1, in induction broth 2
Culture break up 24-48 hour, differentiate the similar blastaea structure with interior embryo group, blastocoele and nutritive layer cell composition dry
Cell clone embryo；
Induction broth 1 is formulated, by volume
Induction broth 2 is formulated, by volume
(4) animal cloning is bred：The artificial embryology that step (3) builds is transplanted to receiving for estrus synchronization according to routine techniques
In body animal uterus, it is possible to which obtaining has and stem cell heredity identical cloned animal offspring.
The mammal includes experimental animal, domestic animal, the mankind.
The experimental animal refers to mouse, rat, rabbit, primate；Domestic animal refers to ox, sheep, pig, horse, deer.
Ovum or body early embryo in the step (1), collection, family can be put to death for experimental animal ovum or body early embryo
Poultry ovum can live body collection or using butcher dam ovary gather.
The mould that the method that the above-mentioned mammalian artificial's embryo's structure-animal cloning based on stem cell is bred is obtained
Type.
The beneficial effects of the invention are as follows：Mammalian artificial embryo is built based on stem cell, animal cloning is proposed
The new technology model bred, is the weight of non-animal breeding the characteristics of this technology model has easy to operate, extremely low production cost
Technological means is wanted, while having important medical model application value.
Brief description of the drawings
Fig. 1 is the method that mammalian artificial embryo structure-animal cloning of the present invention based on stem cell is bred
Flow chart.
Specific embodiment
Below according to accompanying drawing, the present invention is described in detail：
The present invention includes two Key Technologies Designs and following four implementation steps, two Key Technologies Designs be respectively with
Stem cell versatility is to the differentiation capability application of initial stage embryo, the body early embryo of the stem cell-ovum oolemma complex of structure
Induction.Main four technical steps are preparation, the preparation of oocyte or body early embryo oolemma, the artificial embryo of stem cell
Tire builds breeds with cell differentiation induction, animal cloning.
The present invention utilizes the versatility or totipotency feature of stem cell, animal ovum with mammal, including humans
Sub- oolemma or the basic stem cell receptor structure built equivalent to the artificial synthesized structure artificial embryo of oolemma, insert dry
It is set to induce differentiation into mammal preimplantation embryos structure (8cell stage-Morula after cell under in vitro conditions
stage and Blastocyst stage).Three kinds of property stem cells can be used for the induction structure of artificial embryology：One kind is embryo
Stem cell (Embryonic SC), is for second the upper strata embryonic stem cell (Epiblast SC) of body early embryo, and the third comes
From the somatic stem cell iPS (Somatic SC-iPS) of adult tissue.In order to stem cell build artificial embryology during
Cell differentiation be monitored, in the stem cell for using carry marked cells versatility or totipotency Oct-4, Sox2 or Nanog
The green protein GFP of gene connection.But can not use this fluorescence mark in animal breeding industry or Human clinical's application
Note.
It is not included in invention on various mammals stem cell animal establishing techniques including humans
Hold.
As shown in figure 1, the present invention comprises the steps：
(1) preparation of mammalian stem cell：The induction that mammalian stem cell is used for artificial embryology builds, and passes through respectively
Self-control or commercial sources are obtained；
(2) preparation of oocyte or body early embryo oolemma：Carrying out every time, artificial embryology structure is first 24 hours, according to
The quantity of manufacturing artificial embryo, preparation needs the ovum or body early embryo of quantity 120%；The ovum or body early embryo of collection with
Micromanipulation mode is cut oolemma and suctions out ooplasm or cell division ball, is made the oolemma of ghost；
(3) artificial embryology builds and cell differentiation induction：
A. stem cell injects ovum oolemma：Various stem cells are directly injected into the oolemma of ghost and induce people
Work embryo, with (w:W) the stem cell group that 0.5% trypsin treatment is cultivated, then with micromanipulation mode to each animal
40-60 stem cell of injection in the oolemma of the ghost of ovum or body early embryo, in stem cell-oolemma complex set,
And carried out in stem cell medium in vitro culture 12-24 hours according to different plant species, stem cell is continued increment and form 60-
80 cell masses, the condition of culture of stem cell-oolemma complex is 5%CO2, 5%O2And 90%N2。
B. the external evoked culture of artificial embryology：The stem cell that step A is obtained-ovum oolemma complex, in 5%CO2
In the condition of culture of 95% air, Fiber differentiation 12-24 hours in induction broth 1, in induction broth 2
Culture break up 24-48 hour, differentiate the similar blastaea structure with interior embryo group, blastocoele and nutritive layer cell composition dry
Cell clone embryo；
Induction broth 1 is formulated, by volume
Induction broth 2 is formulated, by volume
(4) animal cloning is bred：The artificial embryology that step (3) is built is moved according to routine techniques (No operation uterine transplantation)
Plant the receptor intrauterine of estrus synchronization, it is possible to which obtaining has and stem cell heredity identical cloned animal offspring.
The mammal includes experimental animal, domestic animal, the mankind.
The experimental animal refers to mouse, rat, rabbit, primate；Domestic animal refers to ox, sheep, pig, horse, deer.
Ovum or body early embryo in the step (1), collection, family can be put to death for experimental animal ovum or body early embryo
Poultry ovum can live body collection or using butcher dam ovary gather.
The model that the method that above-mentioned mammalian artificial's embryo's structure-animal cloning based on stem cell is bred is obtained
Embodiment 1
Horse stem cell-artificial embryology builds：
1) preparation of stem cell：It is identical with setting up for stem cell line, it is made through division in culture stem cell the last week first
Stop individual layer vegetative cell layer (32-34 days embryo fibroblasts of horse) for the treatment of.The condition of culture of vegetative cell is 5%CO2
Air with 95%, the composition of nutrient solution adds 20% serum for STO.Take the 8000-10000 freezen protective through passing on 16 times
Horse embryonic stem cell, with 2-3 4 well culture plates are inserted after stem cell medium centrifugal treating after defrosting, makes its propagation 3-4
My god, and form the stem cell group of multiple 500-800 cell compositions.The stem cell line is the stem cell from embryo
(Embryonic SC), without carrying any quiding gene for being available for stem cell differentiation marker.
2) preparation of horse ovum oolemma：24 hours before stem cell injection oolemma, according to 50 clone embryos of making
Quantity, collect 70 pieces of horse ovums with the discarded horse ovary in slaughterhouse.The horse ovum of collection cuts oolemma in micromanipulation mode
And ooplasm is suctioned out, it is made 70 empty oolemmas.
3) stem cell injection oolemma：Cell can be directly injected into the oolemma of ghost and induce clone embryos.It is dry thin
The injection mode of born of the same parents is, when stem cell forms the cell mass of 500-800, to do thin with being isolated after 0.5% trypsin treatment
Born of the same parents group, then with micromanipulation mode to 40-60 stem cell of injection in the empty oolemma of each oocyte or body early embryo
Or stem cell group.Total 46 stem cells of successful combination-ovum oolemma complex, injection terminate after stem cell-transparent
In band complex set, and carry out in vitro culture.
4) in vitro culture of stem cell-ovum oolemma complex and cell induction break up：Stem cell-ovum oolemma
The in vitro culture of complex still needs the individual layer vegetative cell layer that division stops treatment, and condition of culture is 5%CO2With 95%
Air, the incubation time in induction broth 1 is 24 hours, and the incubation time in induction broth 2 is 72 hours, is added up to
86 hours Fiber differentiation time.The similar blastaea structure with interior embryo group, blastocoele and nutritive layer cell composition can be differentiated to do
Cell clone embryo 32 (32/46,69.6%).
5) formation and identification of stem cell-artificial embryology：Because the stem cell line is the stem cell from embryo
(Embryonic SC), without carrying any quiding gene for being available for stem cell differentiation marker, therefore utilizes cells and characteristic of stem base
Because of Oct-4 specific immunofluorescence decoration methods, the clone embryos to being come by stem cell differentiation are analyzed identification, wherein 84%
(27/32) the cell function feature of normal fetus is shown：The inner cell mass of the cloned blastocysts for differentiating still shows Oct-4 genes
Green and nutrition confluent monolayer cells due to differentiation lose versatility or totipotency show green or green substantially weaken.
Embodiment 2
Mouse stem cells-artificial embryology builds：
1) preparation of stem cell：It is identical with setting up for stem cell line, it is made through division in culture stem cell the last week first
Stop individual layer vegetative cell layer (13.5 days embryo fibroblasts of mouse) for the treatment of.The condition of culture of vegetative cell is 5%CO2
Air with 95%, the composition of nutrient solution adds 20% serum for STO.Take the 8000-10000 freezen protective through passing on 24 times
Mouse embryo stem cell, with 2-3 4 well culture plates are inserted after stem cell medium centrifugal treating after defrosting, makes its propagation 3-4
My god, and form the stem cell group of multiple 500-800 cell compositions.The stem cell line stem cell line is from early after implantation
The stem cell (Embryonic SC) of embryonic stem cell (Epiblast SC) embryo of phase embryo's upper cell, carries Oct-4 bases
Because of green protein, its differentiation situation directly can be under the microscope detected.
2) preparation of Mouse Eggs oolemma：24 hours before stem cell injection oolemma, embryos are cloned according to making 100
The quantity of tire, 137 pieces of ovums are gathered with 10 mouse of HORMONE TREATMENT from operation on fallopian tube.The Mouse Eggs of collection are with micro- behaviour
Oolemma is cut as mode and suction out ooplasm, 114 for being made sky oolemma.
3) stem cell injection oolemma：The injection mode of stem cell is, when stem cell forms the cell mass of 500-800, to use
Stem cell group is isolated after 0.5% trypsin treatment, then with micromanipulation mode to each oocyte or early embryo
Injection 30-40 stem cell or stem cell group in the empty oolemma of tire.105 stem cell-ovums of total successful combination are transparent
Band complex, injection terminate after in stem cell-oolemma complex set, and carry out in vitro culture.
4) in vitro culture of stem cell-ovum oolemma complex and cell induction break up：Stem cell-ovum oolemma
The in vitro culture of complex still needs the individual layer vegetative cell layer that division stops treatment, and condition of culture is 5%CO2With 95%
Air, the incubation time in induction broth 1 is 24 hours, and the incubation time in induction broth 2 is 24 hours, is added up to
72 hours Fiber differentiation time.The similar blastaea structure with interior embryo group, blastocoele and nutritive layer cell composition can be differentiated to do
Cell clone embryo 87 (87/105,82.9%).
5) formation and identification of stem cell-artificial embryology：To the clone embryos by stem cell differentiation in fluorescence microscope
Observation identification is directly carried out down, it is found that wherein 82% (71/87) stem cell clone embryo shows the cell function of normal fetus
Feature：The inner cell mass of the cloned blastocysts for differentiating still shows Oct-4 genes green and nutrition confluent monolayer cells are lost due to differentiation
Versatility or totipotency do not show green or green substantially weakens.
Embodiment 3
Human stem cells-artificial embryology builds：
1) preparation of stem cell：The 8000-10000 freezen protective hESC through passing on 24 times is taken, is thawed
Afterwards with 2-3 4 well culture plates are inserted after stem cell medium centrifugal treating, make its propagation 3-4 days, and form multiple 500-
800 stem cell groups of cell composition.
2) preparation of mankind's ovum oolemma：24 hours before stem cell injection oolemma, cloned according to making 12-15
The quantity of embryo, 15 discarded ovums are obtained from Test-Tube Baby Lab, are cut oolemma in micromanipulation mode and are suctioned out
Ooplasm, the empty oolemma being made.
3) stem cell injection oolemma：The injection mode of stem cell is, when stem cell forms the cell mass of 500-800, to use
Stem cell group is isolated after 0.5% trypsin treatment, then with micromanipulation mode to note in each ovum sky oolemma
Penetrate 30-40 stem cell or stem cell group.12 stem cells of total successful combination-ovum oolemma complex, injection terminates
Afterwards in stem cell-oolemma complex set, and carry out in vitro culture.
4) in vitro culture of stem cell-ovum oolemma complex and cell induction break up：Stem cell-ovum oolemma
The in vitro culture of complex still needs the individual layer vegetative cell layer that division stops treatment, and condition of culture is 5%CO2With 95%
Air, the incubation time in induction broth 1 is 24 hours, and the incubation time in induction broth 2 is 24 hours, is added up to
72 hours Fiber differentiation time.
5) formation and identification of stem cell-artificial embryology：Due to currently without dry thin using the mankind with fluorescence labeling
Born of the same parents are, similar with the compound of interior embryo group, blastocoele and nutritive layer cell composition that can differentiate mainly by morphological method
Body is judged as stem cell-clone embryos 8 (8/12,66.7%).
Embodiment described above is merely to illustrate technological thought of the invention and feature, in the art its object is to make
Technical staff it will be appreciated that present disclosure and implementing according to this, it is impossible to patent model of the invention is only limited with the present embodiment
Enclose, i.e., equal change or modification that all disclosed spirit is made still fall in the scope of the claims of the invention.
Claims (5)
1. a kind of method that mammalian artificial's embryo's structure-animal cloning based on stem cell is bred, it is characterised in that
Comprise the following steps：
(1) preparation of mammalian stem cell：The induction that mammalian stem cell is used for artificial embryology builds, respectively by self-control
Or commercial sources are obtained；
(2) preparation of oocyte or body early embryo oolemma：Carrying out every time, artificial embryology structure is first 24 hours, according to making
The quantity of artificial embryology, preparation needs the ovum or body early embryo of quantity 120%；The ovum or body early embryo of collection are with micro-
Mode of operation is cut oolemma and suctions out ooplasm or cell division ball, is made the oolemma of ghost；
(3) artificial embryology builds and cell differentiation induction：
A. stem cell injects ovum oolemma：Various stem cells are directly injected into artificial embryo is induced in the oolemma of ghost
Tire, the stem cell group cultivated with the trypsin treatment of mass percent concentration 0.5%, then with micromanipulation mode to each
40-60 stem cell of injection in the oolemma of the ghost of oocyte or body early embryo, stem cell-oolemma complex set
In, and carried out in stem cell medium in vitro culture 12-24 hours according to different plant species, stem cell is continued formation of rising in value
60-80 cell mass, the condition of culture of stem cell-oolemma complex is 5%CO2, 5%O2And 90%N2；
B. the external evoked culture of artificial embryology：The stem cell that step A is obtained-ovum oolemma complex, in 5%CO2With
In the condition of culture of 95% air, Fiber differentiation 12-24 hours in induction broth 1, in induction broth 2
Culture differentiation 24-48 hours, differentiates the similar blastaea structure with interior embryo group, blastocoele and nutritive layer cell composition dry thin
Born of the same parents' clone embryos；
Induction broth 1 is formulated, by volume
Induction broth 2 is formulated, by volume
(4) animal cloning is bred：The artificial embryology that step (3) is built is moved according to the acceptor that routine techniques is transplanted to estrus synchronization
Thing intrauterine, it is possible to which obtaining has and stem cell heredity identical cloned animal offspring.
2. the mammalian artificial's embryo's structure-animal cloning based on stem cell according to claim 1 is bred
Method, it is characterised in that the mammal includes experimental animal, domestic animal, the mankind.
3. the method that the mammalian artificial embryo based on stem cell according to claim 2 builds-clones, it is special
Levy and be, the experimental animal refers to mouse, rat, rabbit, primate；Domestic animal refers to ox, sheep, pig, horse, deer.
4. the mammalian artificial's embryo's structure-animal cloning based on stem cell according to claim 1 is bred
Method, it is characterised in that ovum or body early embryo in the step (1), can locate for experimental animal ovum or body early embryo
Dead collection, domestic animal ovum live body collection or can be gathered using dam ovary is butchered.
5. the mammalian artificial embryo structure-animal cloning based on stem cell as described in claim any one of 1-4
The model that the method bred is obtained.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109735487A (en) * | 2018-12-18 | 2019-05-10 | 陈子江 | A kind of constraint method of lensless Fourier transform holography cell |
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| CN1483817A (en) * | 2002-09-18 | 2004-03-24 | 李建远 | Method for cloning human embryonic stem cell without animal component |
| CN1922307A (en) * | 2003-12-30 | 2007-02-28 | 财团法人首尔大学校产学协力财团 | Embryonic stem cell line and method for preparing the same |
| KR100670616B1 (en) * | 2005-08-25 | 2007-01-17 | 주식회사 메디아나전자 | Artificial blstocyst for cultivating cells using epithelial cell or fibroblast, cultivating apparatus thereof |
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| Title |
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| HIROYUKI HIRAI ET AL.: "Regulation of embryonic stem cell self-renewal and pluripotency by leukaemia inhibitory factor", 《BIOCHEM J.》 * |
| XIHE LI ET AL.: "Horse Embryonic Stem Cell Lines from the Proliferation of Inner Cell Mass Cells", 《STEM CELLS AND DEVELOPMENT》 * |
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| CN109735487A (en) * | 2018-12-18 | 2019-05-10 | 陈子江 | A kind of constraint method of lensless Fourier transform holography cell |
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Application publication date: 20170531 |