WO2011134210A1 - 一种培养基添加剂及其应用 - Google Patents

一种培养基添加剂及其应用 Download PDF

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WO2011134210A1
WO2011134210A1 PCT/CN2010/075551 CN2010075551W WO2011134210A1 WO 2011134210 A1 WO2011134210 A1 WO 2011134210A1 CN 2010075551 W CN2010075551 W CN 2010075551W WO 2011134210 A1 WO2011134210 A1 WO 2011134210A1
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medium
vitamin
cells
reprogramming
oct4
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PCT/CN2010/075551
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English (en)
French (fr)
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裴端卿
陈捷凯
刘晶
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中国科学院广州生物医药与健康研究院
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Priority to JP2012512195A priority Critical patent/JP2012524551A/ja
Priority to EP10850541.3A priority patent/EP2565263A4/en
Priority to US13/263,865 priority patent/US20130040390A1/en
Publication of WO2011134210A1 publication Critical patent/WO2011134210A1/zh

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    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the field of cell culture media, and particularly relates to a medium additive, which can be used for inducing pluripotent dry fines or mammalian fine-pack culture.
  • KOSR serum replacement medium
  • the composition of the system is still not completely clear, such as the bovine serum globulin complex (Albumax) contains a variety of unknown lipids, the effect of these components on reprogramming is unknown.
  • serum replacement cannot support the growth of pre-programmed cells.
  • reprogramming efficiency is not high enough even with serum. Therefore, a serum-free, fully accurate and efficient reprogramming system is particularly important for the study of iPS mechanisms, drug screening and clinical application of iPS.
  • the object of the present invention is to overcome the above technical deficiencies and to provide a component-determined and highly efficient culture system for obtaining IPS cells.
  • the culture system maintains stem cell growth and proliferation in the absence of feeder cells, while accelerating the iPS process and improving iPS efficiency.
  • the medium additive of the present invention includes vitamin C and a glycogen synthase kinase 3 inhibitor.
  • the medium additive of the present invention may also be in addition to vitamin C and glycogen synthase 3 inhibitors, but also includes vitamin B12, insulin, receptor tyrosine kinases and antioxidants.
  • the vitamin C includes ascorbic acid and its derivatives such as sodium ascorbate, a stable form of vitamin C, 2-phosphoric acid ascorbic acid. A preferred form thereof is 2-phosphoric acid-ascorbic acid.
  • the working concentration of vitamin C is 0-10 (H ⁇ g/ml, but not limited to the above concentration, wherein the preferred concentration is 50 ⁇ g/ml.
  • the working concentration of the vitamin B12 is 0-2.8 ⁇ g/ml, but not limited to the above concentration Wherein the preferred concentration is 1.4 ⁇ g/ml.
  • the insulin includes biologically active insulin of various sources synthesized and artificially synthesized, and preferably insulin is human recombinant insulin (SIGMA company;), and the working concentration is 0-50 ⁇ g/ml, but is not limited to the above concentration, wherein preferably The concentration is 20 ⁇ : g / ml.
  • the glycogen synthase kinase 3 inhibitor comprises at least one of Li X, Chir99021, BIO and SB216763. Among them, the preferred form of Li + is LiCl, and the working concentration ranges from 0 to 10 ⁇ g/ml, but is not limited to the above concentration, and a preferred concentration is 5 mmol/ml.
  • Glycogen synthesis kinase is a kind of serine/threonine brick acid kinase, which is involved in the regulation of various signaling pathways.
  • the preferred drug for its inhibitor is CHIR99021, working concentration range is 0-12 micromol/ml, but not limited to the above concentration. Wherein the preferred concentration is 3 ⁇ mol/ml.
  • the receptor gram kinases include basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet growth factor (PDGF), insulin-like growth factor (IGF), and liver. At least one of growth factors (HGF).
  • the drug is a basic fibroblast growth factor, and the working concentration ranges from 0 to 100 ⁇ g/ml, preferably at a concentration of 5 ng/ml.
  • the antioxidant includes at least one of thiamine, superoxide dismutase, catalase, reduced glutathione, vitamin E, acetylated vitamin E, linoleic acid, linolenic acid, and sodium selenite.
  • the working concentration of thiamine ranges from 0 to 36 ⁇ g/ml, but is not limited to the above concentration, wherein the preferred concentration is 9 ⁇ g/ml; the working concentration of superoxide dismutase ranges from 0 to 10 ⁇ g/ml, but is not limited thereto.
  • the above concentration, wherein the preferred concentration is 2.5 ⁇ g/ml.
  • the working concentration of reduced glutathione ranges from 0 to 4 g/ml, but is not limited to the above, and a preferred concentration is 1.5 ⁇ g/ml.
  • the working concentration of vitamin E ranges from 0 to 16 ⁇ g/ml, but is not limited to the above concentrations, and a preferred concentration is 1 ⁇ g/ml.
  • the working concentration of acetylated vitamin E ranges from 0 to 16 ⁇ g/ml, but is not limited to the above concentrations, and a preferred concentration is 1 ⁇ g/ml.
  • the working concentration of linoleic acid ranges from 0 to 4 ⁇ g/ml, but is not limited to the above concentrations, and a preferred concentration is 1 ⁇ g/ml.
  • the working concentration of ethanolamine ranges from 0 to 16 ⁇ g/ml, but is not limited to the above concentrations, and a preferred concentration is 1 ⁇ g/ml.
  • the medium additive of the present invention may further be a mixture of the above two medium additives respectively and a serum cell growth promoter; wherein the serum cell growth promoter comprises albumin hydrolysate, transferrin, and triiodide gland At least one of amino acid, adrenal ketone, lipoic acid, ethanolamine, progesterone, putrescine, and vitamin A.
  • the albumin hydrolysate is a product of hydrolysis of albumin (also known as albumin), and its constituent components are amino acids and polypeptides, and the specific components are specific, and the concentration range is 0-10 mg/ml, but is not limited to the concentration, preferably the concentration. It is 1 mg/ml.
  • the transferrin comprises transferrin containing iron ions and transferrin containing no iron ions, wherein the preferred transferrin is transferrin containing iron ions, and the concentration range is 0-200 micrograms/ml, but Not limited to this concentration, a preferred concentration is 100 ⁇ g/ml.
  • the working concentration of the lipoic acid ranges from 0 to 3.2 ⁇ g/ml, but is not limited to the above working concentration, and a preferred concentration is 0.2 ⁇ g/ml.
  • the working concentration of the vitamin A ranges from 0 to 1.6 ⁇ g/ml, but is not limited to the above working concentration, and a preferred concentration is 0.1 ⁇ g/ml.
  • the present invention also provides a complete medium consisting of one or more of a basal medium, a serum, a serum substitute additive and the above medium medium additives; or a basal medium and the above Medium additive composition.
  • a complete medium consisting of one or more of a basal medium, a serum, a serum substitute additive and the above medium medium additives; or a basal medium and the above Medium additive composition.
  • Table 1 shows the optimized medium additive ingredients and concentrations.
  • the present invention also provides a complete medium for inducing pluripotent stem cells, the complete medium for inducing pluripotent stem cells consisting of one or more of a basal medium, serum, serum substitute additives and the above two medium additives .
  • the basal medium includes, but is not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), F-10, F-12, RPMI 1640, GMEM (Glass ows's Minimal Essential Medium), aMEM (Minimal Essential Medium), Iscove's Modified Dulbecco's Medium and M199. Its preferred form is DMEM.
  • KOSR KonckOut Serum Replacement
  • N2 ⁇ 27
  • ITS Intelligent Transporterin-Selenium Supplement
  • the medium additive was configured according to the ingredients shown in Table 1 and used to add to the base medium leg EM to form a chemically defined complete medium iCD1.
  • Table 1 shows the optimized media additive ingredients and concentrations.
  • Table 2 shows the composition and concentration of the optimized complete medium iCDl.
  • the culture system of the present invention is a serum-free culture system for obtaining IPS cells efficiently from somatic cells without serum and components, and can maintain the growth and proliferation of fibroblasts and stem cells in the absence of feeder cells. At the same time accelerate the iPS process and improve iPS efficiency. It can also be used for eukaryotic cell culture. Table 1
  • Insulin Human, Recombination
  • Palmitic acid ⁇ +01
  • Transferrin (containing iron ions)
  • Figure 1 is a graph showing the results of the comparison of the efficiency of different culture schemes; wherein: Figure la is the four culture conditions of vitamin C, KSR-BN and Basal 3.0 added to mES and mES (the specific components of the four culture conditions are described later), SKO (SOX2, KIF4, and OCT4) Number of reprogrammed clones induced by 20,000 embryonic fibroblasts on day 10 after virus infection; Figure lb shows four cultures of vitamin C, KSR-BN, and Basal 3.0 added at mES and mES The ratio of reprogrammed cells was measured on the 10th day after SKO infection;
  • Figure 2 is a dose effect experiment result of the core component in the additive; wherein: Figure 2a is a concentration of lithium chloride at 0-10 mmol/ml Within the range, the number of reprogrammed clones on the 10th day after SKO infection; Figure 2b is the ratio of reprogrammed cells (green fluorescent cells) on day 10 after SKO infection in the range of 0 - 10 mmol/ml of lithium chloride; Figure 2c is a statistical analysis of the number of reprogrammed clones on the 10th day after SKO infection in the concentration range of 0 - 2.8 ⁇ g/ml of vitamin B12; Figure 2d is the concentration range of vitamin B12 in the range of 0 - 2.8 ⁇ g / ml, after SKO infection Day 10 reprogrammed cell ratio; Figure 2e is the basic fibroblast growth factor in the concentration range of 0 - 7 ng / ml, the number of reprogrammed clones on the 10th day after SKO
  • Figure 3 is a dose effect experiment of antioxidants; wherein, Figure 3a is the antioxidant vitamin E in the concentration range of 0-16 ⁇ g / ml, the number of reprogrammed clones on the 10th day after SKO infection; Figure 3b is the antioxidant vitamin E is in the concentration range of 0-16 4 gram/ml, and the ratio of OCT4-GFP positive cells is measured by flow cytometry on the 10th day after infection; Fig.
  • 3c is the antioxidant superoxide dismutase (SOD) at 0- In the concentration range of 10 4 g / ml, the number of reprogrammed clones on the 10th day after SKO infection;
  • Figure 3d is the antioxidant superoxide dismutase (SOD) in the concentration range of 0-10 ⁇ : g / ml, The ratio of OCT4-GFP positive cells was measured by flow cytometry on the 10th day after infection;
  • Fig. 3e is the reprogrammed clone of the antioxidant reduced glutathione in the range of 0-6 ⁇ g/ml, and the 10th day after SKO infection.
  • Figure 3f shows the ratio of antioxidant-reduced glutathione in the range of 0-6 ⁇ g/ml, and the ratio of OCT4-GFP positive cells was measured by flow cytometry on the 10th day after infection.
  • Figure 3g shows the number of reprogrammed clones on the 10th day after SKO infection in the concentration range of 0-36 ⁇ g/ml;
  • Figure 3h shows the concentration of superoxide dismutase (SOD) at 0-10 ⁇ g/ml. Within the range, the ratio of OCT4-GFP positive cells was measured by flow cytometry on the 10th day after infection;
  • Figure 4 is a dose effect diagram of the growth support, wherein Figure 4a is the growth support vitamin A in the range of 0-16 ⁇ g / ml, the number of reprogrammed clones on the 10th day after SO infection, Figure 4b is the growth support Vitamin A was in the range of 0-16 ⁇ g/ml, and the ratio of OCT4-GFP positive cells was measured by flow cytometry on the 10th day after infection; Figure 4c is the growth support ethanolamine in the range of 0-16 ⁇ g/ml, SKO The number of reprogrammed clones on the 10th day after infection; Figure 4d is the growth support ethanolamine in the concentration range of 0-16 ⁇ g / ml, the ratio of OCT4-GFP positive cells measured by flow cytometry on the 10th day after infection; 4e is the growth support linoleic acid in the concentration range of 0-4 ⁇ g / ml, the number of reprogrammed clones on the 10th day
  • Figure 4g is a statistical analysis of the number of reprogrammed clones on the 10th day after SKO infection with lipoic acid in the range of 0-3.2 ⁇ g/ml
  • Figure 4h is the growth support linoleic acid in the concentration range of 0-4 ⁇ g/ml, infection The ratio of OCT4-GFP positive cells was measured by flow cytometry on the 10th day afterwards;
  • Figure 5 is a graph comparing the effects of the medium formulation iCD1 (Basa B. O added glycogen synthase 3 inhibitor CHIR99021) of the present invention with the more advanced induced reprogramming culture scheme reported;
  • Figure 5a is a comparison of the efficiency of the chemical composition determining medium formulation iCDl shown in the present invention with the reported reprogramming culture protocol;
  • Figure 5b is a reprogrammed clone picture of the eighth day after infection of several different culture protocols shown in Figure a
  • Figure 5c is a graph of the reprogramming efficiency of the KSR-BN culture program (see the description of the ingredients).
  • Figure 5d is a graph showing the reprogramming efficiency of mES after adding vitamin C.
  • Figure 5e is a graph showing the reprogramming efficiency of the mES->KSR culture program. The specific process of mES->KSR culture program can be found in the following instructions);
  • Figure 6 is a graph showing the effects of six components (basic fibroblast growth factor, vitamin C, CHIR9902L lithium chloride, vitamin B12, 3 ⁇ 4 amine) on the reprogramming efficiency and growth of the medium additive of the present invention, wherein
  • Figure 6a shows the effect of the six components on the reprogramming efficiency in the present invention
  • Figure 6b shows the reprogramming image under different culture conditions on the 8th day after infection, a diagram
  • Figure 6c shows the growth curve under the above culture conditions
  • Figure 7 is a graph showing the dose effects of bFGF, vitamin C, CHIR99021 and the time of action of bFGF; wherein, Figure 7a is a dose-effect diagram of basic fibroblast growth factor (bFGF); Figure 7b is an experimental diagram of the action time of bFGF. Figure 7c is a dose effect diagram of vitamin C; Figure 7d is a dose effect diagram of CHIR99021;
  • Figure 7 shows the dose effect of bFGF, vitamin C, CHIR99021, the duration of action of bFGF and other receptor tyrosine kinases.
  • Figure 7a is a dose effect diagram of reduced fibroblast growth factor (bFGF);
  • Figure 7b is a graph of the action time of bFGF;
  • Figure 7c is a dose effect diagram of vitamin C;
  • Figure 7d is the dose of CHIR99021
  • Figure 7d is a graph showing the effect of different receptor tyrosine kinases on reprogramming;
  • Figure 8 is a chemically-defined medium iCD1, ALK5 (a receptor for tumor necrosis factor TGF-beta) inhibitor A83-01, histone methylase inhibitor valproic acid and fetal calf
  • the experimental results of serum can not further improve the efficiency of OKS-mediated reprogramming;
  • Figure 8a shows the chemical composition of the cultured iCD1 after infection of mouse fibroblasts with SKO virus, and the addition of A83-01 (0.5 ⁇ ), valproic acid (1 mM) and 2% fetal bovine serum in iCD1 medium, the number of reprogrammed clones was calculated on the 10th day after infection;
  • Figure 8b is a typical reprogrammed clone in a experiment;
  • Figure 9 is a graph showing the experimental results of the initial planting density affecting the reprogramming efficiency
  • Figure 10 is a diagram showing the results of expression of endogenous pluripotency molecular markers under iCD1 culture conditions
  • Figure 10a shows the expression of endogenous pluripotency molecular markers by real-time fluorescent PCR
  • Figure 10b shows immunofluorescence experiments directly on cell culture plates
  • Figure 10c shows the expression status by immunoblotting
  • Figure 11 is a reprogramming trace diagram of the iCDl culture system
  • Figure 12 is a graph showing the reprogramming efficiency and related molecular marker expression on the eighth day after infection, wherein Fig. 12a shows that Oct4-GFP mouse embryonic fibroblasts are infected with SKO virus and exogenous red fluorescent protein DsRed virus, respectively, at mES
  • Fig. 12a shows that Oct4-GFP mouse embryonic fibroblasts are infected with SKO virus and exogenous red fluorescent protein DsRed virus, respectively, at mES
  • Figure 12b shows SKO virus detected by real-time fluorescent quantitative PCR The expression of pluripotency molecular markers in different cell populations after mES, iCD1 culture for 8 days and iCD1 culture for 8 days after infection;
  • Figure 13 is a process chimeric experiment related result; wherein, Figure 13a is a venom of OSK-induced iPSC cells detected in iCD1 medium by embryo injection; Figure 13b is a statistical analysis of the experiment described in Figure a for different induction days; Figure 13c is a chimeric mouse produced by injection of sputum embryos from iPSC cells obtained on day VIII; this experiment demonstrates that cells infected with SKO can be reprogrammed in iCD1 for 8 days to form pluripotent stem cells capable of producing chimeras.
  • Figure 14 is a graph showing experimental results of OK and OS reprogramming somatic cells in an iCD1 culture system
  • Figure 14a is a photograph of the original clone and passage of the OK and OS reprogrammed somatic cells under iCD1 culture conditions;
  • Figure 14b shows the specific molecular marker expression of the stem cells by immunofluorescence;
  • Figure 14c shows the OK and OS under the iCD1 culture system.
  • Figure 14d is a picture of a teratoma slice; this figure shows that pluripotent stem cells formed by OK and OS reprogramming have the characteristics of embryonic stem cells.
  • Figure 15 is a graph showing the results of related experiments on OCT4 reprogramming MEF under iCD1 conditions; wherein, Figure 15a is a morphological photograph of the original clone and the formed cell line induced by Oct4 on the 33rd day after infection; Figure 15b is a phenotype of Oct4 iPS clone expression.
  • Figure 15c shows real-time quantitative PCR showing that the cloned clones express stem cell-specific pluripotent molecular markers;
  • Figure 15d shows the expression of foreign genes by quantitative rt-PCR;
  • Figure 15e is the insertion identification analysis.
  • Figure 15f is a chimeric mouse that can be produced by injection of embryos from Oct4 iPSC cells
  • Figure 15g is a chimeric mouse produced with germline chimerism ability
  • FIG 16 is a diagram showing the correlation between the use of Oct4-VP16 (VP16 is a viral-derived gene sequence linked to a transcription factor to enhance the activity of a transcription factor), and KCD4 and Sox2 infection, iCDl can improve reprogramming efficiency;
  • Figure 16a shows that Oct4-GFP mouse embryonic fibroblasts were infected with Oct4-vpl6SK, cultured in mES medium, added with vitamin C in mES medium and iCD1 medium, and the number of fluorescent clones was calculated on the 10th day after infection;
  • Figure 16b is a typical photograph in the experiment described in Figure 16a;
  • Figure 17 is a related experimental diagram of Oct4-vpl6/Kif4 and Oct4-vpl6/Sox2 reprogramming somatic cell reprogramming in the iCD1 culture system; wherein, Figure 17a is Oct4 -GFP mouse embryonic fibroblasts were infected with Oct4-vp 16/Kif4 and Oct4-
  • Figure 18 is a related experimental diagram of Oct4-vpl6 reprogramming somatic cells in an iCD1 culture system;
  • Figure 18a is a typical photograph of an iPS clone induced by Oct4-vpl6;
  • Figure 18b is an Oct4-GFP mouse embryonic fibroblast infection.
  • Oct4-vpl6 mES mES was added to culture in vitamin C and iCDl medium, and the number of reprogrammed clones was calculated in different culture days;
  • Fig. 18c is the cloned expression pluripotency molecular marker induced by Oct4-vl6;
  • Fig. 18d is Oct4-vpl6 induced iPS Cloning of injected embryos, pictures of chimeric mice that can be produced;
  • Figure 19 is an experimental diagram of the addition of iCDl core components to mES medium to facilitate reprogramming
  • FIG. 19a shows that Oct4-GFP mouse embryonic fibroblasts are infected with SKO virus and cultured in mES, mES+vitamin C, SmES (mES plus vitamin C, bFGF, CHIR9902K lithium chloride and B27) medium, respectively.
  • the number of reprogrammed clones was calculated in 8 days;
  • Fig. 19b is a typical fluorescent clone photograph on the 8th day of SmES culture conditions; Fig.
  • FIG. 19c is the meta-viscosity of Oct4-GFP mouse embryonic fibroblasts infected with OCT4 virus at mES, mES, respectively The number of fluorescent clones was observed on different days in iCD1 medium;
  • Figure 19d is a photograph of the original clone and passage of OCT4 reprogrammed somatic cells in the SmES culture system.
  • Basic medium artificially prepared containing sugars, amino acids, inorganic salts, vitamins, lipids, etc. A culture of nutrients required for cell growth.
  • the basal medium of the present invention includes Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow's Minimal Essential Medium (GMEM), a Minimal Essential Medium (a MEM), Iscove's Modified Dulbecco's Medium and M199, etc., but not limited to the above basic medium, wherein the preferred basal medium is a high glucose DMEM basal medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • F-10 F-12
  • RPMI 1640 Glasgow's Minimal Essential Medium
  • GMEM Glasgow's Minimal Essential Medium
  • a MEM Minimal Essential Medium
  • Fetal bovine serum A culture that supports the growth of cells in the fetal bovine blood.
  • the specific composition is unknown. It is rich in nutrients and growth factors and supports the growth of many fine cell types.
  • disadvantages such as unclear ingredients, large batch differences, and possible animal pathogens.
  • Serum Substitute A chemical additive that is a commercial alternative to serum.
  • the ingredients are generally determined.
  • the serum replacements described in the present invention include KSR (a commercialized serum-supplemented additive), 2 (a commercial serum replacement additive, components of insulin, transferrin, progesterone, putrescine, selenium) Sodium sulphate, and B27 (a serum-free culture additive for culturing nerve cells) are added to the serum, but are not limited to these.
  • Embryonic stem cells referred to as ES or EK cells.
  • Embryonic stem cells are a type of cells isolated from early embryos (before the gut stage) or primitive gonads. They have the characteristics of in vitro proliferation, self-renewal and multi-directional differentiation in vitro.
  • In vitro culture of mouse embryonic stem cells requires the support of feeder cells and mouse leukemia inhibitory factors to maintain their pluripotent state. The study found that in vitro culture, the combination of glycogen synthesis kinase inhibitor and mitogen inhibitor can inhibit the differentiation of embryonic stem cells, and maintain the survival of the stem cells in the absence of feeder cells and mouse leukemia inhibitors. Energy type.
  • Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine protein kinase, which is an important component of various signal transduction pathways in cells. It is involved in intracellular glucose metabolism and cell proliferation. , a variety of physiological processes such as cell differentiation and apoptosis.
  • Glycogen synthesis kinase inhibitors mainly include CHIR99021, BIO, SB216763 and the like. The priority drug is CHIR99021, the knot
  • iPS cells Abbreviations for induced pluripotent stem cells and induced pluripotent stem cells. The state of the pluripotent stem cells is restored for the differentiated cells through the reprogramming factor treatment. iPS cells have been shown to produce intact mice by injection of tetraploid embryo technology, indicating that iPS cells are pluripotent.
  • the reprogramming factor refers to a factor that restores differentiated cells to a pluripotent state, mostly a nuclear transcription factor, which has been reported for heavy use.
  • the programmed transcription factors include: SOX2, OCT3, KIF4, NONOG, LIN28, c-myc, lin28, esrrb, tbx3, and the like.
  • the transcription factors of the present invention include, but are not limited to, the above transcription factors.
  • the composition of the feeder medium is: high glucose basic medium (DMEM), external force. 10% fetal bovine serum (FBS) (Hyclone), 2 mM glutamic acid (Glutamine X non-essential amino acid (NEAA).
  • DMEM high glucose basic medium
  • FBS fetal bovine serum
  • NEAA Glutamine X non-essential amino acid
  • the composition of the serum-containing ES cell medium is: high glucose basic medium (DMEM is a basic medium named after the inventor's initial letter), 15% fetal bovine serum FBS (GIBCO), 2 mM glutamine (Glutamine), Non-essential amino acids (NEAA), penicillin/streptomycin, beta-mercaptoethanol, sodium pyruvate.
  • DMEM high glucose basic medium
  • GOBCO 15% fetal bovine serum FBS
  • Glutamine 2 mM glutamine
  • NEAA Non-essential amino acids
  • penicillin/streptomycin beta-mercaptoethanol
  • sodium pyruvate sodium pyruvate.
  • mES is a classic stem cell culture medium consisting of: high glucose DMEM medium supplemented with 15% fetal bovine serum, non-essential amino acids, glutamic acid, penicillin/streptomycin, beta-mercaptoethanol, pyruvic acid and leukocyte inhibitory factor (a growth factor that maintains the pluripotent state of mice).
  • KSR serum-free medium KSR is an abbreviation for Knockout Serum Replace, a commercial generation of serum stem cell culture additives.
  • the KSR complete medium used in the examples of the present invention is divided into two types, one is used for the induction process of iPS, and its components are: high glucose basic medium (DMEM), 10% KSR additive, 2 mM glutamic acid (Glutamine). ), non-essential amino acids (NEAA), penicillin/streptomycin, beta-mercaptoethanol, sodium pyruvate.
  • DMEM high glucose basic medium
  • Glutamine 2 mM glutamic acid
  • NEAA non-essential amino acids
  • penicillin/streptomycin beta-mercaptoethanol
  • sodium pyruvate sodium pyruvate
  • KSR serum-free medium for culturing stem cells or iPS clones consisting of: KNOCKOUT DMED (basic medium optimized for stem cell culture with osmotic pressure), 15% KSR additive, 2 mM valley Glutamine, non-essential amino acid (NEAA), penicillin/streptomycin, beta-mercaptoethanol.
  • the murine cytokine inhibitor LIF millopore, trade name ESGRO, a growth factor that inhibits mouse stem cell differentiation
  • ESGRO a growth factor that inhibits mouse stem cell differentiation
  • KSR-BN is the above-mentioned KSR complete medium for iPS induction process.
  • Basal3.0 complete medium refers to one of the mediums of the present invention, specifically adding vitamin C, insulin, lithium chloride, vitamin B12, antioxidant, receptor tyrosine from high sugar basic medium (DMEM). It consists of a kinase growth factor and a series of growth supports. The specific ingredients are the components shown in Table 2, lacking CHIR99021.
  • iCDl Complete Medium One of the media described in the present invention.
  • the additives described in Table 1 are mixed with high-glycemic basic medium (DMEM), glutamic acid, non-essential amino acids, penicillin/streptomycin, beta-mercaptoethanol, sodium pyruvate, etc., wherein the additives described in Table 1 and others
  • DMEM high-glycemic basic medium
  • the composition volume ratio is 1:24.
  • the specific ingredients and concentrations are as described in Table 2.
  • mouse-based somatic cell reprogramming is performed as follows, unless otherwise specified:
  • the somatic cell types used for reprogramming were all OG2 mouse embryonic fibroblasts, and the number of passages was no more than three generations.
  • the OG2 mouse is a transgenic mouse in which a green fluorescent protein (GFP) gene is ligated after the promoter of the stem cell-specific gene Oct4 gene.
  • GFP green fluorescent protein
  • FFP green fluorescent protein
  • OG2 mouse embryonic fibroblasts are activated by endogenous Oct4 gene.
  • green fluorescent protein is accompanied by expression.
  • Reprogramming clones ie, the number of green fluorescent clones or the ratio of green fluorescent cells analyzed by flow cytometry, allows researchers to compare reprogramming efficiencies under different conditions.
  • Cell experiments and virus preparation were as follows: Cells were seeded in 12-well adherent cell culture plates at a planting density of 20000 cells/well. After 6-18 hours of cell growth, the cell density and status were infected with a virus with a mouse reprogramming factor. The infection was carried out in two rounds. A second round of infection was performed 24 hours after the first round of infection, and the virus solution was replaced with various test solutions 24 hours after the second round of infection. The day of the change was recorded as day 0 (D0); at different time points after infection, the number of GFP fluorescent clones in the original well or the ratio of GFP fluorescent cells was analyzed by flow cytometry according to the experiment.
  • D0 day 0
  • the virus is prepared by transfecting the reprogramming factor plasmid cloned into the PMX vector into viral packaging cells (PlatE), transfecting the fresh medium for 12-16 hours after transfection, and collecting the culture solution for 48 hours after transfection. The virus solution was once infected, and the fresh culture solution was added for 24 hours, and then the culture solution was collected as a second infectious virus solution.
  • Platinum viral packaging cells
  • SKO refers to three viruses or three virus-infected cells such as SOX2, KIF4 and OCT4, which are equivalent to the three factors.
  • OK refers specifically to OCT4 and KIF4 viruses or cells infected by two viruses.
  • OS specifically refers to OCT4 and SOX2 viruses or cells infected with both viruses.
  • Example 1 Comparison of reprogramming efficiency of B3.0 with other culture protocols and optimization of component concentration.
  • the SKO virus was mixed with 1: 1: 1 (0.5 ml each) and infected into a total of 20,000 fibroblasts in a well of a 12-well plate.
  • the cells were cultured at 37 °C and 5 % C0 2 respectively.
  • Medium, mES was added to vitamin C, SR-B medium and Basal 3.0 medium.
  • the number of reprogrammed clones was directly counted under a fluorescence microscope on the 10th day after infection, and the ratio of reprogrammed cells was analyzed by flow cytometry.
  • FIG. 2 is the results of the dose-effect experiment of the core component; wherein, Figure 2a is the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the dose range of lithium chloride in the range of 0-2.8 mmol/ml. 2b is the ratio of reprogrammed cells measured for lithium chloride under the corresponding test piece.
  • Figure 2c shows the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the dose range of 0-10 mg/ml for vitamin B12
  • Figure 2d shows the reprogramming of vitamin B12 under the corresponding test piece.
  • Cell ratio Figure 2e shows the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the range of 0-10 ng/ml of basic fibroblast growth factor
  • Figure 2f shows the basic fibroblast growth factor in Figure 2e. The ratio of reprogrammed cells measured under the corresponding test pieces.
  • Figure 2g shows the number of reprogrammed clones induced by Basa.O on the 10th day after infection in the dose range of 0-100 mg/ml of vitamin C
  • Figure 2h shows the reprogramming of vitamin C under the corresponding test piece.
  • Figure 2i shows the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the dose range of 0-50 mg / ml of insulin.
  • the recommended working concentrations are respectively: lithium chloride: 5 mmol/ml, vitamin B 12: 1.4 ⁇ g/ml, basic fibroblast growth factor: 5 ng/ml, vitamin C: 50 ⁇ g/ml, insulin: 50 ⁇ g/ml. However, it is not limited to this concentration range.
  • Figure 3 is a graph showing the results of dose-effect experiments of a series of antioxidants.
  • Figure 3a shows the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the range of 0-16 ⁇ : g/ml of antioxidant vitamin E.
  • Figure 3b shows the reprogrammed cell ratio of vitamin C measured under the corresponding test piece.
  • Figure 3c shows the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the range of 0-10 tg/ml of antioxidant superoxide dismutase (SOD), and
  • Figure 3d shows the antioxidant superoxide. The ratio of reprogrammed cells measured by dismutase (SOD) under the corresponding test piece.
  • Figure 3e shows the number of reprogrammed clones induced by BasaB.O in the concentration range of antioxidant reduced glutathione in the range of 0-6 ⁇ g/ml, on day 10 after infection
  • Figure 3f shows the oxidized reduced glutathione in Figure 3e.
  • Figure 3g shows the number of reprogrammed clones induced by Basal3.0 on the 10th day after infection in the concentration range of 0-36 ⁇ g/ml of antioxidant thiamine.
  • Figure 3h shows the antioxidant thiamine measured under the corresponding test piece. Reprogrammed cell ratio.
  • the recommended working concentrations for the above substances are: Vitamin E: l mg / ml, superoxide dismutase: 2.5 mg / ml, reducing glutathione 1.5 mg / ml, thiamine: 9 mg / ml.
  • Figure 4 shows the results of dose effect experiments for other growth support materials.
  • Figure 4a shows the number of reprogrammed clones induced by Basal 3.0 on the 10th day after infection in the range of 0-1.6 ⁇ g/ml of vitamin A
  • Figure 4b shows the reprogrammed cell ratio measured by vitamin A in the corresponding test piece.
  • Figure 4c shows the number of reprogrammed clones induced by BasaB.O on the 10th day after infection in the concentration range of 0-16 ⁇ g / ml of ethanolamine
  • Figure 4d shows the ratio of reprogrammed cells measured by ethanolamine in the corresponding test piece. .
  • Figure 4e shows the number of reprogrammed clones induced by Basal3.0 on the 10th day after infection in the concentration range of 0-4 ⁇ g/ml of linoleic acid.
  • Figure 4f Reprogrammed cell ratio measured by ethanolamine under the corresponding test piece.
  • the recommended working concentrations for the above substances are: vitamin AO.lt g/ml, ethanolamine: 1 ⁇ g/ml, linoleic acid: 1 ⁇ g/ml, lipoic acid: 0.2 ⁇ g/ml.
  • glycogen synthesis kinase inhibitor substances include CHIR99021, BIO. SB31xxxx was used as a candidate substance for the above iPS test experiment (reprogramming clone number and reprogrammed cell ratio two indexes as evaluation criteria for reprogramming efficiency;).
  • the GSIO- ⁇ inhibitor CHIR99021 significantly improved the reprogramming efficiency of BasaB.O, so the improved Basal3.0 added the glycogen synthase kinase 3 (GSK3-P) inhibitor CHIR99021, but not limited to the chemical composition of CH99021.
  • the defined medium was named iCDl.
  • Figure 5b is a reprogrammed clone picture of the eighth day after infection of several different culture protocols shown in Figure a. The scale shown: 2 mm. The results indicate that the iCDl culture system is clear. It is superior to any other culture system.
  • FIG. 5c shows the KSR-BN culture protocol to improve the reprogramming efficiency.
  • Oct-GFP mouse embryonic fibroblasts (MEFs) were infected with Sox2/Klf4/Oct4 (SKO) virus, mES and KSR were used respectively.
  • Figure 5d is a graph showing the improvement of reprogramming efficiency after adding vitamin C to mES.
  • Figure 5e shows the mES->KSR culture protocol to improve the reprogramming efficiency.
  • Oct4-GFP mouse embryonic fibroblasts (MEF) were infected with SKO virus and cultured with mES and mES conversion KSR culture protocols respectively.
  • the mES conversion KSR culture protocol was The mES medium was used for the first 4 days after infection, and the KSR medium was converted to the fourth day.
  • the ratio of reprogrammed cells in the two culture systems was measured by flow cytometry at different time points.
  • the number of experimental repeats n 2, error bars Represents the standard deviation.
  • the experimental results are consistent with the literature or patent reports. This result indicates that the comparison of iCDl with several other culture protocols is authentic.
  • Example 3 Effect of components in iCDl on reprogramming.
  • Fig. 6a is the six components of the present invention (basic fibroblast growth factor, vitamin (:, CHIR9902 lithium chloride, vitamin B12, strepamine) Effect of reprogramming efficiency; Oct4-GFP mouse embryonic fibroblasts were infected with SKO and cultured with complete, missing a single component and all core components in iCDl medium and classical mES medium.
  • a reprogrammed image under different culture conditions as shown in Figure a, the scale shown, 2 mm) showed vitamins (basic fibroblast growth factor, CHIR99021, lithium chloride, vitamin B12, thiamine) It has a certain influence on the efficiency of reprogramming, among which vitamin C, basic fibroblast growth factor and CHIR99021 have a significant effect on the reprogramming efficiency.
  • the reprogramming factor SKO-infected embryonic fibroblasts are: iCDl, iCDl minus vitamin C, iCDl minus alkaline
  • the growth curve of fibroblast growth factor, iCD1 minus CHIR99021, iCDl minus lithium chloride, iCDl minus vitamin B12, iCDl minus straight amine, iCDl minus all six components and mES were observed. description.
  • 20,000 initial Oct4-GFP mouse embryonic fibroblasts were infected with SKO virus and cultured in the above nine media.
  • the results in Figure 6c indicate that basic fibroblast growth factor (bFGF) and chir99021 have different effects on cell growth, while Vc has no significant effect on cell growth.
  • Figure 7a is a dose effect diagram of basic fibroblast growth factor (bFGF).
  • bFGF basic fibroblast growth factor
  • Figure 7b is a graph showing the effect of the action time of basic fibroblast growth factor (bFGF).
  • bFGF basic fibroblast growth factor
  • FIG. 7d is a dose effect diagram of CHIR99021.
  • Ten thousand Oct4-GFP mouse embryonic fibroblasts were infected with SKO, and cultured in iCD1 medium supplemented with different doses of CH-129021 at 0-12 mmol/ml, and the number of reprogrammed clones was counted on the 10th day after infection.
  • Experimental repetition number n 3. Error bars represent standard deviation.
  • epidermal growth factor was also used in reprogramming efficiency testing experiments.
  • 0ct4-GFP mouse embryonic fibroblasts were infected with SK0 and then cultured in iCD1 medium supplemented with epidermal growth factor (EGF) by adding basal fibroblast growth factor (bFGF) without the addition of receptor tyrosine.
  • Figure 7e is a statistical result of reprogramming clones on day 10 post infection, indicating that other receptor kinases can also promote reprogramming in addition to basic fibroblast growth factor.
  • Example 4 A83-01, valproic acid and fetal bovine serum could not further improve OKS-mediated reprogramming efficiency
  • Figure 8a is a chemically defined medium iCD1 after infection of mouse fibroblasts with SKO virus, and iCDl supplemented with A83-01 (0.5 ⁇ ), valproic acid (1 mM) and 2% fetal bovine serum. The medium was cultured, and the number of reprogrammed clones was counted on the 10th day after infection.
  • ALK5 inhibitor A83-01 histone methylation inhibitor valproic acid (VPA) could not further improve iCDl reprogramming efficiency, and fetal bovine serum even inhibited iCDl reprogramming efficiency to some extent.
  • 8b is a typical reprogrammed clone image from the experiment of Figure 8a. The ruler shown is 2 mm. The results of this experiment indicate that iCDl is a highly optimized culture system for inducing reprogramming.
  • Example 5 Effect of Initial Planting Density on Reprogramming Efficiency
  • Oct4-GFP transgenic mouse embryonic fibroblasts were infected with SKO virus after two rounds of infection, trypsinized, and planted into 96-well plates at different cell densities, specifically 5, 10, 50, 100, 200, 500, 1000, 2000, 5000, 10,000 per square centimeter.
  • the reprogramming efficiency at various planting densities was calculated after 8 days of incubation with iCDl.
  • the reprogramming efficiency was calculated as the ratio of the number of reprogrammed clones on day 8 to the number of initial cell implants.
  • Figure 9 is a graph showing the effect of initial planting density on reprogramming efficiency. The experimental results show that the reprogramming efficiency can reach a higher value when the initial cell density is between about 2,000 and 5,000 per square centimeter.
  • Example 6 Under the condition of iCD1 culture, the expression of endogenous pluripotency molecular markers was rapidly activated, and the exogenous transduction factor was rapidly silenced.
  • Oct4-GFP transgenic mouse embryonic fibroblasts are infected by SKO virus after two rounds of infection, and then cultured in mES medium, KSR-BN medium and iCD 1 culture system to infect the day of infection.
  • mES medium As day 0, culture samples were cultured in mES medium, KSR-BN medium and iCD1 medium for 2 days, 4 days, 6 days and 8 days, respectively, and ribonucleotides of each sample were extracted and quantified by real-time fluorescence.
  • FIG 10b is a graph showing the results of expression of pluripotency molecular markers from protein water.
  • Oct4-GFP mouse embryonic fibroblasts were infected with S O virus and cultured in iCD1 for 8 days, and fixed directly on the cell culture plate for immunofluorescence experiments.
  • the results showed that the pluripotent molecular markers Cdhl, SSEA-1 and Nanog were expressed in the reprogramming clones on the 8th day.
  • Figure 10C shows the total protein extracted from the cultures on days 4 and 8 of mES and iCD1 culture conditions.
  • the expression of Nanog was detected by antibody immunoblotting.
  • the results showed that on the 8th day after infection, Nanog expression was significant under iCDl culture conditions, while Nanog was not expressed under control mES conditions. This result further demonstrates that the reprogramming process under iCD1 culture conditions is significantly accelerated.
  • the sorting flow cytometry will be used in iCD1 culture conditions.
  • the above three types of cell populations in the culture on the 8th day were sorted.
  • Figure 12a shows the ratio of reprogrammed cells (GFP) and red fluorescent cells (DsRed) in two conditions by flow cytometry. It can be seen from Fig. 12a that the reprogrammed cell population is entered under iCD1 culture conditions ( The green fluorescent cell population reached a total of 54.3%, and the reprogramming positive red fluorescent negative cell population reached 34.8%.
  • Figure 12b (This is the result of real-time PCR to detect the expression of pluripotency molecular markers in different cell populations of mES, iCD 1 culture 8 and iCD1 culture for 8 days after SKO virus infection)
  • the extinction of the three types of extracellular diseases is also very thorough: the cell-specific pluripotency of the cell population with positive green fluorescence and red fluorescence 3 is in an intermediate state, and the exogenous Oct4 has not been completely silenced. Red fluorescent positive green fluorescent negative cell population stem cell specific
  • the expression level of pluripotency molecular markers is extremely low, while the expression level of exogenous Oct4 is extremely high.
  • chimeric mice The ability to form chimeric mice is a very critical standard feature of mouse embryonic stem cells.
  • reprogrammed clones were not different from embryonic stem cells, and reprogrammed cells selected by different methods were used to construct chimeric mice.
  • the specific method is shown in Figure 13a: Oct4-GFP transgenic mouse embryonic fibroblasts were infected with three factors (SOX, KIF4 and OCT4) and DS-RED. After infection, cultured in iCDl medium, D8, D11, D14 after infection. In the day, the cells were selected by chiseling and hand selection to construct chimeric mice.
  • the selection method was as follows: the original well cells were digested and centrifuged, and the GFP-positive, DS-RED-negative cells were sorted by flow cytometry to construct chimeric mice.
  • the selection method is as follows: Under the fluorescence microscope, the clones are peeled off to adhere to the wall, the clones are suspended in the medium, and after a certain amount is removed, the medium containing the clones is collected into a 15 ml centrifuge tube, and centrifuged.
  • FIG. 13b is a statistical table of the experiments described in Figure a for different induction days.
  • Figure 13c shows chimeric mice produced by injection of sputum embryos from iPSC cells obtained on day 8. This result strongly proves that reprogramming can be completed on day 8 under iCDl conditions.
  • Example 8 Effect of the medium of the invention on reprogramming in the case of a small factor
  • a different virus combination (ok/os/sk) was used to infect mouse fibroblasts by: injecting OCT4, KIF4 virus into a 12-well plate in a 1:1 mix (0.5 ml each) A total of 20,000 cells in a well, with OCT4, SOX2 virus mixed with 1:1 (0.5 ml each) to infect a hole in a 12-well plate for a total of 30,000 cells, and the KIF and Sox2 viruses have been mixed 1:1 ( Each of the 0.5 ml) infected one well of a twelve-well plate totaled 30,000 cells.
  • Figure 14a is a photograph and passage photograph of the original clone of OK and OS reprogrammed somatic cells under iCD1 culture conditions.
  • the wells cultured in the iCD1 culture system showed 1-6 fluorescent clones/well in 12-15 days, and in the medium infected with OCT4, SOX2, at 18-25.
  • One to three fluorescent clones will appear within the day, while in the conventional mES culture conditions, there are no clones. .
  • Figure 14b is an iPS clone expressing stem cell-specific molecular marker obtained by OK and OS reprogramming. The picked 0K and OS clones were subcultured on feeder cells, and the clones were found to express pluripotency molecular markers such as Nanog and Rexl by immunofluorescence assay.
  • Figure 14c shows the results of Oct4/Kif4 and Oct4/Sox2 reprogramming experiments. The table shows the time and statistical results of reprogramming clones for each experiment. The results of three experiments show that the test has good repeatability.
  • Figure 14d shows the teratoma tissue cuts of OK and OS clones injected into the rats. The tissue scores of the three germ layers in the tissue sections indicate that OK and OS clones have the potential to differentiate into three germ layer cells.
  • OCT4 reprogramming clones were picked and passaged, and the expression of pluripotent molecular markers was detected by immunofluorescence. These clones were found to express pluripotent molecular markers such as nanog, SSEA-l and Rexl. As shown in Figure 15b. The expression levels of endogenous pluripotency molecular markers Oct4, nanog, Dppa3, Rexl, Dnmt31 relative to mouse Oct4-GFP mouse embryonic fibroblasts were detected by real-time fluorescent quantitative PCR. The results in Figure 15C also indicate that these clones express endogenous levels of Oct4, nanog, rexl, Dppa3, Dnmt31 similar to standard embryonic cells Rl.
  • Figure 15d is a graph showing the expression status of a foreign gene by real-time fluorescent quantitative PCR.
  • Oct4-GFP mouse embryonic fibroblasts 4 days after SKO infection served as a positive control.
  • the results showed that these reprogrammed clones were picked and the exogenous transcription factors were silenced.
  • the results of the insertion assay showed (Fig. 15e) that the clones picked were not caused by virus contamination.
  • Figure 15f is a chimeric mouse produced by injection of axillary embryos into Oct4 reprogrammed clonal cells.
  • the resulting chimeric male and white strain mice were given whole black mice, see Figure 15f, which demonstrates that the Oct4 reprogramming clone is a germline chimera.
  • the results demonstrate that rigorously demonstrated that Oct4 reprogramming cells are similar to embryonic stem cells, thus demonstrating that Oct4 can completely reprogram mouse embryonic fibroblasts under iCD1 culture conditions.
  • Oct4-v l6 links a vpl6 sequence behind the Oct4 gene, and their combined expression enhances the transcriptional function of Oct4. It is verified that Oct4-vpl6, Sox2, and Kif4 have higher ability to reprogram somatic cells than Oct4, Sox2, and Kif4.
  • iCDl is superior to other culture conditions in the reprogramming process involved in Oct4-vpl6, Oct4-vpl6, Sox2, if4 infection of 20,000 Oct4-GFP mouse embryonic fibroblasts, respectively, cultured in mES, mES added vitamins C and iCDl culture system. The number of reprogrammed clones was calculated on the 10th day after infection.
  • FIG. 16a The results showed that 2500 reprogrammed clones appeared on day 10 under iCD1 culture conditions, while 650 reprogrammed clones appeared in mES-added vitamin C wells, whereas under mES conditions only Very few clones.
  • Figure 16b is a photograph of a fluorescent clone under three culture conditions. Similarly, we also tested the reprogramming capabilities of Oct4-vpl6, Sox2 and Oct4-vpl6, Kif4 under iCDl conditions.
  • FIG. 17b is a typical photograph of iPS clones induced by Oct4-vpl6/if4 and Oct4-vpl6/Sox2. Immunofluorescence was used to detect the expression of pluripotency molecular markers with antibodies. It was found that Nanog, Oct4, Rexl, SSEA-1 and other pluripotent molecular markers were expressed, and the results are shown in Figure 17c.
  • Figure 17c shows typical pluripotency molecular markers expressed by iPS cells induced by Oct4-vpl6K and Oct4-vpl6S.
  • Oct4 can be reprogrammed into somatic stem cells under iCD1 conditions, but its efficiency is extremely low, and it needs to be subcultured.
  • the characteristics of Oct4-vpl6 make it possible to improve the efficiency of single-factor reprogramming.
  • 30,000 Oct4-GFP transgenic mouse embryonic fibroblasts were infected with Oct4-vpl6 virus and cultured in mES, mES+ vitamin C and iCD1 medium, respectively.
  • mES mES+ vitamin C
  • iCD1 medium iCD1 medium
  • iCD1 vitamin C, lithium chloride, basic fibroblast growth factor, insulin, glycogen synthase 3-b inhibitor CHIR99021
  • SmES Super-mES
  • mES medium and mES were added with vitamin C medium as a control. The results are shown in Fig. 19a.
  • SKO infection there were about 400 clones in the wells of SmES culture, and there were about 20 reprogrammed clones in the wells of vitamin E supplemented with mES.
  • Figure 19b is a photograph of the original well fluorescence on day 8 of SmES culture conditions. Similarly, the reprogramming effect of SmES on Oct4 single factor in SmES medium was also tested. (In the SmES culture system, OCT4 can reprogram somatic cells. Oct4-GFP mouse embryonic fibroblasts were infected with OCT4 virus, respectively, in mES, mES ten Vitamin C, iCDl medium, the number of fluorescent clones observed on different days,)
  • Figure 19c shows the results of three independent experiments showing that Oct4 reprogrammed mouse embryonic fibroblasts under SmES conditions.
  • Figure 19d is a photograph of the original clone and the passage clone.

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Description

说 明 书 一种培养基添加剂及其应用 技术领域
本发明属于细胞培养基领域，具体涉及一种培养基添加剂，该培养基添加剂可以用于诱导多能性 干细^ ^或哺乳动物细包培养。
背景技术
2008年， 日本科学家 Yamanaka及其同事用四个转录因子（Oct4, Kif4, Sox2, c-Myc )成功的 将小鼠的成纤维细胞重编程为胚胎干细胞， 这一技术被称为诱导多能干细胞（iPS )技术。 其后， 人 及其他物种如大鼠， 猴，猪的成纤维细胞也被成功的重编程， 这项技术被认为是再生医学中最为重要 的突破。 目前几乎所有小鼠的重编程过程采用的培养体系含有血清。 采用血清培养体系有如下缺点： 1、 重编程过程緩慢。 血清中被证明含有一些能抑制重编程的物质， 如 TGF-b家族生长因子。 除此之 外，血清中的其他不明成分也极有可能延緩重编程进程。这些经被证明或未被证明的物质使得重编程 效率极其低下。 2、 血清存在批与批之间的不稳定性。 血清的制备过程决定了每批血清的成份都不一 样， 这极易导致实验可重复性的下降。 3、 血清的成分不确定性为研究重编程的机制和药物筛选造成 障碍。 血清成分复杂， 各种有利于或不利于 iPS过程的通路都有可能激活， 这将对 iPS机理的研究带 来极大的背景噪音， 同时降低药物筛选的敏感性。
研究表明血清替代物（KOSR )无血清培养基的发明是细胞培养中的一大进步。 该体系完全不含 血清， 同时体系内绝大数成分均明确， 极大地提高了实验的可重复性。 相对于血清而言， 该体系几乎 不含有分化因子， 对干细胞的自我更新的维持更佳。 其次， 该体系与血清混合显示出比血清单独使用 更高的重编程效率。
然而， 该体系成分仍然不是完全明确， 如牛血清球蛋白复合物（Albumax )中含有多种不明脂类， 这些成分对重编程的影响尚不可知。 其次， 血清替代物 ( KOSR )无法支持重编程前期细胞的生长。 此外， 在小鼠 iPS诱导过程中， 即便与血清混合重编程效率也不够高。 因此一种无血清， 成分完全明 确、 高效的重编程系统对研究 iPS机理， 药物筛选及 iPS的临床应用显得尤为重要。
发明内容
本发明的目的是克服上述技术缺陷，提供一种成分确定的高效获得 IPS细胞的培养体系。该培养 体系可在无饲养层细胞的条件下维持干细胞的生长和增殖， 同时加速 iPS进程， 提高 iPS效率。
为实现上述目的， 采用如下的技术方案：
本发明的培养基添加剂包括维生素 C和糖原合成激酶 3抑制剂。
本发明的培养基添加剂还可以是除了包括维生素 C和糖原合成激酶 3抑制剂，还包括维生素 B12、 胰岛素、 受体酪氨酸激酶和抗氧化剂。
为实现上述目的， 采用如下的技术方案：
所述的维生素 C包括抗坏血酸及其衍生物如抗坏血酸纳盐，维生素 C稳定形式 2-磷酸 -抗坏血酸。 其中优选形式为 2-磷酸 -抗坏血酸。 维生素 C工作浓度为 0-10(H敫克 /毫升,但不限于以上浓度， 其中优 选浓度为 50微克每毫升。 所述维生素 B12的工作浓度为 0-2.8微克 /毫升， 但不限于以上浓度， 其中 优选浓度为 1.4微克 /毫升。
所述胰岛素包括提取和人工重组合成的各种来源的具有生物活性的胰岛素，其中优选胰岛素为人 重组胰岛素（SIGMA公司；)， 工作浓度为 0-50微克 /毫升， 但不限于以上浓度， 其中优选浓度为 20 ^：克 /毫升。 所述糖原合成激酶 3抑制剂包括 Li十 、 Chir99021、 BIO和 SB216763中的至少一种。 其中， Li +的优选形式为 LiCl, 工作浓度范围为 0-10微克 /毫升，但不限于以上浓度， 优选浓度为 5毫摩尔 /毫 升。 糖原合成激酶为一类丝氨酸 /苏氨酸磚酸化激酶， 参与多种信号通路的调节， 其抑制剂的优选药 物为 CHIR99021 , 工作浓度范围为 0-12微摩尔 /毫升，但不限于以上浓度， 其中优选浓度为 3 ^敫摩尔/ 毫升。
所述受体賂氨酸激酶包括碱性成纤维生长因子（bFGF )、 表皮生长因子 （EGF )、 血管内皮生长 因子（VEGF ), 血小板生长因子（PDGF )、 胰岛素样生长因子（ IGF )和肝生长因子（ HGF ) 中的至 少一种。 其中优选药物为碱性成纤维生长因子,工作浓度范围为 0-100钠克 /毫升,优选浓度为 5纳克 / 毫升。
所述抗氧化剂包括硫胺、 超氧化物歧化酶、 过氧化氢酶、 还原型谷胱甘肽、 维生素 E、 乙酰化维 生素 E、 亚油酸、 亚麻酸和亚硒酸钠中的至少一种。 其中硫胺的工作浓度范围为 0-36微克 /毫升， 但 不限于以上浓度， 其中优选浓度为 9微克 /毫升； 超氧化物歧化酶的工作浓度范围为 0-10微克 /毫升， 但不限于以上浓度， 其中优选浓度为 2.5微克 /毫升。 还原型谷胱苷肽的工作浓度范围为 0-6 4敫克 /毫 升，但不限于以上浓度，其中优选浓度为 1.5微克 /毫升。维生素 E的工作浓度范围为 0-16微克 /毫升， 但不限于以上浓度，其中优选浓度为 1微克 /毫升。乙酰化维生素 E的工作浓度范围为 0-16微克 /毫升， 但不限于以上浓度， 其中优选浓度为 1微克 /毫升。 亚油酸的工作浓度范围为 0-4微克 /毫升， 但不限 于以上浓度， 其中优选浓度为 1微克 /毫升。 乙醇胺的工作浓度范围为 0-16微克 /毫升， 但不限于以上 浓度， 其中优选浓度为 1微克 /毫升。
本发明的培养基添加剂还可以是上述两种培养基添加剂分别与代血清细胞生长促进剂的混合物； 其中，代血清细胞生长促进剂包括清蛋白水解物、传铁蛋白、三碘曱状腺原氨酸、 肾上腺酮、硫辛酸、 乙醇胺、 黄体酮、 腐胺和维生素 A中的至少一种。
所述清蛋白水解物为清蛋白（又名白蛋白）水解后产物， 其组成成分为氨基酸和多肽， 具体成分 明确， 使用浓度范围为 0-10毫克 /毫升， 但不限于该浓度， 优选浓度为 1毫克 /毫升。
所述转铁蛋白包括含铁离子的转铁蛋白和不含铁离子的转铁蛋白，其中优选的转铁蛋白为含铁离 子的转铁蛋白，使用浓度范围为 0-200微克 /毫升， 但不限于该浓度， 优选浓度为 100微克 /毫升。
所述硫辛酸的工作浓度范围为 0-3.2微克 /毫升， 但不限于以上工作浓度， 其中优选浓度为 0.2微 克 /毫升。
所述维生素 A的工作浓度范围为 0-1.6微克 /毫升， 但不限于以上工作浓度， 其中优选浓度 0.1微 克 /毫升。
本发明还提供了一种完全培养基， 该完全培养基由基础培养基、血清、代血清添加剂中的一种或 多种与上述几种培养基添加剂组成； 或者由基础培养基与上述几种培养基添加剂组成。 同时， 按照表 1所示成份配置并用于添加到基础培养基 DMEM中形成化学成份确定的完全培养基 iCDl。表 1为优 化的培养基添加剂成分和浓度。
本发明还提供了一种诱导多能干细胞的完全培养基，该诱导多能干细胞的完全培养基由基础培养 基、 血清、 代血清添加剂中的一种或多种与上述两种培养基添加剂组成。
所述基础培养基包括但不限于 DMEM (Dulbecco's Modified Eagle's Medium ),MEM(Minimal Essential Medium), BME ( Basal Medium Eagle), F-10, F-12, RPMI 1640, GMEM ( Glasgow's Minimal Essential Medium), aMEM( Minimal Essential Medium), Iscove's Modified Dulbecco's Medium和 M199。 其优选形式为 DMEM。
代血清添力。剂包括但不限于 KOSR ( KonckOut Serum Replacement )， N2 , Β27 和 ITS(Insulin-Transferrin-Selenium Supplement)。 同时， 按照表 1所示成份配置培养基添加剂并用于添加到基础培养基腿 EM中形成化学成份确定 的完全培养基 iCDl。 表 1为优化的培养基添加剂成分和浓度。 表 2为优化的完全培养基 iCDl的成分 和浓度。
本发明的培养体系是一种用于无血清、 成分确定的高效从体细胞获得 IPS 细胞的无血清培养体 系，可在无飼养层细胞的条件下维持成纤维细胞和干细胞的生长和增殖，同时加速 iPS进程，提高 iPS 效率。 同时还可以用于真核细胞培养方面。 表 1
成分 终浓度 （毫克 /升） 添加剂浓度（毫克 /升） 25X
氯化锂 2.12E+02 5.30E+03
胰岛素 （人源， 重组） 2.00E+01 5.00E+02
转铁蛋白 （人，含铁离子） 1.00E+02 2.50E+03
清蛋白水解物（无脂肪酸） 1.00E+03 2.50E+04
过氧化氢酶 2.50E+00 6.25E+01
还原型谷胱苷肽 1.50E+00 3.75E+01
超氧化物岐化酶 2.50E+00 6.25E+01
三碘代甲状腺原氨酸 2.00E-03 5.00E-02
肾上腺酮 2.00E-02 5.00E-01
孕酮 1.26E-02 3.15E-01
硒 1.60E-02 4.00E-01
亚硒酸钠 1.04E-02 2.60E-01
丙酮酸 1.10E+02 2.75E+03
半乳糖 1.50E+01 3.75E+02
腐胺 3.22E+01 8.05E+02
左旋肉碱 2.00E+00 5.00E+01
乙醇胺 1.00E+00 2.50E+01
2磷酸维生素 C 5.00E+01 1.25E+03
维生素 E 1.00E+00 2.50E+01
乙酰维生素 E 1.00E+00 2.50E+01
生物素 1.00E-01 2.50E+00
维生素 A 1.00E-01 2.50E+00
维生素 B12 1.40E+00 3.50E+01
亚油酸 1.00E-01 2.50E+00
亚麻酸 1.00E-01 2.50E+00
碱性成纤维生长因子 5.00E-03 1.25E-01
糖原合成激酶抑制剂
1.40E+00 3.49E+01
CHIR99021 表 2
无机离子 毫克 /升 氨基酸 晕克 /:升
氣化锂 2. L 精 8. 0E+01
氣化钙 2.00 -02 L"丙氣酸 8. -00
f 1.0 Ε- 01 1.…天冬酰胺 I.32ΪΜ01
水 i酸铁
1 天冬氣酸 S, 33Ε+0ί
飄化钾 4.00S+ 02
L-半胱氛酸 6.30E+01
硫¾« 9.77I:W)1
甘氨酸 Λ.50 † 1
氣化钠 6.40K÷03 谷 a酸 1.47B-I 1
碳酸氣钠 3.70E÷03 L 组氨酸 4, 2ΟΕ - Ι
一水磷酸二氣钠 1.25Ε·¾2 L-异 ¾· 酸 L O E+02
1..·'亮氣酸 1.05K f-02
微量元素 L 賴 S酸 1, 46E-i-()2
L 甲硫银酸 3.0OE+0i
i钒酸铵 3. OO " 苯丙 R酸 6.60K H)1
硫酸铜 ί.25K-0 1- 舰酸 1.35Π+ 1
a化锰 5. ΟΟΚ-Οδ 丝氮酸 6.30Β-ΚΠ
m ϊ.60Ε-02 1 苏¾1酸 9. nOE+01
亚»酸钠 \ .04Ε-Ό2 L 色氨酸 L 60EH-01
能量物质 酪氣酸 1.04E+ 2
L -纈弒酸 9,棚. i'Oi
葡萄糖 4.50Ε÷0
两酮酸钠 i.10ΕΗ)2 维生素
半乳糖 1, 50Ε+0 Ι
抗坏血酸 5. OOE-Oi
脂质 维生素 E
乙酰维生素 1. (》(）
花生四烯酸 2.0ί)ί 00
生物《 1.00E -01
胆固醇 2.2CS}--K>2
泛酸 4. OOE÷00
亚油酸 1.00E-S-0I
氣化胆櫞 <1.00E-00
亚麻酸 1. Ο0Β-Η)!
^ 酸 4, OOK'i-O!)
肉 ¾蔻酸 1. ΟΟί—Ϋ ) 1
肌醇 7.20E-00
油酸 1 , ！
ι 烟酰胺 4. OOE-OO
油羧 1. O0E->O:S 维生素 Β6
棕榈酸 ], ΟΟΕ+01
核黄 *
聚.醚 18 1. ΟΟΚί'05
硫胺 4. OOE-00
硬 11酸 J 维生素 A L OOE-Ol
Figure imgf000006_0001
维生素 B12 L 0E-00
細子 /蛋 其他物质
胰岛絷 人， ®组
腐胺
转铁蛋白 （含铁离子）
00fi÷0(l
清蛋白水解物 00Ε-Ϊ-0 左旋肉碱
过氣化氢藤 501·÷00 乙醇胺
贿红 50Κ÷01
还原塑谷胱苷肽 -00 CHTR 9021 401; Η)0
超氧化物岐化瞎 oOtvi-O 巯基乙醇 ί.71ΐ-;-00
三碘代甲状腺原氨酸 0E-03
肾上腺 S 00E-02
穩 261； -02
碱性成纤维细胞生长因子
小鼠:白细胞抑制因子 0OK— 03
(小鼠千细舰培养均露添.加)
附图说明
图 1是不同培养方案效率比较实验结果图； 其中： 图 la是在 mES、 mES添加维生素 C、 KSR-BN 和 Basal3.0四种培养条件下（四种培养条件具体成分见后说明）， SKO(SOX2、 KIF4和 OCT4)病毒感 染后第 10天由两万胚胎成纤维细胞诱导出的重编程克隆数； 图 lb是在 mES、 mES添加维生素 C、 KSR-BN和 Basal3.0的 4种培养条件下， SKO感染后第 10天测得重编程细胞比率；
图 2是添加剂中核心成分的剂量效应实验结果； 其中： 图 2a是氯化锂在 0-10毫摩尔 /毫升浓度 范围内， SKO感染后第 10天重编程克隆数统计结果； 图 2b是在氯化锂 0 - 10毫摩尔 /毫升浓度范围 内， SKO感染后第 10天重编程细胞（绿色荧光细胞） 比率； 图 2c是维生素 B12在 0 - 2.8微克 /毫 升的浓度范围内， SKO感染后第 10天重编程克隆数统计结果； 图 2d是维生素 B12在 0 - 2.8微克 / 毫升的浓度范围内， SKO感染后第 10天重编程细胞比率； 图 2e是碱性成纤维细胞生长因子在 0 - 7 纳克 /毫升的浓度范围内， SKO感染后第 10天重编程克隆数统计结果； 图 2f是碱性成纤维细胞生长 因子在 0 - 7纳克 /毫升的浓度范围内， SKO感染后第 10天重编程细胞比率； 图 2g是维生素 C在 0 - 100 ^：克 /毫升的浓度范围内， SKO感染后第 10天重编程克隆数计算结果； 图 2h是维生素 C在 0 - 100微克 /毫升的浓度范围内， SKO感染后第 10天重编程细胞比率； 图 2i是胰岛素在 0 - 50微克 / 毫升的浓度范围内， SKO感染后第 10天重编程克隆数计算结果。
图 3是抗氧化剂的剂量效应实验结果； 其中， 图 3a是抗氧化剂维生素 E在 0-16微克 /毫升浓度 范围内， SKO感染后第 10天重编程克隆数统计结果； 图 3b是抗氧化剂维生素 E在 0-16 4啟克 /毫升浓 度范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率； 图 3c是抗氧化剂超氧化物 岐化酶（ SOD )在 0-10 4敖克 /毫升浓度范围内， SKO感染后第 10天重编程克隆数统计结果； 图 3d是 抗氧化剂超氧化物岐化酶（ SOD )在 0-10 ^：克 /毫升浓度范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率； 图 3e 是抗氧化剂还原型谷胱甘肽在 0-6微克 /毫升浓度范围内， SKO感染 后第 10天重编程克隆数统计结果； 图 3f是抗氧化剂还原型谷胱甘肽在 0-6微克 /毫升浓度范围内， 感 染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率。 图 3g抗氧化剂疏胺在 0-36微克 /毫升浓度 范围内， SKO感染后第 10天重编程克隆数统计结果； 图 3h是超氧化物岐化酶（ SOD )在 0-10微克 / 毫升浓度范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率；
图 4是生长支持物的剂量效应图， 其中， 图 4a是生长支持物维生素 A在 0-16微克 /毫升范围内， S O感染后第 10天重编程克隆数统计结果， 图 4b是生长支持物维生素 A在 0-16微克 /毫升范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率； 图 4c是生长支持物乙醇胺在 0-16微克 / 毫升浓度范围内， SKO感染后第 10天重编程克隆数统计结果； 图 4d是生长支持物乙醇胺在 0-16微 克 /毫升浓度范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率； 图 4e是生长支持 物亚油酸在 0-4微克 /毫升浓度范围内， SKO感染后第 10天重编程克隆数统计结果； 图 4f是是生长 支持物亚油酸在 0-4微克 /毫升浓度范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比 率；
图 4g是硫辛酸在 0-3.2微克 /毫升浓度范围内， SKO感染后第 10天重编程克隆数统计结果； 图 4h是 生长支持物亚油酸在 0-4微克 /毫升浓度范围内， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细 胞比率；
图 5是本发明所述培养基配方 iCDl ( BasaB.O添加糖原合成酶 3抑制剂 CHIR99021 ) 与已报道 的较先进的诱导重编程培养方案的效果比较图； 其中，
图 5a是本发明所示的化学成份确定培养基配方 iCDl与已报道的重编程培养方案的效率比较图； 图 5b为 a图所示几种不同培养方案感染后第八天的重编程克隆图片； 图 5c是 KSR-BN培养方案提高重 编程效率图（成分见后续说明） 图 5d是 mES添加维生素 C后提高重编程效率图； 图 5e是 mES->KSR 培养方案提高重编程效率图， （mES->KSR培养方案具体过程见后续说明）；
图 6为本发明培养基添加剂中六种成份（碱性成纤维生长因子、 维生素 C、 CHIR9902L 氯化锂、 维生素 B12、 ¾胺）对重编程效率和生长的影响， 其中，
图 6a为本发明中的六种成分对重编程效率的影响； 图 6b为感染后第 8天， a图所述不同培养条件下 的重编程图像； 图 6c为上述培养条件下生长曲线；
图 7是 bFGF、 维生素 C、 CHIR99021的剂量效应和 bFGF的作用时间的实验结果图； 其中， 图 7a是碱性成纤维生长因子 （ bFGF ) 的剂量效应图； 图 7b是 bFGF的作用时间实验图； 图 7c 是维生 素 C的剂量效应图； 图 7d是 CHIR99021的剂量效应图；
图 7是 bFGF、 维生素 C、 CHIR99021的剂量效应， bFGF的作用时间和其它受体酪氨酸激酶的 实猃结果图； 其中， 图 7a是减性成纤维生长因子（bFGF )的剂量效应图； 图 7b是 bFGF的作用时 间实验图； 图 7c是维生素 C的剂量效应图； 图 7d是 CHIR99021的剂量效应图； 图 7d是不同受体 酪氨酸激酶对重编程的效果图；
图 8是在化学成分限定的培养基 iCDl中， ALK5 (肿瘤坏死因子 TGF-beta的一个受体）抑制剂 A83-01 , 组蛋白甲基化酶抑制剂丙戊酸（valproic acid )和胎牛血清不能进一步提高 OKS介导的重编 程效率的实验结果图； 其中， 图 8a是将小鼠成纤维细胞用 SKO病毒感染后分别在化学成分确定的培 养基 iCDl , 以及添加有 A83-01 (0.5μΜ)、 丙戊酸（ImM)和 2%胎牛血清的 iCDl培养基中培养， 感染后第 10天计算重编程克隆数； 图 8b是 a实验中典型的重编程克隆；
图 9是细胞初始种植密度影响重编程效率的实验结果图；
图 10是 iCDl培养条件下， 内源多能性分子标记的表达的结果图； 其中，
图 10a是用实时荧光 PCR检测内源性多能分子标记的表达； 图 10b是直接在细胞培养板上固定进行 免疫荧光实验； 图 10c是用免疫印迹检测表达状况；
图 11是 iCDl培养体系下重编程追踪图；
图 12是感染后第八天重编程效率及相关分子标记物表达图，其中， 图 12a是 Oct4-GFP小鼠胚胎 成纤维细胞用 SKO病毒和外源红色荧光蛋白 DsRed病毒感染后，分别在 mES和 iCDl培养基中培养 感染后的第八天， 检测两种状况下重编程细胞（绿色荧光细胞）和未重编程细胞（红色荧光细胞）的 比率； 图 12b是用实时荧光定量 PCR检测 SKO病毒感染后分别在 mES ,iCDl培养 8天及 iCDl培养 8天分选处的不同细胞群的多能性分子标记的表达情况；
图 13是过程嵌合实验相关结果； 其中， 图 13a是通过胚胎注射检测在 iCDl培养基中 OSK诱导 的 iPSC细胞的多能性； 图 13b是在不同诱导天数实施的 a图所述实验的统计状况； 图 13c是由第八 天得到的 iPSC细胞注射嚢胚产生的嵌合体小鼠； 该实验说明感染了 SKO的细胞在 iCDl中重编程 8 天即可形成能产生嵌合体的多能干细胞。
图 14是在 iCDl培养体系中， OK和 OS重编程体细胞的实验结果图； 其中，
图 14a是在 iCDl培养条件下， OK和 OS重编程体细胞的原始克隆照片和传代照片； 图 14b是免疫荧 光检测其干细胞特意的分子标记表达情况； 图 14c是 iCDl培养体系下， OK和 OS重编程实验实施情 况和效率统计； 图 14d是畸胎瘤切片图片； 该图表明 OK和 OS重编程所形成的多能干细胞具有胚胎 干细胞的特征。
图 15是 iCDl条件下 OCT4重编程 MEF的相关实验结果图； 其中， 图 15a是感染后第 33天由 Oct4诱导的原始克隆及形成的细胞系的形态照片； 图 15b是 Oct4 iPS克隆表达多能性分子标记； 图 15c是实时荧光定量 PCR表明挑取克隆均表达干细胞特异的多能型分子标记； 图 15d是通过定量 rt-PCR方法分析外源基因的表达状况； 图 15e是插入鉴定分析确定挑取的克隆的确为 Oct4单因子诱 导而来， 而非污染； 图 15f是由 Oct4 iPSC细胞注射嚢胚可产生的嵌合体小鼠； 图 15g是产生的嵌合 体小鼠具有生殖系嵌合的能力；
图 16是釆用 Oct4-VP16(VP16为病毒来源的基因序列， 其与转录因子连接可增强转录因子的活 性)、 Klf4和 Sox2感染条件下， iCDl可提高重编程效率的相关实验图； 其中， 图 16a是 Oct4-GFP小 鼠胚胎成纤维细胞被 Oct4-vpl6SK感染后， 分别培养在 mES培养基， 添加维生素 C的 mES培养基 以及 iCDl培养基中培养， 感染后第 10天计算荧光克隆数； 图 16b是图 16a所述实验中的典型照片； 图 17是在 iCDl培养体系中， Oct4-vpl6/Kif4和 Oct4-vpl6/Sox2重编程体细胞重编程的相关实验 图； 其中， 图 17a是 Oct4-GFP小鼠胚胎成纤维细胞被 Oct4-vp 16/Kif4和 Oct4-vpl6/Sox2分别感染后 培养在 iCDl体系下， 分别在不同天数计算重编程克隆数； 图 17b是由 Oct4-vpl6K和 Oct4-vpl6S诱 导出的 iPS克隆的典型照片； 图 17c是由 Oct4-vpl6K和 Oct4-vpl6S诱导出的 iPS细胞表达典型的多 能性分子标记；
图 18是在 iCDl培养体系中， Oct4-vpl6重编程体细胞的相关实验图； 图 18a是由 Oct4-vpl6诱 导出的 iPS克隆的典型照片； 图 18b是 Oct4-GFP小鼠胚胎成纤维细胞感染 Oct4-vpl6后分别在 mES,mES添加维生素 C和 iCDl培养基中培养， 在不同培养天数计算重编程克隆数； 图 18c是 Oct4-v l6诱导出的克隆表达多能性分子标记； 图 18d是 Oct4-vpl6诱导的 iPS克隆注射嚢胚， 可产 生的嵌合体小鼠的图片；
图 19是 iCDl核心成分添加到 mES培养基中促进重编程的实验图； 其中，
图 19a是 Oct4-GFP小鼠胚胎成纤维细胞感染 SKO病毒后，分别在 mES、 mES+维生素 C、 SmES(mES 添加维生素 C、 bFGF、 CHIR9902K 氯化锂和 B27 )培养基中培养， 在感染后第 8天计算重编程克隆 数； 图 19b是 SmES培养条件下第 8天典型的荧光克隆照片； 图 19c是 Oct4-GFP小鼠胚胎成纤维细 胞感染 OCT4病毒后， 分别在 mES、 mES十维生素〔、 iCDl培养基中在不同天数观察荧光克隆数； 图 19d是 SmES培养体系下， OCT4重编程体细胞的原始克隆照片和传代照片。
具体实施方式
为使本发明更加容易理解， 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用 于说明本发明而不用于限制本发明的范围。
需要说明的是， 在下列实例中， 如未进行特殊说明， 以下术语和培养基的组分如下所述： 基础培养基： 为人工制备的含有糖类、 氨基酸、 无机盐、 维生素、 脂质等细胞生长所需营养物质 的培养品。 本发明所述基础培养基包括 Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow's Minimal Essential Medium (GMEM), a Minimal Essential Medium ( a MEM), Iscove's Modified Dulbecco's Medium和 M199等，但不仅限于上述基础培养基，其中优选的基础培养基为高糖 DMEM基础培养基。
胎牛血清： 胎牛血液中分离提取得到的支持细胞生长的培养物， 具体成分未知， 含有丰富的营养 物质和生长因子， 可支持多种细类型胞的生长。但有成分不明、批次差异大及可能含有动物病原体等 缺点。
血清替代物： 为一类商品化代替血清的培养物添加剂， 成分一般确定。 本发明中所述的血清替代 物包括 KSR (—种商业化的带血清培养添加剂）、 2 (一种商业化的血清替代添加剂， 成分为胰岛素， 转铁蛋白， 孕酮， 腐胺， 亚硒酸钠。）和 B27 (—种用于培养神经细胞的无血清培养添加剂）等代血 清培养添加基， 但不限于这几种。
胚胎干细胞： 简称 ES或 EK细胞。 胚胎干细胞是早期胚胎（原肠胚期之前）或原始性腺中分离 出来的一类细胞， 它具有体外培养无限增殖、 自我更新和多向分化的特性。 小鼠的胚胎干细胞体外培 养需要饲养层细胞和小鼠白血病抑制因子的支持维持其多能性状态。 研究发现， 在体外培养过程中， 糖原合成激酶抑制剂和丝裂晦抑制剂联合使用可抑制胚胎干细胞的分化，可在无饲养层细胞和小鼠白 血病抑制因子情况下维持胜胎干细胞的多能型。
糖原合成激酶 3 ( glycogen synthase kinase-3 , GSK-3 )是一种多功能的丝氨酸 /苏氨酸蛋白激酶， 是细胞内多种信号传导通路的重要成分， 参与细胞内糖代谢， 细胞增殖、 细胞分化和凋亡等多种生理 过程。 糖原合成激酶抑制剂主要包括 CHIR99021、 BIO、 SB216763等。 其中优先药物为 CHIR99021, 其结
Figure imgf000009_0001
iPS细胞： induced pluripotent stem cell的缩写及诱导多能性干细胞。 为分化细胞经过重编程因子 处理回复为多能干细胞的状态。 目前已经通过注射四倍体嚢胚技术证明 iPS细胞可产生完整的小鼠， 表明 iPS细胞具有多能型。
重编程因子指能将分化细胞回复为多能性状态的因子， 多为核转录因子， 目前已有报道的用于重 编程的转录因子包括： SOX2、 OCT3、 KIF4、 NONOG、 LIN28、 c-myc、 lin28、 esrrb、 tbx3等。 本发 明所述转录因子包括但不仅限于上述转录因子。
饲养层细胞培养基 ( feeder medium )组成成分为： 高糖基础培养基 ( DMEM ), 外力。 10%胎牛血 清（FBS ) (Hyclone), 2mM谷氨酸 ( Glutamine X 非必需氨基酸（NEAA )。
含血清的 ES细胞培养基成分为： 高糖基础培养基 ( DMEM为以发明人首字母命名的一种基础培 养基）、 15%胎牛血清 FBS (GIBCO)、 2mM谷氨酰胺（ Glutamine )、 非必需氨基酸（NEAA )、 青霉素 /链霉素、 beta-巯基乙醇、 丙酮酸钠。
mES是经典的干细胞培养基，其组分为： 高糖 DMEM培养基添加 15%胎牛血清、非必须氨基酸、 谷氨酸、 青霉素 /链霉素、 beta-巯基乙醇、 丙酮酸和白细胞抑制因子 (一种维持小鼠多能形状态的生长 因子)。
KSR无血清培养基： KSR为 Knockout Serum Replace的缩写， 为一种商品化代血清干细胞培养 添加剂。 本发明实施例中所用的 KSR完全培养基分为两种， 一种用于 iPS的诱导过程 ， 其组成成分 为： 高糖基础培养基（DMEM ), 10%KSR添加剂， 2mM谷氨酸（ Glutamine )，非必需氨基酸（ NEAA ), 青霉素 /链霉素， beta-琉基乙醇， 丙酮酸钠。 另一种用于培养干细胞或 iPS克隆的完全 KSR无血清培 养基， 其组成成分为： KNOCKOUT DMED (—种渗透压经过优化的适于干细胞培养的基础培养基）， 15%KSR添加剂， 2mM谷氨酸（ Glutamine ), 非必需氨基酸（ NEAA ), 青霉素 /链霉素， Beta-巯基乙 醇。 所有 iPS过程与克隆培养基都添加鼠白细胞抑制因子 LIF ( millipore, 商品名为 ESGRO , 为一种 抑制小鼠干细胞分化的生长因子；)， 添加的终浓度为 1000U/ml。
KSR-BN即上述用于 iPS诱导过程的 KSR完全培养基添加碱性成纤维生长因子和 N2 (一种商业 化的血清替代添加剂， 成分为胰岛素， 转铁蛋白， 孕酮， 腐胺， 亚硒酸钠。）
Basal3.0完全培养基： 指本发明所述培养基中的一种， 具体由高糖基础培养基（ DMEM )添加维 生素 C、 胰岛素、 氯化锂、 维生素 B12、 抗氧化剂、 受体酪氨酸激酶生长因子和一系列生长支持物等 组成。 具体成分为表 2所示成分缺 CHIR99021。
iCDl完全培养基： 本发明所述培养基中一种。 为表 1所述添加剂与高糖基础培养基（DMEM )， 谷氨酸， 非必须氨基酸， 青霉素 /链霉素， beta-巯基乙醇， 丙酮酸钠等混合组成， 其中表 1所述添加 剂与其他成分体积比为 1 :24。 具体成分和浓度如表 2所述。
另外， 如无特殊说明， 基于小鼠的体细胞重编程均采用如下方式进行：
重编程采用的体细胞类型均为 OG2小鼠胚胎成纤维细胞， 传代数不超过三代。 OG2小鼠为在干 细胞特异表达基因 Oct4基因的启动子后连有绿色荧光蛋白 （GFP )基因的转基因小鼠。 在重编程后 期， 当 OG2小鼠胚胎成纤维细胞内源 Oct4被激活时， 绿色荧光蛋白伴随表达， 在荧光显^:镜下， 可 见细胞或重编程克隆呈现绿色，通过在荧光显微镜下直接统计重编程克隆即绿色荧光克隆数或釆用流 式细胞仪分析绿色荧光细胞的比率， 研究者可以比较不同条件下的重编程效率。
细胞实验和病毒的制备过程如下： 细胞种植在 12孔贴壁细胞培养板内， 种植密度为 20000细胞 / 孔。 细胞种植 6-18小时后， 视细胞密度和状态用带有小鼠重编程因子的病毒进行感染。 感染分两轮 进行， 第一轮感染后 24小时后进行第二轮感染， 第二轮感染后 24小时将病毒液更换成各种待测培养 液。 换液当天记为第 0天（D0 ); 感染后不同时间点， 按实验需要在原孔内数 GFP荧光克隆数或采用 流式细胞仪分析 GFP荧光细胞的比率。
病毒的制备过程为： 将克隆到 PMX载体上的重编程因子质粒转染病毒包装细胞（PlatE ), 转染 后 12-16小时， 更换新鲜培养液， 用转染后 48小时收集培养液作为第一次感染用病毒液， 添加新鲜 培养液 24小时后再收集培养液作为第二次感染病毒液。
此外， 如无特殊说明， 一些缩写形式按如下方式理解：
SKO特指 SOX2、 KIF4和 OCT4等三个病毒或三种病毒感染过的细胞， 与三因子含义等同。 OK 特指 OCT4和 KIF4两种病毒或两种病毒感染过的细胞， OS特指 OCT4和 SOX2两种病毒或两种病毒 感染过的细胞。 实施例 1 : B3.0与其他培养方案的重编程效率比较与组分浓度优化。
将 SKO病毒以 1 : 1 : 1混合（每种 0.5ml )后感染到十二孔板的一孔共计 2万个成纤维细胞中， 在 37度， 5 % C02的条件下分别培养在 mES培养基， mES添加维生素 C， SR-B 培养基和 Basal3.0 培养基中。 感染后第 10天在荧光显微镜下直接统计重编程克隆数， 并用流式细胞仪分析重编程细胞 的比率。 图 l a是在 mES、 mES添加维生素 C、 KSR-BN和 Basal3.04种培养条件下感染后第 10天由 每两万 Oct4-GFP转基因胚胎成纤维细胞诱导出的重编程克隆数； 图示中每个实验组重复数 n=3 , 误 差线代表标准偏差。结果表明，感染后第 10天，在 Basal3.0培养条件下，可出现 1278个重编程克隆， 而在 KSR-BN和 mES添加维生素 C的培养条件下分别只有 180和 113个重编程克隆， 而在 mES培 养条件下， 几乎不出现重编程克隆。 图 lb是在 mES、 mES添加维生素 C、 KSR-BN和 Basal3.0的 4 种培养条件下， 感染后第 10天由流式细胞仪测得 OCT4-GFP阳性细胞比率； 试验重复数 n=3 , 误差 线代表标准偏差。 结果表明， 感染后第 10天， 在 Basal3.0培养体系下， 有 35.2%的细胞发生重编程， 而在 KSR-BN条件和 mES添加维生素 C条件下， 分别只有 14.25%和 3.9%的细胞发生重编程。 这两 种实验结果均表明 Basal3.0是一种非常优秀诱导三因子（ OCT4，Sox2，Kif4 )重编程的培养体系。
为了确定 Basal3.0 中， 各成分在三因子感染条件下对重编程的影响以及其最适工作浓度范围， BasaB .O中的不同物质采用不同的浓度被用于 iPS效率测试实验。 重编程克隆数或重编程细胞比率两 种评价指标被采用， 以便更准确的判断测试物质对重编程的影响。 图 2为核心成分的剂量效应实验结 果； 其中， 图 2a是氯化锂在 0-2.8毫摩尔 /毫升的剂量范围内， 感染后第 10天， Basal3.0诱导形成的 重编程克隆数， 图 2b为氯化锂在相应试验件下测得的重编程细胞比率。 图 2c是维生素 B 12在 0-10 毫克 /毫升的剂量范围内， 感染后第 10天， Basal3.0诱导形成的重编程克隆数， 图 2d为维生素 B12 在相应试验件下测得的重编程细胞比率。图 2e是碱性成纤维细胞生长因子在 0-10纳克 /毫升剂量范围 内， 感染后第 10天， Basal3.0诱导形成的重编程克隆数， 图 2f为碱性成纤维细胞生长因子在相应试 验件下测得的重编程细胞比率。 图 2g是维生素 C在 0-100毫克 /毫升的剂量范围内， 感染后第 10天， Basa .O诱导形成的重编程克隆数， 图 2h为维生素 C在相应试险件下测得的重编程细胞比率。 图 2i 是胰岛素在 0-50毫克 /毫升的剂量范围内， 感染后第 10天， Basal3.0诱导形成的重编程克隆数。 以上 试验中，各组试验重复数 n=3，误差线代表标准偏差。该系列物质在测试过程中对重编程影响比较显著， 因此归为 Basal3.0中影响重编程的核心物质， 分别为推荐的工作浓度分别为氯化锂： 5毫摩尔 /毫升， 维生素 B 12: 1.4微克 /毫升，碱性成纤维细胞生长因子： 5纳克 /毫升， 维生素 C: 50微克 /毫升， 胰岛 素： 50微克 /毫升。 但不限于此浓度范围。
图 3为一系列抗氧化剂的剂量效应实验结果图， 图 3a是抗氧化剂维生素 E在 0-16 ^：克 /毫升浓 度范围内， 感染后第 10天， Basal3.0诱导形成的重编程克隆数， 图 3b为维生素 C在相应试验件下测 得的重编程细胞比率。 图 3c是抗氧化剂超氧化物歧化酶（ SOD )在 0-10 t克 /毫升浓度范围内， 感染 后第 10天， Basal3.0诱导形成的重编程克隆数， 图 3d为抗氧化剂超氧化物歧化酶（SOD )在相应试 验件下测得的重编程细胞比率。 图 3e是抗氧化剂还原型谷胱甘肽在 0-6微克 /毫升浓度范围内， 感染 后第 10天， BasaB.O诱导形成的重编程克隆数， 图 3f为氧化剂还原型谷胱甘肽在相应试验件下测得 的重编程细胞比率。 图 3g是抗氧化剂硫胺在 0-36微克 /毫升浓度范围内， 感染后第 10天， Basal3.0 诱导形成的重编程克隆数， 图 3h为抗氧化剂硫胺在相应试验件下测得的重编程细胞比率。 以上实验 每组重复数 n=3.误差线代表标准偏差。 以上物质推荐的工作浓度分别为： 维生素 E: l毫克 /毫升， 超 氧化物岐化酶： 2.5毫克 /毫升,还原性谷胱苷肽 1.5毫克 /毫升， 硫胺： 9毫克 /毫升。
图 4为其他一些生长支持物质的剂量效应实验结果。 图 4a是维生素 A在 0-1.6微克 /毫升浓度范 围内， 感染后第 10天， Basal3.0诱导形成的重编程克隆数， 图 4b为维生素 A在相应试验件下测得的 重编程细胞比率。 图 4c是乙醇胺在 0-16 ^敫克 /毫升浓度范围内， 感染后第 10天， BasaB.O诱导形成 的重编程克隆数， 图 4d为乙醇胺在相应试验件下测得的重编程细胞比率。 图 4e是亚油酸在 0-4微克 /毫升浓度范围内， 感染后第 10天， Basal3.0诱导形成的重编程克隆数， 图 4f乙醇胺在相应试险件下 测得的重编程细胞比率。 图 4g是硫辛酸在 0-3.2 t克 /毫升浓度范围内， 感染后第 10天， Basal3.0诱 导形成的重编程克隆数， 图 4h为硫辛酸在相应试验件下测得的重编程细胞比率。 以上实验每组重复 数 n=3.误差线代表标准偏差。 以上物质推荐的工作浓度分别为： 维生素 AO.l t克 /毫升， 乙醇胺： 1 微克 /毫升，亚油酸： 1微克 /毫升， 硫辛酸： 0.2微克 /毫升。
实施例 2: 对 BasaB.O的改进
在 Basal3.0浓度测试过程中，氯化锂对重编程的显著影响让我们猜测抑制糖原合成激酶可能是其 促进重编程的原因， 因此， 一系列糖原合成激酶抑制剂物质包括 CHIR99021，BIO,SB31xxxx均被作为 候选物质进行了上述 iPS测试实验（重编程克隆数和重编程细胞比率两种指标作为重编程效率的评价 标准；)。结果， GSIO-β抑制剂 CHIR99021对 BasaB.O重编程效率有显著提升， 因此将改进的 Basal3.0 即添加糖原合成激酶 3(GSK3-P)抑制剂 CHIR99021 , 但不仅限于添加 CH99021的化学成分限定的培 养基命名为 iCDl。
通过上述的 iPS效率测试实验， 我们比较了 iCDl和目前所有已报道的较先进的培养方案的重编 程效率， 其中包括： mES添加维生素 C, mES转换 KSR (即在感染后第 4天由由经典的胚胎干细胞培 养基 ( mES )培养基转换到商业化的无血清培养基 ( KSR ), KSR-BN。 图 5a是本发明所示的化学成 份确定培养基配方 iCDl与其他已报道的重编程培养方案的效率比较图。 在上述几种培养方案下， 统 计感染后第 8天， 由每 2万胚胎成纤维细胞诱导出的重编程克隆数。 图示中每个实验组重复数 n=2, 误差线代表标准偏差。 结果显示在 iCDl培养条件下， 有 1467.3个重编程克隆， 而在 mES添加维生 素 C， mES转换 KSR, KSR-B 条件下分别只有 32.3,20.7， 68.3个重编程细胞出现。 图 5b为 a图所 示几种不同培养方案感染后第八天的重编程克隆图片。 所示标尺： 2毫米。 该结果表明 iCDl培养体 系明显优于其它任何一种培养体系。
为了更客观的比较这几种不同培养条件下的效率差别，我们按照文献或专利报道重复了 KSR-BN, mES添加维生素 C, mES转换 KSR这三种条件诱导重编程效率的实验， 不同天数的重编程效率被统 计， 图 5c是 KSR-BN培养方案提高重编程效率图， Oct4-GFP 小鼠胚胎成纤维细胞（ MEFs )被 Sox2/Klf4/Oct4 (SKO)病毒感染后， 分别用 mES和 KSR-BN培养基培养，在不同时间点用流式细胞仪 检测两种培养体系下重编程细胞的比率。 实验重复数 n=2， 误差线代表标准偏差。 图 5d是 mES添加 维生素 C后提高重编程效率图， Oct4-GFP小鼠胚胎成纤维细胞（ MEF )被 SKO病毒感染后， 分别用 mES和 mES添加维生素 C两种培养基培养，在不同时间点用流式细胞仪检测两种培养体系下重编程 细胞的比率。 实验重复数 n=2 , 误差线代表标准偏差。 图 5e是 mES->KSR培养方案提高重编程效率 图， Oct4-GFP小鼠胚胎成纤维细胞（ MEF )被 SKO病毒感染后， 分别用 mES和 mES转换 KSR培 养方案培养， mES转换 KSR培养方案为感染后前 4天采用 mES培养基，第四天转换为 KSR培养基； 分别在不同时间点用流式细胞仪检测两种培养体系下重编程细胞的比率； 实验重复数 n=2， 误差线代 表标准偏差。 实验结果均能与文献或专利报道吻合。 该结果表明 iCDl与其它几种培养方案的比较结 果是真实可信的。
实施例 3: iCDl中各组分对重编程的影响。
为了评价 iCDl中主要成分对重编程效率的影响， 维生素 C，碱性成纤维细胞生长因
子, CHIR99021, 氯化锂， 维生素 B12,硫胺分别或全部从 iCDl中减除来实施 ips效率测试实验， iCDl 培养基作为全对照， mES培养基作为基础参照。 转染后第八天， 重编程克隆数（图 6a, 图 6a为本发 明中的六种成分（碱性成纤维生长因子、 维生素 (：、 CHIR9902 氯化锂、 维生素 B12、 石克胺）对重 编程效率的影响； Oct4-GFP小鼠胚胎成纤维细胞用 SKO感染后分别用完全的、 缺失单独某种成分以 及缺失所有核心成分的 iCDl培养基及经典的 mES培养基培养。 感染后的第 8天分别计算不同培养 条件下每 2万细胞诱导出的重编程克隆数。 图示中每组实验重复数 n=3.误差线代表标准偏差。）和同 期重编程克隆照片 （图 6b为感染后第 8天， a图所述不同培养条件下的重编程图像， 所示标尺， 2毫 米）结果显示维生素（、 碱性成纤维细胞生长因子、 CHIR99021、 氯化锂、 维生素 B12、 硫胺均对重 编程效率有一定影响， 其中维生素 C、碱性成纤维细胞生长因子、 CHIR99021对重编程效率的影响尤 其明显。 在不添加该 6种成分的情况下， 几乎无重编程克隆出现。 为了确定上述 6种物质对重编程的影响是细胞生长依赖型还是细胞生长非依赖型， 重编程因子 SKO感染后的胚胎成纤维细胞分别在： iCDl、 iCDl减除维生素 C、 iCDl减除碱性成纤维细胞生长 因子、 iCDl减除 CHIR99021、 iCDl减除氯化锂、 iCDl减除维生素 B12、 iCDl减除直胺、 iCDl减 除所有该六种成分和 mES等 9种培养状态下的生长曲线被描述。 2万初始 Oct4-GFP小鼠胚胎成纤维 细胞被 SKO病毒感染后， 分别在上述 9种培养基中培养。 分别在感染换液当天（DO天）及感染后的 第 3天和第 6天， 将细胞消化， 计算每孔细胞数。 每组实验重复数 n=3， 误差线代表标准偏差。 图 6c 的结果表明碱性成纤维生长因子（bFGF )和 chir99021对细胞生长有不同程度的影响， 而 Vc则对细 胞生长无明显影响。
为了进一步确定碱性成纤维细胞生长因子影响重编程的最佳浓度。不同剂量的碱性成纤维细胞生 长因子被添加到 iCDl 中来检测其对重编程的作用， 图 7a为碱性成纤维生长因子（bFGF )的剂量效 果图。 1万 Oct4-GFP小鼠胚胎成纤维细胞被 SKO病毒感染后， 分别在添加 0-10纳克 /毫升的不同剂 量 bFGF的 iCDl培养基中培养， 感染后的第 10天计算重编程克隆数。 实验重复数 n=3。 误差线代表 标准偏差。 结果表明， 当终浓度高于 5纳克 /毫升时， 继续提高碱性成纤维细胞生长因子的浓度对重 编程效率的提高作用不显著。图 7b为碱性成纤维生长因子（ bFGF )的作用时间的效果图。 1万 Oct4-GFP 小鼠胚胎成纤维细胞被 SKO感染后用 iCDl培养基培养，分别在 0至 10天的不同时间段添加 bFGF, 并 在感染后的第 10天计算荧光克隆数。每组实验重复数 n=6,误差线代表标准偏差。该图在重编程过程 的不同时间段添加碱性成纤维细胞生长因子的实验结果表明， 在 iCDl培养条件下， 碱性成纤维细胞 生长因子主要作用在重编程的前 0-8天， 继续延长作用时间对重编程效率的进一步提高作用较轻微。
同样， 为了确定在改进条件即 iCDl条件下， 维生素 C和糖原合成激酶抑制剂 CHIR99021的最 佳作用浓度， 不同剂量的维生素 C和糖原合成激 抑制剂 CHIR99021分别被添加到 iCDl中，来确定 它们的最适作用浓度。 图 7c是维生素 C的剂量效应图。 2万 Oct4-GFP小鼠胚胎成纤维细胞被 S O 感染后， 分别在添加有 0-100微克 /毫升不同剂量的维生素 C的 iCDl培养基中培养， 感染后的第 10 天统计重编程克隆数。 实验重复数 n=3.误差线代表标准偏差。结果表明，当维生素 C或磷酸化维生素 C (维生素 C的稳定形式）的浓度超过 25微克每毫升时， 继续提高维生素 C的浓度对重编程效率无 明显提高。 图 7d是 CHIR99021的剂量效应图。 1万 Oct4-GFP小鼠胚胎成纤维细胞被 SKO感染后， 分别在添加 0-12毫摩尔 /毫升不同剂量 CHIR99021的 iCDl培养基中培养， 感染后的第 10天统计重 编程克隆数。 实验重复数 n=3.误差线代表标准偏差。
结果表明糖原合成激酶抑制剂的最佳作用浓度在 3毫摩尔左右，高剂量的糖原合成激酶抑制剂对重编 程效率反而有一定抑制作用。 以上实验结果均为本发明中相应物质的工作浓度提供参考。
为了证明其它受体酪氛酸激酶家族生长因子也对重编程起作用，表皮生长因子也被用于重编程效 率测试实验。 0ct4-GFP小鼠胚胎成纤维细胞用 SK0感染后分别用不添加受体酪氨酸激， 添加碱性成 纤维细胞生长因 （bFGF ), 添加表皮细胞生长因子（EGF)的 iCDl培养基中培养， 图 7e是感染后第 10 天重编程克隆统计结果，该结果表明除碱性成纤维细胞生长因子外， 其它受体 氨酸激酶也可促进重 编程。
实施例 4 A83-01、 丙戊酸和胎牛血清不能进一步提高 OKS介导的重编程效率
为了进一步提能高 iCDl重编程效率， 先前已报道的几种提高重编程效率的物质， 如 ALK5抑制 剂 A83-01、组蛋白甲基化酶抑制剂丙戊酸 (VPA)以及在传统小鼠胚胎干细胞中使用的胎牛血清被添加 到 iCDl进行重编程效率测试。 图 8a是将小鼠成纤维细胞用 SKO病毒感染后， 分别在化学成分确定 的培养基 iCDl , 以及添加有 A83-01 (0.5μΜ)、 丙戊酸（ImM)和 2%胎牛血清的 iCDl培养基中培 养， 感染后第 10天计算重编程克隆数。 结果表明， ALK5抑制剂 A83-01,组蛋白甲基化 ϋ抑制剂丙戊 酸 (VPA)均不能进一步提高 iCDl重编程效率，而胎牛血清甚至对 iCDl重编程效率有一定程度的抑制， 图 8b是图 8a实验中典型的重编程克隆图像。 所示标尺 2毫米。 该实验结果表明 iCDl是一种十分优 化的诱导重编程的培养体系。
实施例 5初始种植密度对重编程效率的影响 为了探讨初始细胞密度对重编程效率的影响， Oct4-GFP转基因小鼠胚胎成纤维细胞被 SKO病毒 经过两轮感染后， 用胰酶消化， 分别按不同细胞密度种植到 96孔板内，具体为 5、 10、 50、 100、 200、 500、 1000、 2000、 5000、 10000个每平方厘米。 用 iCDl培养 8天后计算各种不同种植密度下的重编 程效率，重编程效率的计算方式为第 8天的重编程克隆数与初始细胞种植数的比值。实验重复数 n=6, 误差线代表标准偏差。 图 9为细胞初始种植密度影响重编程效率图， 实验结果表明， 当初始细胞密度 在约 2000到 5000个每平方厘米之间时， 重编程效率可达到较高值。
实施例 6 iCDl培养条件下， 内源性多能分子标记的表达迅速激活， 外源转因子则迅速沉默
为了从分子水平上证明 iCDl培养体系优于其他培养体系， 干细胞特异表达的多能型性分子标记 物 Nanog和内源性 Oct4 的相对表达量被用于评价细胞整体重编程进程的快慢。 具体实施方法为， Oct4-GFP转基因小鼠胚胎物成纤维细胞被 SKO 病毒经过两轮感染后， 分别培养在 mES培养基， KSR-BN培养基和 iCD 1培养体系下，以感染换液的当天作为第 0天，分别收取在 mES培养基， KSR-BN 培养基和 iCDl培养基培养 2天， 4天， 6天和 8天培养物样品， 提取各样品的核糖核苷酸， 用实时 荧光定量聚合 ϋ链式反应（RT-PCR )检测 nanog和内源 Oct4的表达量。 实验结果如图 10a所示， 在 iCDl培养条件下， nanog和内源 Oc4的激活明显要快于 mES和 KSR-BN组。 该实验每组实验重复 数 n=3.误差线代表标准偏差。
图 10b为从蛋白水品上验证多能性分子标记物的表达结果图。 Oct4-GFP小鼠胚胎成纤维细胞被 S O病毒感染后在 iCDl中连续培养 8天， 直接在细胞培养板上固定进行免疫荧光实验。 结果表明， 第 8天在原盘出现的重编程克隆其多能性分子标记物 Cdhl,SSEA-l和 Nanog均有表达。 所示标尺， 100微米。 图 10 C为将 mES和 iCDl培养条件下第 4天和第 8天的培养物提取总蛋白， 采用蛋白免 疫印记方法， 用抗体检测 Nanog的表达状况， 实验结果表明， 感染后第 8天， 在 iCDl培养条件下， nanog表达显著， 而在对照组 mES条件下， Nanog则未表达。 该结果也进一步证明了 iCDl培养条件 下重编称过程显著加快。
外源转录因子的沉默是重编程后期的一个重要事件，也是判断完全重编程的一个指标。 为了更直 观的追踪外源转录因子的沉默， 我们采用红色荧光蛋白 Ds-Red用于模拟外源转录因子的沉默。 具体 实施方法为将红色荧光蛋 Ds-Red和 SKO病毒一起感染 Oct4-GFP转基因小鼠胚胎物成纤维细胞，在 iCDl培养基下培养， 用荧光显微镜跟踪观察细胞绿光（指示内源多能性分子标记 Oct4的表达 )和红 光（指示外源病毒的表达状况）的表达状况。 图 11为 iCDl培养体系下的重编程追踪， 如图 11所示， 在感染后的第 3天， 部分细胞开始出现绿色荧光细胞， 但同时红光也表达， 表明其内源性 Oct4基因 已经开始激活，但外源基因还没有沉默。感染后第 6天，绿色荧光显著加强， 而红色荧光则开始变弱， 表明内源性 Oct表达显著提高， 而外源病毒已经开始沉默，预示着细胞开始进入完全重编程的后期阶 段。 感染后的第 8天， 多数克隆只表达重编程而不表达红色荧光， 表明重编程过程已经基本完成。
为了进一步证明上述绿色荧光阳性红色荧光阴性的细胞群比绿色红色双阳性的细胞群及绿色荧 光阴性红色荧光阳性的细胞群重编程程度更高， 用分选式流式细胞仪将在 iCDl培养条件下第 8天的 培养物中的上述三类细胞群分选出。 图 12a是用流式细胞仪分别检测两种状况下重编程细胞 ( GFP ) 和红色荧光细胞 (DsRed)的比率，从图 12a中可以看出在 iCDl培养条件下， 进入重编程的细胞群（绿 色荧光细胞群）总共达到 54.3%, 其中重编程阳性红色荧光阴性的细胞群达到 34.8%。 而 mES培养 体系下仅有 0.3%的细胞表现出重编程阳性， 绿色荧光阳性红色荧光阴性的细胞则无法检测到。 将分 选出的三类细胞群提取核糖核苷酸，用实时荧光定量聚合酶链式反应来检测三类细胞群中多能性分子 标记物内源性 Oct4，nanog,Dppa3，Rexl的表达及外源 Oct4的沉默状态。 mES和 iCDl第 8天的未分选 培养物作为整体对照。 由图 12b (是用实时荧光定量 PCR检测 SKO病毒感染后分别在 mES,iCD 1培 养 8及 iCDl培养 8天分选处的不同细胞群的多能性分子标记的表达情况）结果可以看出此三类细胞 外源病 的 默也十分彻 ：而绿色突光和红色焚光3又阳性的细胞群干细胞特异的多能性分^表达水 平则处于中间状态， 且外源 Oct4还未彻底沉默。 红色荧光阳性绿色荧光阴性的细胞群干细胞特异的 多能性分子标记表达量则极低， 而外源 Oct4表达量则极高。 这些实验结果有力的证明了外源沉默是 完全重编程的重要条件。 同时也证明 iCDl培养条件可加速重编程过程。
实例 7 iCDl培养条件下， 第 8天可将小鼠胚胎成纤维细胞完全重编程的实验
能够形成嵌合鼠是小鼠胚胎干细胞的一个十分关键的标准特征。为了更严格的验证得到重编程克 隆与胚胎干细胞没有差别，通过不同方法挑选出的重编程细胞被用于构建嵌合鼠。具体方法见图 13a: 用三因子（ SOX,KIF4和 OCT4 )和 DS-RED混合感染 Oct4-GFP转基因小鼠胚胎成纤维细胞，感染后 培养 iCDl培养基中， 在感染后的 D8，D11，D14天， 分别用机选和手选的方法挑选细胞构建嵌合鼠。 机选的方式为， 将原孔细胞消化， 离心， 采用流式细胞方法分选出 GFP阳性， DS-RED阴性的细胞 来构建嵌合鼠。 手选方法为： 在荧光显微镜下， P遣机将克隆剥落去贴壁， 使克隆悬浮于培养基中， 剥 取一定量后， 将包含有克隆团的培养基收集到 15ml离心管中， 离心收集克隆团， 用 PBS清洗后， 离 心去上清， 加入 200ul0.25%胰蛋白酶， 37°C孵育 3-5min，血清终止后， 离心， 用 mES培养基重悬， 然 后按常规打嵌合鼠操作构建嵌合鼠。 图 13b为不同诱导天数实施的 a图所述实验的统计表。 图 13c为 由第 8天得到的 iPSC细胞注射嚢胚产生的嵌合体小鼠。 该结果有力的证明了， 在 iCDl条件下， 重 编程可在第 8天完成。
实施例 8: 本发明的培养基在少因子情况下对重编程的影响
在 iCDl培养条件下， SKO三个转录因子极高的重编程效率预示着更少的转录因子也有可能重 编程成体细胞。基于这一猜想不同的病毒组合 (ok/os/sk)被用来感染小鼠成纤维细胞， 具体方法是： 将 OCT4, KIF4病毒以 1 : 1混合（每种 0.5ml )感染十二孔板的个孔共计两万个细胞， 用 OCT4, SOX2 病毒以 1 : 1混合（每种 0.5ml )感染十二孔板的一个孔共计三万个细胞， 将 KIF和 Sox2病毒已 1 :1混 合（每种 0.5ml )感染十二孔板的一个孔共计三万个细胞。 分别将上述三种感染的细胞培养在 iCDl 培养基或常规 mES培养基中， 连续培养观察。 图 14a是在 iCDl培养条件下， OK和 OS重编程体细 胞的原始克隆照片和传代照片。 分别地， 在感染 OCT4, KIF4的 MEF中， 在 iCDl培养体系下培养 的孔在 12-15天内出现 1-6个荧光克隆 /孔， 而在感染 OCT4， SOX2的培养基中， 在 18-25天内将会 出现 1-3个荧光克隆， 而在常规 mES培养条件下， 则无任何克隆。。 在 Kif4和 Sox2感染的孔中， 无 论是 iCDl还是在 mES培养条件， 25天内均无任何重编程克隆出现。 图 14b是 OK和 OS重编程获得 的 iPS克隆表达干细胞特意性分子标记。 挑取的 0K和 OS克隆继代培养在饲养层细胞上， 用免疫荧 光检测发现挑去的克隆均其表达 Nanog和 Rexl等多能性分子标记。 图 14c为 Oct4/Kif4和 Oct4/Sox2 重编程实验结果统计，表中给出了每次实验计算重编程克隆的时间和统计结果，三次试验结果表明试 验具有较好的重复性。 图 14d为 OK和 OS克隆注射棵鼠长出的畸胎瘤组织切边， 组织切片中明显的 三个胚层的组织分表明 OK和 OS克隆具备向三个胚层细胞分化的潜能。
实例 9在 iCDl培养条件下, Oct4可使小鼠胚胎成纤维细胞重编程
OK和 OS在 iCDl中均能产生重编程克隆的事实表明 Oct4在重编程过程中起关键作用，那么 Oct4 单独能否在 iCDl中产生重编程克隆就值得尝试了。 因此三万 0ct4-GFP转基因小鼠胚胎成纤维细胞 被 Oct4病毒经过两轮感染后， 在 iCDl中培养， 连续培养近 30天后， 仍未见有荧光克隆出现， 将培 养物用胰酶消化传代种植到饲养层细胞上， 用 iCDl继续培养， 在传代后的第 5天左右， 发现有绿色 重编程克隆出现， 如图 15a所示。 将这些 OCT4重编程克隆挑取传代， 釆用免疫荧光的方法， 用抗体 检测其多能性分子标记物的表达， 发现这些克隆均表达多能性分子标记如 nanog,SSEA-l 和 Rexl，结 果如图 15b所示。，用实时荧光定量 PCR检测内源多能性分子标记 0ct4、 nanog、 Dppa3、 Rexl、 Dnmt31 相对于小鼠 Oct4-GFP小鼠胚胎成纤维细胞的表达水平。 图 15C结果也表明这些克隆表达内源 Oct4、 nanog, rexl , Dppa3、 Dnmt31的水平与标准的胚胎千细胞 Rl类似。 图 15d是通过实时荧光定量 PCR 方法分析外源基因的表达状况。 SKO感染后 4天的 Oct4-GFP小鼠胚胎成纤维细胞作为阳性对照。 结 果显示挑取的这些重编程克隆， 外源转录因子均已沉默。 插入鉴定检测结果表明 （图 15e)，挑取的克 隆不是因为病毒污染引起。 图 15f为 0ct4重编程克隆细胞注射嚢胚产生的嵌合体小鼠。 产生的嵌合 体公鼠与白色品系小鼠产下全黑色小鼠， 见图 15f，该结果证明 Oct4重编程克隆为生殖系嵌合。 上述 结果证明严格证明 Oct4重编程细胞与胚胎干细胞类似， 从而证明了在 iCDl培养条件下， Oct4可将 小鼠胚胎成纤维细胞完全重编程。
实施例 10 iCDl培养基在 Oct4-vpl6重编程中的的应用
Oct4-v l6即在 Oct4基因后面连结一段 vpl6序列,它们的联合表达可增强 Oct4的转录功能。 实 验证明 Oct4-vpl6，Sox2，Kif4重编程体细胞的能力比 Oct4，Sox2，Kif4要高。 为了证明在 Oct4-vpl6参与 的重编程过程中, iCDl也优于其他培养条件， Oct4-vpl6,Sox2, if4感染 2万 Oct4-GFP小鼠胚胎成纤 维细胞后， 分别培养在 mES、 mES添加维生素 C和 iCDl培养体系下。 感染后第 10天计算重编程克 隆数， 实验每组重复数 n=3，误差线代表标准偏差。结果见图 16a,该结果发现，在 iCDl培养条件下， 第 10天出现了 2500个重编程克隆， 而在 mES添加维生素 C的孔中则出现了 650个重编程克隆， 而 在 mES条件下只有极少数克隆。 图 16b是三种培养条件下的荧光克隆照片。 同样， 我们还检测了在 iCDl条件下， Oct4-vpl6, Sox2 以及 Oct4-vpl6, Kif4的重编程能力。 具体方法是： 3万小鼠胚胎成 纤维细胞被 Oct4-vpl6， Sox2 以及 Oct4-vpl6， Kif4病毒分别感染后培养在 iCDl条件下， 在不同天数 计算重编程克隆数。 每个实验组重复数 n=3，误差线代表标准偏差。 图 17a的结果表明， 在 iCDl培养 条件下， Oct4-vpl6, Sox2 以及 Oct4-vpl6, Kif4均能使小鼠胚胎成纤维细胞重编程。 感染后的第 15 天， Oct4-vpl6， Sox2 重编程的效率约为 1%, Oct4-vpl6, if4 重编程效率约为 0.15%。 图 17b 为 Oct4-vpl6/ if4和 Oct4-vpl6/Sox2诱导出的 iPS克隆的典型照片。 采用免疫荧光的方法， 用抗体检测 其多能性分子标记的表达， 发现 Nanog，Oct4,Rexl，SSEA-l等多能性分子标记均有表达，结果如图 17c 所示。 图 17c是由 Oct4-vpl6K和 Oct4-vpl6S诱导出的 iPS细胞表达典型的多能性分子标记。
Oct4在 iCDl条件下能重编程成体干细胞， 但其效率极低， 且需分盘继代培养。 Oct4-vpl6的特 性使得其是否能提升单因子重编程效率变得备受关注。 为了检测这个结果， 用 Oct4-vpl6病毒感染 3 万 Oct4-GFP转基因小鼠胚胎物成纤维细胞，分別在 mES,mES+添加维生素 C及 iCDl培养基中培养。 结果发现在感染后的第 10天左右， 在 iCDl培养基培养的孔中出现绿色重编程克隆。 如图 18a所示。 而在 mES和 mES添加维生素 C的孔中则没有。 感染后的第 17天， 在 iCDl培养的孔内， 每空约有 10个左右重编程克隆， 而在 mES和 mES添加维生素 C的孔中则没有见到明显重编程克。 统计结果 如图 18b。 将挑取的克隆继代培养， 釆用免疫荧光的方法， 用抗体检测其多能性分子标记物的表达， 结果如图 18c所示， 为 Oct4-vpl6诱导出的克隆表达多能性分子标记， )这些挑取的重编程克隆均表 达 Nanog，SSEA-l等多能性分子标记物。 将挑取的克隆注射到嚢胚内， 再植入代孕小鼠子宫， 结果成 功的构建出嵌合体小鼠， 见图 18d。 该结果严格的证明 Oct4-vpl6在 iCDl中得到的重编程细胞与胚 胎干细胞具有相似的多能性。
以上实验有力的证明了在使用 Oct4-vpl6重编程条件下， iCDl培养基与其它培养基相比具有明显 优势。
实施例 11 mES条件下， iCDl核心成分的应用
为了测试 iCDl中的核心成分在血清中的作用， 将 iCDl中的核心成分 (维生素 C, 氯化锂， 碱性 成纤维生长因子， 胰岛素， 糖原合成酶 3-b抑制剂 CHIR99021 )添加到 mES中， 由此得到的培养基 命名为 SmES(Super-mES)。 SKO和普通 Oct4在 SmES中的重编程效果被检测。 mES培养基及 mES 添加维生素 C培养基作为对照。 结果如图 19a所示， 在 SKO感染后的第 8天， 在 SmES培养的孔内 约有 400个左右的克隆， 而在 mES添加维生素 C的孔内有 20个左右重编程克隆。 图 19b为 SmES 培养条件下， 第 8天原孔荧光照片。 同理， SmES对 Oct4单因子在 SmES培养基中的重编程效果也 被测试。 （在 SmES培养体系下， OCT4可重编程体细胞。 Oct4-GFP小鼠胚胎成纤维细胞感染 OCT4 病毒后， 分别在mES,mES十Vitamin C，iCDl培养基， 在不同天数观察荧光克隆数，） 图 19c显示的是 三次独立实验的结果， 该结果表明， 在 SmES条件下， Oct4将小鼠胚胎成纤维细胞重编程。 图 19d 为原始克隆和传代克隆照片。

Claims

权 利 要 求 书
1、 一种培养基添加剂， 其特征在于， 包含维生素 C和糖原合成激酶 3抑制剂。
2、 根据权利要求 1 所述的培养基添加剂， 其特征在于， 所述糖原合成激酶 3抑制剂为锂离子、 Chir99021、 BIO和 SB216763中的至少一种。
3、 根据权利要求 1所述的培养基添加剂， 其特征在于， 所述培养基添加剂还包括维生素 B12、 胰 岛素、 受体酪氨酸激酶和抗氧化剂。
4、 根据权利要求 3所述的培养基添加剂， 其特征在于， 所述受体酪氨酸激酶为碱性成纤维生长因 子 （bFGF )、 表皮生长因子（EGF )、 血管内皮生长因子（VEGF )， 血小板生长因子（ PDGF )、 胰岛素 样生长因子（IGF )和肝生长因子（HGF ) 中的至少一种。
5、 根据权利要求 3所述的培养基添加剂， 其特征在于， 所述抗氧化剂为硫胺、 超氧化物歧化酶、 过氧化氢酶、还原型谷胱甘肽、 维生素 E、 乙酰化维生素 E、 亚油酸、 亚麻酸和亚励酸纳中的至少一种。
6、 根据权利要求 1或 3所述的培养基添加剂， 其特征在于， 所述培养基添加剂还包括代血清细胞 生长促进剂。
7、 根据权利要求 6所述的培养基添加剂， 其特征在于， 所述代血清细胞生长促进剂为清蛋白水解 物、 转铁蛋白、 三碘甲状腺原氨酸、 肾上腺酮、 硫辛酸、 乙醇胺、 黄体酮、 腐胺和维生素 A中的至少一 种。
8、 一种完全培养基， 其特征在于， 由基础培养基、 血清、 代血清添加剂中的一种或多种与权利要 求 1、 3或 6所述的培养基添加剂组成。
9、 一种完全培养基， 其特征在于， 由基础培养基与权利要求 1、 3或 6所述的培养基添加剂组成。
10、 权利要求 1、 3或 6所述的培养基添加剂或权利要求 8或 9所述的完全培养基在诱导多能性干 细胞或真核细胞培养中的应用。
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