WO2004111206A1 - Procede d'isolation et de culture de cellule souche embryonnaire humaine - Google Patents

Procede d'isolation et de culture de cellule souche embryonnaire humaine Download PDF

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Publication number
WO2004111206A1
WO2004111206A1 PCT/CN2003/000459 CN0300459W WO2004111206A1 WO 2004111206 A1 WO2004111206 A1 WO 2004111206A1 CN 0300459 W CN0300459 W CN 0300459W WO 2004111206 A1 WO2004111206 A1 WO 2004111206A1
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Prior art keywords
embryonic stem
stem cells
human pluripotent
cells
pluripotent embryonic
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PCT/CN2003/000459
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English (en)
Chinese (zh)
Inventor
Guangxiu Lu
Ge Lin
Changqing Xie
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Changsha Hui-Lin Life Technology Co. Ltd
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Priority to PCT/CN2003/000459 priority Critical patent/WO2004111206A1/fr
Priority to CN03826612.1A priority patent/CN1788079A/zh
Priority to AU2003246148A priority patent/AU2003246148A1/en
Publication of WO2004111206A1 publication Critical patent/WO2004111206A1/fr

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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
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the invention relates to a method for isolating human pluripotent embryonic stem cells and culturing them in vitro. Background technique:
  • 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".
  • 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:
  • 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.
  • human embryos 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.
  • ectotrophoblast cells 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.
  • 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.
  • 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.
  • mEFs mouse embryonic fibroblasts isolated from mouse early embryos on the word cell culture layer
  • FBS fetal bovine serum
  • rh-LIF fetal bovine serum
  • Serum-free culture system is a new culture system for mouse embryonic stem cells.
  • Serum replacement 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.
  • FBS fetal bovine serum
  • rh- bFGF recombinant human basic fibroblast growth factor
  • 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.
  • the blastocyst 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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;
  • Lane 1 PCR reaction of chHES-4 cells without reverse transcriptase
  • Lane 3 c / zHES-4 cells, ⁇ -actin internal control.
  • Lane 6 2000 cAHES-4, reaction after telomerase heat inactivation
  • 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 ⁇ -Mercaptoethanol, 1% non-essential amino acids (GIBCO-BRL) and human recombinant basic fibroblast growth factor (bFGF, GIBCO-BRL);
  • Embryoid body culture medium 80% DMEM, 20% fetal bovine serum, ImM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, 1% non-essential amino acids (GIBCO-BRL company);
  • Fibroblast culture medium 85% DMEM, 15% fetal bovine serum;
  • 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.
  • Mitomycin C Prepare 1mg / ml with PBS solution and store at 4 ° C until use. 7. Anti-SSEA-1 monoclonal antibody (Hybridoma Bank).
  • 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.
  • mice embryo fibroblast feeder layer treated with mitomycin C Aspirate the spent culture medium and add mitomycin C (final concentration 1 ⁇ g / 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.
  • 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;
  • the inner cell mass grows and the outline is obvious.
  • 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
  • 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 ⁇ -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).
  • Detection of ES cell undifferentiation It is detected by indirect immunofluorescence cytochemistry.
  • 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
  • ES cell karyotype analysis Human ES cells grown on the trophoblast layer were cultured to the
  • Telomerase activity Telomerase activity is measured according to the method provided by the kit (see Figure 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,
  • 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.

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Abstract

L'invention concerne l'isolation et la culture de cellule souche embryonnaire humaine, selon les étapes suivantes : (1) implantation de blastula humaine dans la couche d'alimentation de fibroblaste embryonnaire; (2) extraction de masse de cellule interne (MCI) lorsque la croissance externe correspondante est achevée, puis élimination de la couche cellulaire de différenciation périphérique, dispersion de MCI en morceaux, et réimplantation dans la nouvelle couche d'alimentation de fibroblaste embryonnaire. Ce procédé permet d'omettre l'opération d'immunité et simplifie le processus d'isolation.
PCT/CN2003/000459 2003-06-13 2003-06-13 Procede d'isolation et de culture de cellule souche embryonnaire humaine WO2004111206A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN2003/000459 WO2004111206A1 (fr) 2003-06-13 2003-06-13 Procede d'isolation et de culture de cellule souche embryonnaire humaine
CN03826612.1A CN1788079A (zh) 2003-06-13 2003-06-13 一种分离和培养人类多能胚胎干细胞的方法
AU2003246148A AU2003246148A1 (en) 2003-06-13 2003-06-13 A method for isolation and culture of human embtyonic stem cell

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GB0911060D0 (en) * 2009-06-26 2009-08-12 Ge Healthcare Uk Ltd Methods for predicting the toxicity of a chemical
CN113234809A (zh) * 2021-05-08 2021-08-10 杭州憶盛医疗科技有限公司 一种多能性干细胞的表观遗传稳定性研究方法

Citations (2)

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Publication number Priority date Publication date Assignee Title
CN1284993A (zh) * 1997-11-25 2001-02-21 Arc基因组研究公司 多能胚胎干细胞以及获得它们的方法
CN1404526A (zh) * 2000-02-21 2003-03-19 威斯康星校友研究基金会 从灵长类动物胚胎干细胞制备胚状体的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1284993A (zh) * 1997-11-25 2001-02-21 Arc基因组研究公司 多能胚胎干细胞以及获得它们的方法
CN1404526A (zh) * 2000-02-21 2003-03-19 威斯康星校友研究基金会 从灵长类动物胚胎干细胞制备胚状体的方法

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