WO2004111206A1

WO2004111206A1 - A method for isolation and culture of human embtyonic stem cell

Info

Publication number: WO2004111206A1
Application number: PCT/CN2003/000459
Authority: WO
Inventors: Guangxiu Lu; Ge Lin; Changqing Xie
Original assignee: Changsha Hui-Lin Life Technology Co. Ltd
Priority date: 2003-06-13
Filing date: 2003-06-13
Publication date: 2004-12-23
Also published as: AU2003246148A1; CN1788079A

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 isolating and culturing human pluripotent embryonic stem cells

The invention relates to a method for isolating human pluripotent embryonic stem cells and culturing them in vitro. Background technique:

The successful isolation and culture of human embryonic stem cells (Embryoni c stem c l l, ES cells) Establishing lines is an important progress in life sciences today. Human ES 'cells are a type of stem cells isolated from early human embryos and able to remain undifferentiated under appropriate conditions in vitro. It has the potential to differentiate into all cellular tissues of the body (totipotency of differentiation) and can be used as "seed cells". One can obtain a large number of different types of cells from induced ES cells to differentiate into cells for transplantation, tissue replacement and Organ cloned cell donors provide an unlimited source of cells for future treatment of many refractory diseases in humans. Therefore, it has broad prospects in future clinical applications, which is the main future use of human ES cells. Other uses of human ES cells include:

1. As a vector for gene therapy, which can reduce the potential harm of the original viral vector and improve the safety of gene therapy;

2. It can be used as a model for studying the developmental biology of mammals. In the process of ES cell differentiation in vitro, it must pass through a certain precursor cell stage. This provides an ideal experimental system for studying the origin and characteristics of certain precursor cells. It can qualitatively and quantitatively study the effects of certain cytokines, extracellular matrix and other factors on cell growth and differentiation, avoiding and reducing overall embryo research. Complexity of various endogenous factors.

3. Pharmacological research: Human ES cells can provide normal human cells of any tissue type, provide a large number of samples for the development of new drugs, and can be used to screen drugs, identify target gene sites for new drug effects, and screen genes for tissue regeneration gene therapy.

See reference 1-11 for the establishment of human ES cell lines. When human embryos develop into blastocysts in vitro, they differentiate into outer ectotrophoblast cells and internal inner cell masses. The former forms the placenta when the embryo is implanted in the uterus, while the latter forms a fetus. Embryonic stem cells are derived from the inner cell mass of the blastocyst. Earlier researchers tried to isolate mouse embryonic stem cells by directly planting human blastocysts on mouse embryo fibroblast serving cells to isolate human embryonic stem cells, but they were unsuccessful. It is generally believed that the isolation of human embryonic stem cells is different from that of mice. After the entire blastocyst is planted, the presence of ectotrophoblast cells will induce the differentiation of parts of the inner cell mass, which is not conducive to the isolation of human embryonic stem cells. In the literatures that are currently published and routinely cultured in vitro, researchers have basically adopted the same method: human blastocysts are obtained by in vitro culture, and the inner cell mass of the blastocysts is separated by immunosurgery. The ectotrophoblast antibody is cultured and then transferred to the guinea pig complement. The antibody-complement complex will attack and destroy the cell membrane of the ectotrophoblast cells, resulting in the disintegration and shedding of the ectotrophoblast cells. After beating, the ectotrophoblast cells spread out, thus dividing Isolate the inner cell mass located inside the blastocyst. The inner cell mass was planted on mouse embryonic fibroblasts (mEFs) isolated from mouse early embryos on the word cell culture layer, and DMEM medium + fetal bovine serum (FBS) + recombinant human leukemia inhibitory factor was used. (rh-LIF) culture system, the human embryonic stem cell line can be isolated by continuous expansion and passage.

Preparation method of mouse embryo fibroblast feeder layer (see reference 3)

Serum-free culture system is a new culture system for mouse embryonic stem cells. Serum replacement (SR) is mainly used to replace the often added fetal bovine serum (FBS) components in the culture medium, which can effectively prevent different production batches of FBS from supporting the embryonic stem cell growth significantly. (See Reference 4). Shows that using a serum-free culture system, that is: knock-out DMEM + Knock-out SR + recombinant human basic fibroblast growth factor (rh- bFGF), the cloned human embryonic stem cells have a significantly higher cell growth rate For conventional culture systems using FBS. However, there have been no reports of the use of serum-free culture systems for the isolation of human embryonic stem cells. This study complies with China's "Regulations on the Management of Assisted Reproductive Technology". Patients who received conventional IVF-embryo transfer voluntarily signed an informed consent, indicating that after the successful pregnancy, they voluntarily donated the remaining frozen embryos for scientific research, or voluntarily abandoned the remaining embryos to be frozen after embryo transfer for scientific research. The fresh or thawed embryos were cultured to the blastocyst stage with G1.2 and G2.2 sequential culture media. Summary of the Invention

The technical scheme of the present invention is: directly planting human blastocysts into the embryonic fibroblast word trophoblast, after the inner cell mass grows out, pick out the inner cell mass, remove the peripherally wrapped differentiated cell layer, and transfer the inner cells The clumps are dispersed into small pieces and replanted on a new embryonic nutrient layer.

The present invention provides purified human pluripotent embryonic stem cells, which are obtained by the above method.

The blastocysts used can be obtained from in vitro culture of fresh or frozen thawed embryos. Allow the blastocysts to hatch on their own, or remove the zona pellucida by digestion with protease. The outer layer of the blastocyst is wrapped with a zona pellucida, similar to the "egg shell". Before further processing of the blastocyst, the outer zona pellucida must be removed. When the blastocyst is cultured in vitro, it can sometimes break out of the "shell" by itself, ie hatch ; Or remove the zona pellucida by protease digestion to help the blastocysts hatch. This is a common procedure when setting up a department.

The embryo fibroblast feeder layer for direct blastocyst implantation can be a mouse embryo fibroblast feeder layer. Rat embryo fibroblast feeder layer is treated with Mi tomyc i n C for mitotic inactivation to prevent fibroblasts from continuing to divide and grow, which is also a common step in establishing a line.

A method for preparing a mouse embryo fibroblast feeder layer has been disclosed in the prior art.

The mouse embryonic fibroblast word feeder used to isolate human ES cells from the remaining human blastocysts and to passage human ES cells is preferably prepared the day before use. Replace with fresh human ES before use Cell culture medium.

Directly planted blastocysts grow on their own wall.

The inner cell mass is the internal structure of the blastocyst. After adherent growth, the "inner cell mass" structure will grow and differentiate at the same time. It appears as a layer of differentiated cells around the periphery, like a "peel". When picking, you can only pick them together, and then peel off the "skin" of surrounding differentiated cells.

When the inner cell mass grows, the outline is obvious; when differentiated endoderm-like cells surround the periphery, while the inner undifferentiated cell mass has not yet stratified, differentiated, or cystically changed, the inner cell mass is picked out and removed The peripherally differentiated cells are dispersed.

When dispersing the internal cell clusters, try to tear them into small monolayer cell masses, preferably into 50-200 cell monolayer cell masses, in order to destroy its globular structure and promote its flat laying on fresh rat embryos. Fibroblasts serve on the feeder layer. Globules are not easy to spread on mouse embryo fibroblasts and grow on the feeder layer, and they often differentiate after adhering.

The medium used can be DMEM + fetal bovine serum, KNOCK OUT DMEM + KNOCK-0UT SR + rh-bFGF. The applicant has found that KNOCK-OUT DMEM + KNOCK-OUT SR + rh-bFGF is more suitable than DMEM + fetal bovine serum for isolating human embryonic fetal stem cell lines directly from human blastocysts that have not undergone immunosurgery. The preferred concentration ratio is 85% Knock-out DMEM + 15% nock-out Serum Replacer + 4 ng / ml rh-bFGF.

Human pluripotent embryonic thousand cells isolated by the above method can be cultured by conventional methods. The medium used can be DMEM + fetal bovine serum, KNOCK-OUT DMEM + KNOCK-0UT.SR + rh-bFGF. The two media also contain 1% non-essential amino acid stock solution (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 2raM L-Glutatnine (Gibco), 50IU / ml penicillin (Sigma), 50ug / ml Streptomyces (Sigma). Applicants have found that KNOCK- OUT DMEM + KNOCK-0UT SR + rh-bFGF is more suitable than DMEM + fetal bovine serum for isolating human embryonic stem cell lines directly from human blastocysts that have not undergone immunosurgery. The preferred concentration ratio is 85% Knock-out DMEM + 15% nock-out Serum Replacer + 4 ng / ml rh-bFGF.

The human pluripotent embryonic stem cells isolated by the above method can be used to establish a human pluripotent embryonic stem cell line according to a conventional method.

The present invention successfully uses mouse embryo fibroblasts as feeder cells, and successfully isolates human embryonic stem cells HES-4 directly from adherently cultured human blastocysts. It has been expanded in vitro for 16 generations, and the positive markers of the undifferentiated state of cAHES-4 cells, SSEA-4, TRA-1-60, and AKP, have been tested for positive expression. Granzyme is positive and maintains a normal karyotype. This proves that stable human embryonic stem cell lines can be obtained with this separation method. Compared with the previous method, this method of isolation eliminates the need for immunosurgery and simplifies the procedure. Brief description of the drawings:

Figure 1 Human blastocyst (40X) planted on feeder layer of mouse embryo fibroblasts;

Figure 2 The inner cell mass portion of the blastocyst (100X); Figure 3 The inner cell mass of the selected blastocyst, after removing the outer differentiated cells (100

X);

Figure 4 cMiES-4 clone (100X) growing on mouse embryo fibroblast feeder layer; Figure 5 c alkaline phosphatase test (100X) of IES-4 cells: The positive result is that the cell clone is stained red and fibroblasts Cell feeder layer is not stained;

Figure 6 SSEA-4 antigen detection of cells (200X): positive clones appeared apple green under 450nm excitation light;

Figure 7 TRA-1-60 test (200X) of cMIES-4 cells: positive clones appeared apple green under 450nm excitation light;

Figure 8 Cell-like embryoid body (40X);

Figure 9 RT-PCR analysis of OCT-4 expression in c // HES-4 cells.

Lane 1, PCR reaction of chHES-4 cells without reverse transcriptase;

2 lanes, cAHES-4 cells.

Lane 3, c / zHES-4 cells, β-actin internal control.

Right (R) lane, pUC Mix Marker, 8.

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

Lane 6, 2000 cAHES-4, reaction after telomerase heat inactivation;

Lanes 7-9, TRAP responses of 500, 1,000, 2,000 cells;

Examples

Material

1. ES cell culture medium · 80% KN0CK-OUT DMEM (K0-DMEM), 20% KN0CK-0UT serum substitute (K0-SR, provided by GIBCO- BRL), ImM L-glutamine, 0. ImM β -Mercaptoethanol, 1% non-essential amino acids (GIBCO-BRL) and human recombinant basic fibroblast growth factor (bFGF, GIBCO-BRL);

2. Embryoid body culture medium: 80% DMEM, 20% fetal bovine serum, ImM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids (GIBCO-BRL company);

3. Fibroblast culture medium: 85% DMEM, 15% fetal bovine serum;

4. 10% DMS0 cryoprotective solution: Set frozen n-tube fibroblasts (the amount of liquid in each cryotube is 1ml), the total liquid volume is n ml, and the required DMSO amount is n / 10 ral, add 4n / 10ml of K0-SR, mixed hook or DMS0 content is 20%. Resuspend the frozen cells with n / 2 ml 10% K0-SR DMEM, and then add the above 20% DMS0 solution.

5. Preparation of 0.1% gelatin: Weigh 0.1 g of gelatin and dissolve it in 100 ml of distilled water. Autoclave sterilization. Store in 4 ° C refrigerator for future use.

6. Mitomycin C: Prepare 1mg / ml with PBS solution and store at 4 ° C until use. 7. Anti-SSEA-1 monoclonal antibody (Hybridoma Bank).

8. Anti-SSEA-4 and Anti-TRA-1-60 monoclonal antibodies (blessed by Professor Peter Andrews of Sheffield University). (See reference 13)

9. FITC-labeled rabbit anti-mouse IgG (Dako).

10. Telomerase activity detection kit was purchased from integen; total RNA extraction kit was purchased from Gentra; mRNA extraction kit was purchased from Qiagen; one-step RT-PCR kit was purchased from Qiagen.

(Two) methods

1.Isolation, culture and passage of mouse embryo fibroblasts

(1) Females with 8 to 15 days of pregnancy were killed by cervical leukoplakia;

(2) After soaking the female rats in 70% alcohol for 1 to 3 minutes, place them on a sterile basin, lift the abdominal wall with forceps, cut the abdominal wall to fully expose the internal organs, cut the peritoneum to expose the uterus, and lift the uterus with small forceps At the base, the uterus is separated, and the uterus is placed in a 100 mm petri dish containing 10 ml of D-PBS;

(3) Cut the uterine wall to release the embryos, and transfer the embryos with intact fetal membranes into fresh D-PBS. Use ophthalmic tweezers to break the fetal membrane to release the embryo, and go to the stereo microscope to remove the head and neck of the mouse embryo. Carefully peel off the visceral tissue of the mouse embryo and treat each embryo as described above. Wash the stripped embryos twice with D-PBS;

(4) After cutting the treated embryo with scissors, add 2 ml of 0.05% trypsin-EDTA, digest at 37 ° C for 5 minutes, and pipette into a cell suspension. The trypsin digestion was neutralized with 5 ml of DMEM solution containing 5% newborn calf serum. Transfer the neutralized liquid into _: 15ml centrifuge tube;

(5) Allow to stand for 2 minutes to allow large pieces of tissue that have not been sufficiently shredded and digested to sink to the bottom of the tube. 'Aspirate the supernatant and transfer the supernatant to a T-175 culture flask. Depending on the number of embryos obtained, determine the number of culture flasks used. Generally, 6-8 embryo-treated cells can be planted in one T-175. Culture flask. Transfer to 37 ° C 5% C02 incubator. Change the liquid after 24 hours to remove dead cell residue;

(6) When the cells grow 80% -90%, the expanded cells must be passaged. Aspirate the spent media before passaging and wash with D-PBS once to eliminate the effect of serum on trypsin in the spent media. Add 37Ό of pre-warmed trypsin-EDTA and digest at 37 ° C for 3-10 minutes. The trypsin digestion was neutralized with DMEM solution containing 5% newborn calf serum, and the cells were detached and pipetted into a single cell suspension by pipetting. It is generally passaged at 1: 3 or 1: 5. The cells are cultured for about 6-7 days. The cells can confluence and need to be passaged again for expansion. Feeder cells for human embryonic stem cells were prepared using cells 2-3. At the same time, the cultured cells and culture medium are routinely tested for mycoplasma and bacteria.

2. Preparation of mouse embryo fibroblast feeder layer: treated with mitomycin C Aspirate the spent culture medium and add mitomycin C (final concentration 1 μg / ml). After moving into a CO2 incubator to process cells for 2.5 hours, aspirate the treatment solution and add a certain amount of D-PBS to wash it. The treatment solution and cleaning solution must be sealed with waste tubes and discarded. Add pre-warmed trypsin-EDTA digestion solution to digest cells, digest at 37 ° C for 3-10 minutes. When part of the cells fall off and most of the adherent cells are round, the fibroblast culture medium is used to neutralize the digestion of the enzyme. Pipette the non-walled cells while dispersing the cells into a single cell suspension. cell counts. Centrifuge at 150g for 5 minutes, remove the supernatant, and wash the cells twice with the culture solution to remove the residual mitomycin C. Finally, add an appropriate amount of culture medium according to the cell density, and plate the cells at 4 or 6 as required before. The culture was continued in the wells for quality control. , Isolation and Culture of Human Embryonic Stem Cells

(1) Mouse embryo fibroblast feeder cells used to isolate human ES cells from human residual blastocysts and to passage human ES cells are prepared one day before use. Replace with fresh human ES cell culture medium before use;

(2) the fresh or thawed embryo is cultured in sequential medium to the blastocyst or hatched blastocyst stage;

(3) the un-hatched blastocysts are digested and removed by protease (Pronase, Sigma) to remove the zona pellucida, or the blastocysts are hatched and directly planted on the mouse embryo fibroblast feeder layer and allowed to attach themselves;

(4) The inner cell mass grows and the outline is obvious. When the periphery is surrounded by differentiated endoderm-like cells, and the internal undifferentiated cell clusters have not yet stratified, differentiated, or become cystic, pick up the inner cell clusters in about 5 to 10 days (see Figures 1 and 2). The key is: (1) tear off the peripheral differentiated cell layer, (2) mechanically disperse the internal cell clusters, and tear as much as possible into a single layer of cell mass to destroy its spherical structure (see Figure 3) and promote its tiling On the fresh mouse embryo fibroblast word feeder layer; spherical small pieces are not easy to grow on the mouse embryo fibroblast feeder layer, and often differentiate after adherence; (3) K0-DMEM + K0 -SR + rh-bFGF, while DMEM + FBS + rh-LIF, which is generally used to isolate human embryonic stem cells

(5) After the cells begin to expand, during routine passage: Aspirate the medium, add 1mg / ml collagen IV, and digest at 37 ° C for 5min. Observe the digestive effect under a stereo microscope. After the ES cell colonies are rolled up, remove the supporting layer and differentiated cells, remove them together with the collagenase solution, add fresh medium, and gently blow with a pipette to clump the adherent cells. Pipetting becomes about 50-100 cells. The digested cells were planted on a freshly prepared mouse embryonic fibroblast feeder layer, observed daily (see Figure 4), and the fluid was changed the next day. Passage once every 4 ~ 5 days.

4. Alkaline phosphatase (AKP) detection of ES cells: After fixation with cold 80% ethanol for 2-18 hours, wash with distilled water twice and stain with freshly prepared dyes. When the staining is satisfactory, use steam Wash twice with distilled water, and then soak with D-PBS to observe the staining results. AKP detection staining solution: 25mg Fast Red TR + 5mg α-naphthol + 44.6ml distilled water + 0.3ml 10% MgCl ₂ + 5nil 4.5% sodium borate. Most clones were positive for AKP (positive results were that cell clones were stained red, fibroblast feeder layers were not stained, and differentiated cell clones were not stained) (see Figure 5).

5. Detection of ES cell undifferentiation: It is detected by indirect immunofluorescence cytochemistry.

Separately (1 uses anti-SSEA-1, ant i-SSEA-4 (1: 3-4) and anti-TRA- 1-60 (1: 3- 4) as primary antibodies, and FITC-labeled rabbit antibodies Mouse IgG (1:50) was used as a secondary antibody to detect cultured ES cells. SSEA-1 and SSEA-1

The TRA-1-60 test was fixed with 100% ethanol, while the SSEA-4 test was fixed with distilled water containing 90% acetone. After fixation, the non-specific antigen was blocked with normal goat serum, washed twice with PBS for 5 minutes each, added with primary antibody, incubated for 30 minutes, and washed twice with PBS for 5 minutes each. Add secondary antibody, incubate for 30 minutes, and wash twice with PBS. Observe the detection results of cell clones under a fluorescence microscope (450nm). SSEA- 4 and TRA- 1-60

(Positive clones appeared apple green under 450nm excitation light) were positive (see Figures 6 and 7), and SSEA-1 was negative. This indicates that ES cells grown on human feeder cell layers can remain undifferentiated. 6. ES cell karyotype analysis: Human ES cells grown on the trophoblast layer were cultured to the

5-6 days, remove the original medium, add colchicine 0.02g / L for 2 hours. The ES cell clones were picked out with a fine needle under a stereo microscope. Digest and collect the cells with 0.05% trypsin-EDTA at 37 ° C, add hypotonic solution KC1 (0.0075%) and incubate at 37 ° C for 30 minutes, and fix repeatedly with methanol: glacial acetic acid (3: 1) 3 times. Drop the slices and bake in the oven at 60 ° C for about 4-5 hours. The prepared chromosome smears were digested with trypsin to show bands, and then stained with freshly configured Giemsa solution, rinsed under running water, blown dry, and sealed with gum, and the results were observed under an oil microscope.

7. Telomerase activity: Telomerase activity is measured according to the method provided by the kit (see Figure 8).

8. The OCT-expression detection method was RT-PCR. Total RNA was extracted using PURESCRIPT RNA isolation kit provided by Gentra, and then mRNA was extracted using Oligotex mRNA kit provided by Qiagen. One step RT- provided by Qiagen was used. The PCR kit is used for detection, and the operation steps are performed according to the method provided by the kit. The primer design is as follows: OCT-4: sense,

5 '-GACAACAATGAGAACCTTCAGGAGA -3'; antisense, 5, -TTCTGGCGCCGGTTACA-GAACCA -3 '; the β-actin primers used as controls are: sense, 5'-TTCTCCTTGATGTCACGCAC-3, antisense, 5 '-CGCACCACTGGCATTGTCAT-3. After electrophoresis of the product on a 1.5% agar gel, ethidium bromide staining was observed (see Figure 9). 2. Preparation of embryoid bodies: Digest the cells as in the conventional method of passage of human ES cells, and culture the human ES cells in a bacterial culture dish without word culture cells. The medium was embryoid body medium. We have found that: Human ES cells can form embryoid bodies in suspension culture after detachment from the trophoblast (see Figure 10).

references

1.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.

5. Xue Qingshan, chief editor. Principles and techniques of in vitro culture (first edition). Beijing: Science Press. 2001, 446-447.

6.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.

Methods Enzymol 1993, 225: 58-76.

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

Cooper, 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

Biol 1991, 115: 843-850.

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

11. C, Hans i si, J. A. Grifo and L. C. Krey, Oct— 4 expression in inner cell mass and trophectoderm of human blastocysts. Mol Hum Reprod

2000, 6 (11): 999-1004

12. Bongso A, Fong CY ₃ Ng SC, et al. Isolation and culture of inner cell mass cells from human blastocysts. Hum Reprod. 1994; 9 (11): 2110-7.

13.P. W. Andrews, J. Oosterhuis, I. Dam janov, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. Robertson, Ed. (IRL, Oxford, 1987), pp. 207-248.

Claims

Rights request

1. A method for isolating human pluripotent embryonic stem cells, comprising the following steps: (1) directly planting human blastocysts into the feeder layer of embryonic fibroblasts; (2) after the inner cell mass has grown out, the inner cell mass is selected Remove the peripherally wrapped differentiated cell layer, disperse the inner cell clumps into small pieces, and re-plant them on the new embryonic fibroblast feeder layer.

2. The method for isolating human pluripotent embryonic stem cells according to claim 1, wherein the embryonic fibroblast feeder layer is a mouse embryo fibroblast feeder layer.

3. The method for isolating human pluripotent embryonic stem cells according to claim 1, wherein the embryonic fibroblast feeder layer is prepared one day before use.

4. The method for isolating human pluripotent embryonic stem cells according to claim 1, wherein when the internal cell cluster is dispersed, it should be torn as small as possible into a single monolayer cell cluster.

5. The method for isolating human pluripotent embryonic stem cells according to claim 1, wherein when the internal cell mass is dispersed, it is dispersed into a monolayer cell mass of 50-200 cells.

6. The method for isolating human pluripotent embryonic stem cells according to claim 1, wherein the culture medium used is KNOCK- OUT DMEM + NOCK-0UT SR + rh- bFGF.

7. The method for isolating human pluripotent embryonic stem cells according to claim 6, characterized in that the preferred concentration ratio of the culture medium used is 85% Knock-out DMEM + 15% Knock-out Serum Replacer + 4 ng / ml rh-bFGF.

8. A method for culturing human pluripotent embryonic thousand cells, characterized in that the human pluripotent embryonic stem cells obtained by one of claims 1 to 7 are cultured in a culture medium.

9. The method for culturing human pluripotent embryonic stem cells according to claim 8, characterized in that the medium is KNOCK-OUT DMEM + KNOCK-0UT SR + rh-bFGF.

10. The method for culturing human pluripotent embryonic stem cells according to claim 9, characterized in that the preferred concentration ratio of the culture medium is 85% Knock-out DMEM + 15% Knock-out

Serum Replacer + 4 ng / ml rh-bFGF.

1 1. A human pluripotent embryonic stem cell line, which is established from the human pluripotent embryonic stem cells obtained by one of claims 1 to 7.