CN1788079A - Method for separating and cultiviting human multifunctional embryo stem cell - Google Patents

Abstract

The present invention relates to the isolation and culture of human embryonic stem cell. Which comprises the following steps: (1) planting the human blastula in embryo fibroblast feeder layer; (2) picking out the inner cell mass (ICMs) when its outward growing are finished, then removing the peripheral differentiation cell layer, dispersing the ICMs to pieces, and replanting it in new embryo fibroblast feeder layer. The said method omits the step of immunity operation, and makes the isolation process simply.

Description

Method for separating and cultiviting human multifunctional embryo stem cell

A kind of method and technology field for separating and cultivating mankind's pluripotent embryonic stem cells

The present invention relates to the separation of mankind's pluripotent embryonic stem cells, the method for in vitro culture.Background technology：

HESC（Embryoni c stem c l l, ES cells）Be successfully separated the impressive progress that culture is current life science.People's ES' cells be from human embryo separation, can in vitro under felicity condition holding undifferentiated state a kind of stem cell.It has the potential for being divided into all cell tissues of body（The totipotency of differentiation）It can be used as " seed cell ", people can obtain the cell donor that substantial amounts of different types of cell is used as cell transplantation, tissue substitute and organ cloning from induction ES cell directionals differentiation, and unlimited cell derived is provided for many refractory diseases for the treatment of in the future mankind.Therefore in the clinical practice in future prospect extensively, this is the main application in people ES cell future.Other purposes of people's ES cells include：

1st, as the carrier of gene therapy, so as to reduce the potential hazard of original viral vector and improve the security of gene therapy；

2nd, the model for studying mammalian developmental biology can be used as.In ES cells in vitro atomizations, certain precursor stage is necessarily first passed through.This provides ideal experimental system for the origin and characteristic for studying some precursors, energy influence that is qualitative or even quantitatively studying the factor such as some cell factors, extracellular matrix cell growth and differentiation, it is to avoid and reduce the complexity of various endogenous factors interference in overall embryo's research.

3rd, pharmacological research：People ES cells can provide the Normal human cells of any organization type, and a large amount of samples are provided for developing new drug, and available for screening medicine, the target gene site of identification new drug effect, screen gene for regeneration gene therapy.

Built on Human ES cells is referring to bibliography 1-11.When human embryos develop blastaea in vitro, the outer embryo trophocyte of outer layer and internal inner cell mass part can be divided into.In Embryo implantation, row is into placenta when behind uterus for the former, and the latter then forms fetus.Embryonic stem cell just derives from the inner cell mass part of blastaea.Early stage has researcher to attempt the separation method using mouse embryo stem cell, i.e., it is direct the blastaea of the mankind is planted to watch in mouse embryo desmocyte human embryo stem cell is separated on foster layer, but not successfully.Generally believe that the separation situation of hESC is different from mouse, after whole blastaea plantation, the presence of outer embryo trophocyte can induce the differentiation of inner cell mass part cell, so as to be unfavorable for therefrom separating human embryo stem cell.In the document of presently disclosed and external cellar culture, researcher substantially employs identical method：In vitro culture obtains mankind's blastaea, inner cell mass part inside blastaea is separated by Immunosurgery method, first it is placed in resist in outer embryo trophoderm antibody i.e. by blastaea and cultivates, transfer in guinea pig complement and cultivate, the complement complex of antibody one can attack the cell membrane for destroying outer embryo trophocyte, cause disintegrating, coming off for outer embryo trophocyte.After piping and druming, outer embryo trophocyte scatters, so that point From the inner cell mass obtained inside blastaea.Inner cell mass is planted in the fibroblast separated by Mouse Embryos（Mouse embryoni c fi brobl ast, abbreviation mEFs) word support cellular layer on, using DMEM culture mediums+hyclone（FBS the cultivating system of)+recombinant human leukemia inhibitory (rh-LIF), constantly amplification passage, you can isolated human embryonic stem cell.

The preparation method of mouse embryo desmocyte feeder layer（See reference document 3)

Serum free culture system is to be used for a kind of new cultivating system of mouse embryo stem cell in the recent period.Mainly with serum substitute（Serum Replacement, SR) replace the hyclone often added in culture medium（FBS) composition, can effectively prevent the FBS of different production batch from notable difference occurring to the supporting function that embryonic stem cell grows,（See reference document 4).Display uses serum free culture system, i.e.,：Knock-out DMEM+Knock-out SR+recombination human basic fibroblast growth factor（Rh- bFGF), the cell monoclonal growth rate of the human embryo stem cell of built system will be apparently higher than the cellar culture system using FBS.But there is no is used for the report that human embryo stem cell is separated using serum free culture system.This research meets Chinese " auxiliary procreation technology management regulation " regulation.The patient for receiving routine in vitro insemination-Embryo Transplant for Treatment voluntarily signs informed consent form, shows that voluntary donations/contributions residue frozen embryo is used for scientific research after successful pregnancy, or voluntarily abandoned after embryo transfer by remaining embryo cryopreservation, for scientific research.Embryo after fresh or defrosting is through G1. 2 and the sequential culture bases of G2. 2（Vi trol i fe) cultivate to blastocyst stage.The content of the invention

The technical scheme is that：Directly mankind's blastaea is planted and supports layer in embryo fibroblast word, after after the growth of inner cell mass evagination, choose inner cell mass, remove the noble cells layer of its periphery parcel, internal cell mass is dispersed into fritter, new embryo's word is planted in again and is supported on layer.

The invention provides mankind's pluripotent embryonic stem cells of purifying, the pluripotent embryonic stem cells are obtained by the above method.

The blastaea used can be obtained by the In vitro culture after fresh or freeze-thaw.Treat that blastaea is voluntarily hatched, or oolemma is removed with protease digestion.Blastaea outer layer has oolemma parcel, similar " eggshell ", before further processing blastaea, it is necessary to remove the oolemma of outer layer；When blastaea is cultivated in vitro, it can voluntarily break " shell " sometimes and go out, that is, hatch；Or oolemma is removed by protease digestion, help blastaea to hatch.This is to build general procedure when being.

Embryonic fibroblast for directly plantation blastaea can use mouse embryo desmocyte feeder layer.The Mi tomyc i n C processing of mouse embryo desmocyte feeder layer, carries out mitosis inactivation, prevents fibroblast from keeping merisis, is also to build the general procedure for being.

The preparation method of mouse embryo desmocyte feeder layer is disclosed in the prior art.

Preferably prepare for the separation Human ES cells from the mankind's remaining blastaea and by the foster layer of the mouse embryo desmocyte word of people's ES passages using the previous day.Fresh mankind ES is changed before use Cell culture medium.

The blastaea directly planted voluntarily adherent tiling growth.

Inner cell mass is the structure inside blastaea, after adherent growth, and " inner cell mass " structure can grow and break up simultaneously, shows as periphery and wraps one layer of noble cells, just as " pericarp ".It can only together choose during picking, the noble cells " skin " on periphery is torn off again afterwards.

When inner cell mass block length goes out, profile is obvious；Periphery is surrounded with the endoderm-like cells of differentiation, and internal neoblast is rolled into a ball not yet when there is layering, differentiation or cystis degeneration, and internal cell mass is chosen, the noble cells of removal periphery wrapping, and disperses.

When internal cell mass is disperseed, it should try one's best and be torn into small cell monolayer agglomerate, preferably be dispersed into the cell monolayer agglomerate of 50-200 cells, to destroy its chondritic, promote it to be laid in fresh mouse embryo desmocyte and watch on foster layer.Spherical fritter is difficult to be laid in mouse embryo desmocyte and watch to grow on foster layer, and often breaks up again after adherent.

Culture medium used can be DMEM+ hyclones, KNOCK OUT DMEM+KN0CK-0UT SR+rh- bFGF.It is found by the applicant that separating the cell line of Human embryo thousand in blastaeas of the KNOCK-OUT DMEM+KN0CK-OUT SR+rh-bFGF than DMEM+ hyclone more suitable for not receiving Immunosurgery from the mankind directly.Preferred concentration ratio be the ng/ml rh-bFGF of+15% nock-out Serum Replacer of 85% Knock- out DMEM+4.

Conventional method culture can be used by the isolated cell of mankind's pluripotent embryonic thousand of the above method.Culture medium used can be DMEM+ hyclones, KNOCK-OUT DMEM+KN0CK-0UT .SR+rh- bFGF.Also contain 1% nonessential amino acid stock in two kinds of culture mediums jointly（), Gibco 0. 2-mercaptoethanols of lmM (Sigma), 2raM L-Glutatnine (Gibco), 50IU/ml penicillin（), Sigma 50ug/ml streptomysins（ Sigma ).It is found by the applicant that separating human embryonic stem cell in blastaeas of the KNOCK- OUT DMEM+KN0CK-0UT SR+rh-bFGF than DMEM+ hyclone more suitable for not receiving Immunosurgery from the mankind directly.Preferred concentration ratio be the ng/ml rh-bFGF of+15% nock-out Serum Replacer of 85 % Knock-out DMEM+4.

Mankind's pluripotential embryonic cell line can be set up according to conventional methods by the isolated mankind's pluripotent embryonic stem cells of the above method.

The present invention has successfully directly separated human embryo stem cell HES-4 successfully using mouse embryo desmocyte as feeder cells from mankind's blastaea of adhere-wall culture.Expanded for 16 generations in vitro, significant antigen SSEA-4, TRA-l-60 and AKP of the undifferentiated state of cAHES-4 cells detection of expression are positive, expressed the distinctive transcripton Oct-4 of multipotential stem cell, Telomerase positive keeps normal caryogram.Stable human embryonic stem cell can be obtained under this separation method so as to demonstrate.For more conventional, the separation method eliminates Immunosurgery link, easy separable programming.Brief description of the drawings：

Fig. 1 is planted in mankind's blastaea on mouse embryo desmocyte feeder layer（40X);

The inner cell mass part of Fig. 2 blastaeas（100X); After the inner cell mass part for the blastaea that Fig. 3 chooses, the noble cells for removing outer layer（100

X );

The cMiES-4 clones (100X) that Fig. 4 grows on mouse embryo desmocyte feeder layer;The alkaline phosphatase detection of Fig. 5 c IES-4 cells（100X ) ：Positive findings is that cell clone is dyed to red, and fibroblast feeder layer is not colored；

The antigens of SSEA- 4 detection of Fig. 6 cells（ 200X ):Positive colony shows apple green under 450nm exciting light；

The TRA- 1- 60 of Fig. 7 cMIES-4 cells pick up survey（ 200X ):Positive colony shows apple green under 450nm exciting light；

The plastidogenetic embryoid bodies of Fig. 8 (40X)；

Fig. 9 RT-PCR analyze expression s of the OCT-4 in c//HES-4 cells

1st swimming lane, the PCR reactions of the chHES-4 cells of no reverse transcriptase；

2 swimming lanes, cAHES-4 cells

3rd swimming lane, c/zHES-4 cells, β-actin internal references

It is right（R) swimming lane, pUC Mix Marker, 8.

The Telomerase of Figure 10 cAHES-4 cells（TRAP) detect；

6th swimming lane, 2000 cAHES-4 react after Telomerase heat inactivation；

7th 9 swimming lane, respectively 500, the TRAP reactions of 1,000,2,000 cell；

Embodiment

Material

1st, ES cell culture mediums 80%KN0CK-OUT DMEM (K0-DMEM), 20%KN0CK-0UT serum replacements（K0- SR, GIBCO- BRL companies provide）, ImM l-GLUTAMINEs, 0. ImM beta -mercaptoethanols, 1% nonessential amino acid（GIBCO- BRL companies）With human recombinant basic fibroblast growth factor（BFGF, GIBCO- BRL companies）；

2nd, embryoid body culture medium：80%DMEM, 20% hyclone, ImM l-GLUTAMINEs, 0. ImM beta -mercaptoethanols, 1% nonessential amino acid（GIBCO- BRL companies）；

3rd, fibroblast culture medium：85%DMEM, 15% hyclone；

4th, 10%DMS0 frozen solutions：Setting freezing n pipe fibroblasts（The amount of liquid filled per cryovial is lml), total amount of liquid is n ml, and required DMSO amounts are the ral of n/10, add 4n/10ml K0- SR, mixed hook is that DMS0 content is 20%.The cell of freezing needed for being resuspended with n/2 ml 10%K0- SR DMEM, add it is above-mentioned contain 20% DMS0 match somebody with somebody liquid.

5th, 0.1% gelatin is prepared：0. 1 grams of gelatin are weighed, are dissolved in 100 milliliters of distilled water.Autoclaving.4 °C of refrigerators are put to save backup.

6th, mitomycin C:Lmg/ml concentration is made into PBS liquid, 4 °C save backup. 7th, Anti- SSEA-l monoclonal antibodies（Hybridoma Bank).

8th, Anti-SSEA-4 and Anti- TRA- 1-60 monoclonal antibodies（Peter professors Andrews of Sheffield universities give）.（See reference document 13)

9th, the rabbit anti-mouse IgG (Dako) of FITC marks.

10th, telomerase activation detection kit is purchased from integen companies；Total serum IgE extraction agent box is purchased from Gentra companies；MRNA extraction agents box is purchased from Qiagen companies；One-step method RT- PCR kits are purchased from Qiagen companies.

(two）Method

1st, MEC separation, culture and passage

(1) the de- dams for putting to death pregnant 8 ~ 15 days in vain of cervical vertebra；

(2) immersion dams are in 70% alcohol after 1 one 3 minutes, it is contained on sterile basin, lift stomach wall with pliers, cut off stomach wall, fully exposure internal organ, cut off peritonaeum exposure uterus, lift uterus base portion with pincet, in separating uterus, the culture dish that uterus is put into the 100mm containing 10ml D-PBS；

(3) uterine wall is cut off to discharge embryo, the embryo with complete fetal membrane is moved into fresh D-PBS.Fetal membrane, which is stabbed out, with ophthalmic tweezers discharges embryo, the viscera tissue of go down in stereoscope mice embryonic neck, carefully stripping removal mice embryonic, operation handles each embryo more than.The embryo being stripped clean is washed two times with D-PBS；

(4) shredded after treated embryo, added in the trypsase-EDTA of 2ml 0.05% with scissors, 37 °C digest 5 minutes, and suction pipe blows and beats into cell suspension.With the digestion in DMEM liquid of the 5ml containing 5% newborn calf serum with trypsase.Liquid after neutralization is moved into_:In 15ml centrifuge tube；

(5) 2 minutes are stood, the larger tissue block for fully not shredding and digesting is sunk to ttom of pipe.' draw upper liquid, supernatant is moved into the blake bottles of T- 175, depending on obtain embryo's quantity number come the blake bottle number used in determining, the cell numbers of general 6-8 pieces of processing embryo can be planted in 1 blake bottle of T- 175.Move into 37 °C of 5%C02 incubators and cultivate.Liquid is changed after 24 hours, dead cell residue is removed；

(6) when cell growth 80% -90%, it is necessary to pass on amplifying cells.Waste and old culture medium is first exhausted before passage, 1 time is washed with D-PBS to eliminate influence of the serum to trypsase in waste and old culture medium.Trypsase-the EDTA of 37 preheatings is added, is digested 3-10 minutes in 37 °C of environment.With being acted in the DMEM liquid containing 5% newborn calf serum with Trypsin Induced, cell is set to take off wall and into single cell suspension with pipette piping and druming.It is general to press 1:3 or 1:5 passages.Cell is about cultivated 6-7 days, and cell, which can converge, need to pass on amplification again.It is used for preparing the feeder cells of hESC using 3 generations of 2- cell.Conventional cell and culture medium to culture carries out mycoplasma and Bacteria Detection simultaneously.

2nd, prepared by mouse embryo desmocyte feeder layer：Handled using mitomycin C Waste and old nutrient solution is absorbed, adds and presses mitomycin C (final concentration of 1 μ g/ml).Move into processing cell after 2. 5 hours in C02 incubators, absorb treatment fluid, add a certain amount of D-PBS cleanings, treatment fluid and cleaning fluid need to useless pipe sealing it is good after abandon it.Trypsase-EDTA digestive juice the vitellophags of preheating are added, 37 °C digest 3-10 minutes.When part cell detachment, when adherent cell is largely rounded, using the digestion in Fibroblast culture solution with enzyme.The cell for not taking off wall is blown and beaten with pipette, while cell is dispersed as into single cell suspension.Cell count.150g is centrifuged 5 minutes, removes supernatant, then wash cell twice with nutrient solution, to remove the mitomycin C of residual, appropriate culture medium is added finally according to cell density, according to needing to continue to cultivate as cell is layered in 4 or 6 orifice plates by preceding method, quality control is carried out and picks up survey., separation and culture hESC

(1) it is used to separate Human ES cells from the remaining blastaea of the mankind and prepares the mouse embryo desmocyte feeder cells of people's ES passages using the previous day.Fresh Human ES cells' culture medium is changed before use；

(2) fresh or defrosting embryo cultivates to blastaea in sequential culture base or hatches blastocyst stage；

(3) blastaea is not hatched through protease（Pronase, Sigma) digestion is removed after oolemma, or blastaea is hatched, directly it is planted on mouse embryo desmocyte feeder layer, allows it voluntarily to attach；

(4) inner cell mass block length goes out, and profile is obvious.Periphery is surrounded with the endoderm-like cells of differentiation, and internal neoblast group is not yet layered, broken up or during cystis degeneration, about 5 10 days, timely picking inner cell mass（See Fig. 1,2).Key is：（1) the noble cells layer of its periphery is torn off,（2) by internal cell mass mechanical dispersion, and it is torn into the cell block of individual layer to destroy its chondritic as far as possible（See Fig. 3), promote it to be laid in fresh mouse embryo desmocyte word and support on layer；Spherical fritter, which is difficult to be laid on mouse embryo desmocyte feeder layer, to be grown, and is often broken up again after adherent；（3) culture medium should select K0-DMEM+K0-SR+rh-bFGF, and be generally used for separating the DMEM+FBS+rh-LIF of human embryo stem cell

(5) cell starts after amplification, during routine passage：Culture medium is sucked, lmg/ml IV Collagenase Types are added, 5min is digested at 37 °C.Digestion effect is observed under stereomicroscope, treats that ES cell colonies periphery is rolled, foster layer and noble cells are watched in removal, together with the one piece of absorption of collagen enzyme liquid, fresh culture is added, suction pipe is gently blown and beaten, adherent cell mass piping and druming is turned into 100 cells of 50- or so.By the cell seeding of digestion on freshly prepared MEC feeder layer, daily observation（See Fig. 4), liquid is changed every other day.Passage in 45 days is once.

4th, the alkaline phosphatase of ES cells（AKP) detect：After cold 80% ethanol fixes 2-18 hours, distillation washing 2 times, with the dyeing of Fresh, when dyeing relatively satisfactory, with steaming Distilled water is washed 2 times, then observes coloration result with D- PBS immersions.AKP detects dyeing liquor：The 3ml 10%MgCl of+44. 6ml distilled water of 25mg Fast Red TR+5mg alpha-Naphthols+0.₂The Boratexes of+5nil 4. 5%.It is most of to be cloned in the AKP positives（Positive findings is that cell clone is dyed to red, and fibroblast feeder layer is not colored, and noble cells clone is also not colored）（See Fig. 5).

5th, the cell undifferentiated property detections of ES：Detected using indirect immunofluorescence cytochemical methods.

Divide another (1 use anti-SSEA-1, ant i-SSEA-4 (1:3-4) and anti-TRA- 1-60 (1:3- 4) as primary antibody, the rabbit anti-mouse IgG (1 marked using FITC:50) the ES cells of culture are detected as secondary antibody.Wherein SSEA-1 and

TRA-1-60 detections are fixed using 100% ethanol, and the detections of SSEA- 4 steam hydropexis using two containing 90% acetone.After fixation, heterogenetic antigen is closed with Normal Goat Serum, is washed with PBS twice, it is every all over 5 minutes, primary antibody is added, is incubated 30 minutes, PBS is washed twice, it is every all over 5 minutes.Secondary antibody is added, is incubated 30 minutes, PBS is washed twice.Under fluorescence microscope（450nm) observe the testing result of cell clone.SSEA- 4 and TRA- 1-60

(positive colony shows apple green under 450nm exciting light）Detection on be positive (see Fig. 6,7), SSEA- 1 is detected as feminine gender.Illustrate that the ES cells grown in people's feeder layer can keep undifferentiated state.6th, ES cells karyotyping：People's ES cells for growing on cellular layer are supported in word, culture to the

5-6 days, original culture medium is removed, the 02g/L of demecolcine 0. is added and handles 2 hours.ES cell clones are chosen with fine needle under stereoscope.Cell is collected with digestion under 0. 05%-EDTA37 °C of trypsase, hypotonic medium KC1 (0. 075%) is added and is placed in 37 °C of effect 30min, use methanol：Glacial acetic acid（3 :1) piece is dripped after fixing 3 times repeatedly, puts and agreement that contracts a film or TV play to an actor or actress is baked in 60 °C of baking boxs 4 ~ 5 hours.Dyed, flowing water is rinsed well, dried up, gummy mounting, in oily Microscopic observation result with the Gi emsa solution of fresh configuration after the Chromosome spreads prepared are digested into aobvious band through pancreatin.

7th, telomerase activation：Telomerase activation provides method detection according to kit（See Fig. 8).

8th, OCT- detection of expression method is RT-PCR, the PURESCRIPT RNA isolation kit that total serum IgE extracting is provided using Gentra companies are carried out, the Oligotex mRNA kit that extracting mRNA is provided with Qiagen companies afterwards are carried out, the One step RT-PCR kits provided using Qiagen companies are detected that operating procedure provides method by kit and carried out.Design of primers is as follows： OCT-4: sense,

5 '-GACAACAATGAGAACCTTCAGGAGA -3 '；Antisense, 5,-TTCTGGCGCCGGTTACA-GAACCA -3 '；It is as the β-actin primers of control：Sense, 5'-TTCTCCTTGATGTCACGCAC-3,； antisense, 5 '-CGCACCACTGGCATTGTCAT-3,.Product is on 1.5 % agar gels after electrophoresis, and ethidium bromide staining is observed（See Fig. 9)., embryoid body prepare：Such as routine passage people's ES cellular processes vitellophags, by people ES cell culture in the Micro-Organism Culture Dish of cell is supported without word.Culture medium is embryoid body culture medium.We have found that：The suspension culture after the foster layer of word is departed from of people ES cells can form embryoid body（See Figure 10).

Bibliography

1st, Thomson J A, Itskovitz-Eldor J, Shapiro SS, et a J. Embryoic stem cell lines derived from human blastocysts. Science. 1998,282:1145-7.

2、 Reubinoff BE, Pera MF, Chui-Yee Fong, et al. Embryonic stem cell lines from human blastocysts : somatic differentiation in vitro.

Nature Biotech 2000, 18(4) :399-404.

3、 Robertson EJ. Embryo— derived stem cell lines. In " Teratocarcinoma and embryonic stem cells- A Practical Approach " , IRL Press, Washington, DC. 1987, 77 - 78

4、 Am it M_} Carpenter MK, Inokuma MS, et aL Clonally derived human embryonic stem cell lines mai tain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000, 227 : 271-278.

5th, Xue Qing philanthropist compiles.The philosophy and technique of in vitro culture（The first edition）.Beijing：Science Press., 446-447 in 2001.

6th, Carpenter UK, Inokuma MS, Denham J, et aL Enrichment of nerons and neural precursors from human embryonic stem cells. Exp Neuro 2001,172:383-397.

7、 Buehr M, Mclaren A. Isolation and culture of primordial germ cells.

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8th, Chunhui Xu, Inokuma MS, Denham J, et al. Freeder-f ree growth of undifferentiated human embryonic stem cells. Nature Biotech 2001,19:971-974.

9th, Cooper is beautiful, Ta ura RN_} Quaran ta V. The major laminin receptor of mouse embryonic stem cells is a novel isof orni of the 6 β 1 integrin. J Cell

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10、 Belkin AM and Stepp MA. Integrins as receptors for laminins, .' Micro Res Tech 2000, 51:280-301.

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Claims (1)

1. Claim

   1st, the method for separating mankind's pluripotent embryonic stem cells, it comprises the following steps：（1) directly mankind's blastaea is planted in embryonic fibroblast；（2) after after the growth of inner cell mass evagination, choose inner cell mass, remove the noble cells layer of its periphery parcel, internal cell mass is dispersed into fritter, is planted in again on new embryo fibroblast feeder layer.

   2nd, the method for separation mankind's pluripotent embryonic stem cells according to claim 1, it is characterised in that embryo fibroblast feeder layer used is mouse embryo fibroblast feeder layer.

   3rd, the method for separation mankind's pluripotent embryonic stem cells according to claim 1, it is characterised in that embryo fibroblast feeder layer used is prepared using the previous day.

   4th, the method for separation mankind's pluripotent embryonic stem cells according to claim 1, it is characterised in that when internal cell mass is disperseed, should try one's best and be torn into small cell monolayer agglomerate.

   5th, the method for separation mankind's pluripotent embryonic stem cells according to claim 1, it is characterised in that when internal cell mass is disperseed, be dispersed into the cell monolayer agglomerate of 50-200 cells.6th, the method for separation mankind's pluripotent embryonic stem cells according to claim 1, it is characterised in that culture medium used is KNOCK- OUT DMEM+ N0CK-0UT SR+rh- bFGF.

   7th, the method for separation mankind's pluripotent embryonic stem cells according to claim 6, it is characterised in that culture medium used it is preferred ' concentration ratio is the ng/ml rh-bFGF of 85 % Knock-out DMEM+15%Knock-out Serum Replacer+4.

   8th, the method for the cell of culture mankind pluripotent embryonic thousand, it is characterised in that cultivate mankind's pluripotent embryonic stem cells that one of claim 1 to 7 is obtained in the medium.

   9th, the method for culture mankind's pluripotent embryonic stem cells according to claim 8, it is characterised in that culture medium KNOCK-OUT DMEM+KN0CK-0UT SR+rh-bFGF.

   10th, the method for culture mankind's pluripotent embryonic stem cells according to claim 9, it is characterised in that the preferred concentration ratio of culture medium is 85 % Knock- out DMEM+15%Knock- out

   Serum Replacer + 4 ng/ml rh - bFGF。

   11, mankind's pluripotential embryonic cell line, it is that the mankind's pluripotent embryonic stem cells obtained by one of claim 1 to 7 are set up.