WO2015013875A1 - 一种全能干细胞培养方法 - Google Patents

一种全能干细胞培养方法 Download PDF

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WO2015013875A1
WO2015013875A1 PCT/CN2013/080370 CN2013080370W WO2015013875A1 WO 2015013875 A1 WO2015013875 A1 WO 2015013875A1 CN 2013080370 W CN2013080370 W CN 2013080370W WO 2015013875 A1 WO2015013875 A1 WO 2015013875A1
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stem cells
cells
culture dish
totipotent stem
culture
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PCT/CN2013/080370
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English (en)
French (fr)
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韩雪
林慧盈
王猛
刘兴宇
竺亚斌
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杭州微基质生物科技有限公司
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Priority to PCT/CN2013/080370 priority Critical patent/WO2015013875A1/zh
Publication of WO2015013875A1 publication Critical patent/WO2015013875A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
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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
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • ESCs embryonic chimeric cells
  • iPSCs pluripotent thousand cells
  • Thousand cells are cells that are capable of differentiating into multiple cell types. Thousands of cells derived from the embryonic stage and immense thousand-cell iPSCs of embryogenic phase produced by re-induction can differentiate into the vast majority of cell types of biological individuals. These two cells can be infinitely added in vitro to provide basic materials for regenerative medicine research and industry. However, both ESCs and iPSCs are prone to death in cell culture, especially for primates such as human cells. This death occurs especially in the process of cell passage, whether it is in a mechanical block or by enzymatic digestion.
  • matrigel has been used as a plating material in the culture system of embryonic thousand cell ESCs and induced omnipotent thousand cell iPSCs.
  • Matrigel has problems such as uncertain chemical composition and unstable batch quality. These problems will bring many ills to both basic research and clinical use. Therefore, it is necessary to find a substitute for the extracellular matrix that can replace Matrigel's chemical composition and is easy to produce and preserve.
  • the extracellular basement membrane is an extracellular matrix that defines the boundaries of tissues in the body, usually present in the epithelial cell layer, the endothelial cell layer and the lower part of the nerve cells, and has a wide range of physiological functions.
  • the structural basis of the basement membrane is formed by the interaction of different kinds of extracellular matrix proteins to form a network cross-linking structure. The most important of these are laminins and type IV collagen.
  • the basement membrane structures based on these two major proteins are highly conserved and are widely found in invertebrates and vertebrate tissues. In the structure. From the perspective of development, the first generation of the basement membrane of the vertebrate is the embryo sac.
  • ICM formation causes ICM to continue to differentiate into two layers of cells - hypoblast and epiblast.
  • the two layers of cells are separated by a basement membrane and are facing away from each other.
  • Epiblast can be used to derive embryonic thousand cells, and also represents the omnipotent thousand cells in the final period of the individual's natural development.
  • the versatile thousand cells successfully seeded in vitro have completely similar structural characteristics to epithelial cells. A cell with a polar distribution is connected by a seal.
  • the present invention provides a versatile thousand cell culture method.
  • the omnipotent thousand cell culture method includes the following steps:
  • the amino acid sequence of the polypeptide described in the step 1) is NCKHQCTC I DGAVGC I PLCP (SEQ ID NO: 1).
  • the concentration of the polypeptide is 0. 01mg/ml ⁇ 20mg/ml; the preferred concentration is 0. l-10mg / ml, a further more preferred concentration is 0. 2- 5-2 ⁇ / ⁇ Further, a further preferred concentration is 0. 5-2mg / ml.
  • the aqueous solution of the polypeptide is used as an extracellular matrix, and is applied to the surface of the cell culture dish by using a pipette to take 0.1 ml of the aqueous solution of the polypeptide and transferring it into a cell culture dish. Shake, shake well, evenly distributed on the bottom of the culture dish, and then place the cell culture dish covered with the aqueous solution of the polypeptide at 37 ° C ⁇ 1 ° C for 3 to 12 hours.
  • the polypeptide is immobilized on the culture surface by adsorption means such as covalent bond, static electricity, or hydrophobic action.
  • step 2) the following treatment steps are performed before the omnipotent thousand cells are inoculated:
  • the pluripotent thousand cells are detached from the original culture surface using cleavage enzyme or EDTA.
  • the pluripotent thousand cell mass obtained after the treatment was seeded on the surface of the cell culture dish.
  • the feeder-free medium in the step 3) comprises the following components: water, minerals, vitamins, sphagnum fibroblast growth factor, TGF- ⁇ , transferrin, insulin or insulin-like growth factor.
  • the omnipotent cell culture temperature is from 36 ° C to 37. 5 ° C, and the feeder-free medium is replaced once a day, once every 7 days.
  • the omnipotent thousand cells are embryonic thousand cells or induced omnipotent thousand cells.
  • the polypeptide used in the present invention supports the amplification of ESCs/iPSCs in a suitable medium (e.g., mTeSR, B27 medium, or conditioned medium) and has not been studied.
  • a suitable medium e.g., mTeSR, B27 medium, or conditioned medium
  • the polypeptide of the NCKHQCTCIDGAVGCIPLCP (SEQ ID NO: 1) sequence is capable of supporting the expansion of pluripotent thousand cells and maintaining the cell content of 0ct4+ cells at 80% or more. After at least 40 days (6 passages, about 40 cell doubling) culture, the cells still highly express the various markers of pluripotent thousand cells. After the expansion of Oct4, the karyotype remains consistent.
  • the present invention enables the requirements of the basal membrane of the omnipotent thousand cells in the trophoblast-free system to fully meet the clinical use standard, and the substances involved can be artificially large-scale and inexpensive, without artificially containing any animal cells. Synthetic in the environment greatly simplifies the basement membrane composition of the omnipotent thousand cell culture system.
  • Figure 1 shows a clonal morphology map after continuous culture for 30 days on the NCKHQCTCIDGAVGCIPLCP (SEQ ID NO: 1) sequence.
  • the scale is 200um.
  • Figure 2 is a graph showing the comparison of amplification magnifications of embryonic cells in the course of six consecutive passages of the polypeptide of the present invention with and without serum.
  • Figure 3 is a plot of the undifferentiated probability and the amount of ROCK inhibitor added.
  • PSC omnipotent cells
  • a base polypeptide that supports the growth adhesion of ESCs/iPSCs cells was obtained.
  • One or more of these polypeptides have also been shown to support the growth of ESCs/iPSCs and are in an undifferentiated state. Only consecutive times (more than 10 passages) and can Substrate coverings in which the thousand cells are in an undifferentiated state can be defined to form the core polypeptide sequences described in the present invention. This matrix is sufficient to support ESCs/iPSCs in an undifferentiated state forever in the culture medium.
  • 0ct4 and Alkaline phosphatase are proteins specifically expressed by embryonic cells, and in the present invention are multiples for characterization of the undifferentiated state of the thousand cells and the amplification of the quantitative thousand cells, respectively.
  • the expression of the pluripotent thousand cell marker after 5 passages (day 35) was further tested by chemically binding the polypeptides which apparently supported the pluripotent chimeric (PSCs) adhesion amplification to the culture surface.
  • PSCs pluripotent chimeric
  • TeSRl media was compared to the best culture conditions previously (in our case, using conditioning media). It was found that TeSRI medium can maintain human ES cells in such an undifferentiated state that more than 90% of the cells continue to be positive for 0ct4 even after long-term culture. The test results are shown in Figure 3.
  • FIG 3 shows the relationship between the probability of undifferentiation and the amount of ROCK inhibitor added.
  • ROCK inhibitor Y27632 can reduce the dependence of human omnipotent cells on extracellular matrix. We found that l-5 mM Y27632 can further increase the degree of undifferentiation of pluripotent thousand cells on the above polypeptides.
  • Example 1 Growth, passage and differentiation of human embryonic stem cell lines under the present culture system
  • the polypeptide sequence as an extracellular matrix is: NCKHQCTCIDGAVGCIPLCP (SEQ ID NO: 1) having a polypeptide aqueous solution concentration of 1 mg/ml. Pipette 1 mL of the peptide aqueous solution, transfer it into a 10 cm 2 culture dish, shake slowly, shake well, distribute it evenly on the bottom of the culture dish, and then cover the culture dish covered with the aqueous solution of the peptide at 37 ° C ⁇ 1 ° C. Place it for 3 hours to allow sufficient adsorption or reaction.
  • the polypeptide is immobilized on the culture surface by adsorption means such as covalent bond, static electricity, or hydrophobic action.
  • step 2) the human embryonic cells before inoculation are subjected to the following treatment steps: 1) Thawing. Embryonic cells that have been in continuous culture do not need to be thawed. 2) Digestion. The cells are detached from the culture surface using cleavage enzyme or EDTA. It is then inoculated on a new culture surface to which the above polypeptide is attached. The treated embryos are seeded in a Petri dish of the aforementioned step 1).
  • the feeder-free medium in step 3) includes the following components: water, minerals, vitamins, amino
  • the embryonic cell culture environment was 37 ° C, 5% C0 2 incubator. The medium was changed once a day and passaged once every 5-7 days.
  • the cell line CN hESCs8/22 was normal in karyotype on the culture surface of this polypeptide after 5 passages.
  • the above cell lines can form teratomas after 10 days of culture on the surface of the polypeptide, demonstrating that the human embryonic cells that have been cultured for a long time remain versatile.
  • the medium without any feeder cells and without the conditioning medium maintains the undifferentiated growth level of human ES cells to 80% or more of the cells in the culture to maintain the degree of undifferentiation at all stages.
  • the addition of the apoptosis inhibitor Y27632 (2.5 mM) increased the number of undifferentiated (0ct4-positive markers) embryos to more than 90%.
  • Example 2 Growth, passage and differentiation of induced human pluripotent stem cell lines under the present culture system
  • the polypeptide sequence as an extracellular matrix is: NCKHQCTCIDGAVGCIPLCP (SEQ ID NO: 1).
  • the concentration of the aqueous solution of the polypeptide was 2 mg/ml. Pipette 1 mL of the peptide aqueous solution, transfer it into a 10 cm 2 culture dish, shake slowly, shake well, distribute it evenly on the bottom of the culture dish, and then cover the culture dish covered with the aqueous solution of the peptide at 37 ° C ⁇ 1 ° C. Leave it for 12 hours.
  • the polypeptide is immobilized on the culture surface by adsorption means such as covalent bond, static electricity, or hydrophobic action.
  • step 2) the induced pluripotent thousand cells before inoculation are subjected to the following treatment steps: 1) Thawing. The induced pluripotent thousand cells that have been in continuous culture do not need to be thawed. 2) Digestion. The cells are detached from the culture surface using cleavage enzyme or EDTA. It is then inoculated on a new culture surface to which the above polypeptide is attached.
  • the treated induced pluripotent thousand cell pellet was seeded in the Petri dish of the aforementioned step 1).
  • the medium in step 3 including the following components: water, minerals, vitamins, amino acids, glucose, fibroblast growth factor, pipecolic acid, TGF-i5, transferrin, insulin or insulin-like growth factor (insulin-like growth factor).
  • the induction omnipotent thousand cell culture temperature was 37 ° C, and the medium was changed once a day and once every 7 days.
  • the cell line MMHPS-iPSCs-001 had a normal karyotype after 5 passages of the culture system. It was confirmed that the cell line MMHPS-iPSCs-001 formed a teratoma after 6 passages.
  • a medium that does not contain any feeder cells and does not contain a conditioned medium maintains the undifferentiated growth level of human ES cells to the extent that more than 80% of the cells in the culture maintain undifferentiation at all stages.
  • the addition of the apoptosis inhibitor Y27632 (2.5 mM) increased the number of undifferentiated (using 0ct4 positive marker) iPSCs to more than 85%.

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Abstract

提供了一种全能干细胞的培养方法。用人工合成的多肽作为细胞外基质，将其铺于细胞培养皿表面，将全能干细胞接种在前述培养皿的表面，注入无饲养层培养基，再将接种后的全能干细胞培养传代。提供的方法能够支持全能干细胞的扩增，并保持Oct4+细胞的细胞含量始终维持在80%以上。经过至少40天培养，即，传代6次，约40次细胞翻倍后，细胞仍高度表达全能干细胞的各种标记物，且扩增培养后，核型仍然保持一致。

Description

一种全能干细胞培养方法 技术领域
本发明的目的在于提供一种全能千细胞培养的方法， 特别是提供了一种胚胎 千细胞 （ESCs ) 或者诱导全能千细胞 （iPSCs ) 的培养方法。
冃 不
千细胞是能够分化为多种细胞类型的细胞。 来自于胚胎期的千细胞和通过再 诱导方式产生的类似胚胎期的全能千细胞 iPSCs , 能够分化成为生物个体绝大部 分细胞类型。 这两种细胞可以在体外培养中无限增值， 为再生医学研究和产业提 供基础材料。 然而， 无论是 ESCs还是 iPSCs在细胞培养中都容易死亡， 尤其是灵 长类动物如人的千细胞。 而这种死亡尤其发生在细胞的传代的过程中， 无论传代 是以机械块状的方式还是通过酶消化分散的方式， 都难以避免。
另一方面， 在胚胎千细胞 ESCs和诱导产生的全能千细胞 iPSCs的培养系统中 一直使用基质胶 （Matrigel ) 作为作为铺板材料。 但是 Matrigel具有化学成分不 确定， 批次质量不稳定等问题。 这些问题对于无论是基础科研和临床使用都将带 来很多弊病。 因此有必要寻找一种能够替代 Matrigel的化学组分确定， 且易于生 产保存的细胞外基质的替代品。
Nature Biotechnology描述了采用重组 laminin ( Nat Biotechnol. 2010 Jun ; 28 (6) : 611-5 ) 和采用从 Vitronectin提取的多肽 （Nat Biotechnol. 2010 Jun ; 28 (6) : 606-10. ) 来代替 Matrigel用于以上描述的千细胞培养的方式。 但是 重组 laminin成本仍然极其高昂； 而来源于 Vitronectin的两种测试的多肽被提 示能够特异性的提供整合素 alpha V beta 3的粘合， 从而使千细胞生长。
细胞外基底膜是定义体内组织边界的细胞外基质， 通常存在于上皮细胞层， 内皮细胞层和神经细胞的下部， 具有广泛的生理学功能。 基底膜的结构基础是由 不同种类的细胞外基质蛋白相互作用形成网状交联结构而形成。 其中最重要的为 层粘连蛋白 （laminins ) 和四型胶原蛋白 （type IV collagen ) ， 以这两种主要 蛋白为基础的基底膜结构是高度保守的， 广泛地存在于无脊椎动物和脊椎动物组 织结构中。 从发育学来看， 脊椎动物的个体发育第一次形成基底膜是在胚囊 ( blastocyst) 期的内细胞群 （inner cell mass ， ICM)中， ICM的形成使得 ICM 继续分化成两层细胞 -内胚层 （hypoblast ) 和外胚层 （epiblast ) 。 这两层细 胞以基底膜隔开， 背向相对。 Epiblast 可以用于衍生出胚胎千细胞， 也代表了在 个体的自然发育过程中的最后时期的全能千细胞。 成功接种在体外的全能千细胞 完全具有和上皮细胞高度相似的结构特性一具有极性分布的细胞通过密封连接
( tight junction) 连接起来。
发明内容
为了克服现有技术的不足， 本发明提供了全能千细胞培养方法。
全能千细胞培养方法， 包括以下步骤：
1 ) 用人工合成的包含氨基酸序列为 NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) 的 多肽水溶液作为细胞外基质， 铺于细胞培养皿表面；
2 ) 将全能千细胞接种于所述的细胞培养皿表面；
3 ) 加入无饲养层培养基， 进行全能千细胞的培养传代。
作为本发明的一种优选方案， 步骤 1 ) 中所述的多肽的氨基酸序列为 NCKHQCTC I DGAVGC I PLCP ( SEQ ID NO: 1 ) 。
作为本发明的另一种优选方案， 所述的多肽水溶液浓度为 0. 01mg/ml〜 20mg/ml ; 优选的浓度为 0. l-10mg/ml ， 进一步更为优选的浓度为 0. 2-5mg/ml， 再进一步更为优选的浓度为 0. 5-2mg/ml。
作为本发明的又一种优选方案， 所述的多肽水溶液作为细胞外基质， 铺于细 胞培养皿表面方法为， 用移液枪吸取 0. lmL的所述的多肽水溶液， 移入细胞培养 皿中， 晃动， 摇匀， 均匀分布于培养皿底部， 然后将所述的多肽水溶液覆盖的细 胞培养皿在 37°C ± 1 °C下放置 3〜12小时。 多肽通过共价键、 静电、 疏水作用等 吸附方式固定于培养用表面上。
作为本发明的进一步的优选方案， 步骤 2 ) 中， 全能千细胞接种前经过以下 处理步骤：
1 ) 解冻； 或已经在连续的培养之中的全能千细胞不需要解冻；
2 ) 使用剪切酶或 EDTA使全能千细胞脱离原培养表面。 处理后得到的全能千 细胞团接种于所述的细胞培养皿表面。
作为本发明的再一个的优选方案， 步骤 3 ) 中的所述的无饲养层培养基， 包 括以下组分： 水、 矿物质、 维生素、 氨茅 成纤维细胞生长因子、 TGF- β , 转铁蛋白、 胰岛素或胰岛素样生长因子。
优选的， 全能千细胞培养温度为 36°C〜37. 5 °C， 每天更换所述的无饲养层培 养基 1次， 7天传代一次。
优选的， 所述的全能千细胞是胚胎千细胞或诱导全能千细胞。
本发明中的使用的多肽在适当培养基 （如 mTeSR, B27培养基， 或者条件培养 基） 中支持 ESCs/iPSCs的扩增没有被研究过。 我们发现， 以上以
NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) 序列的多肽能够支持全能千细胞的扩增 并保持 0ct4+细胞细胞含量始终维持在 80%以上。 经过至少 40天 （6次传代， 约 40次细胞翻倍 doubling) 培养， 细胞仍高度表达全能千细胞的各种标记物 0ct4 扩增培养后， 核型仍然保持一致。
本发明的有益效果： 本发明使得在无滋养层系统中全能千细胞对于基底膜的 要求可以完全达到临床使用标准， 并且所涉及的物质可大规模廉价的通过人工方 式在不含有任何动物细胞的环境中合成， 极大的简化了全能千细胞培养系统的基 底膜成分。
附图说明
下面结合附图和实施例对本发明进一步说明。
图 1展示了在 NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) 序列上连续培养 30天以 后的克隆形态图。 图中， 比例尺为 200um。
图 2展示了本发明的多肽在和无血清下 Matrigel连续 6次传代过程中， 胚胎千细 胞的扩增倍数的比较图。
图 3是未分化概率和加入 ROCK inhibitor量的关系图。
具体实施方式
传统的培养胚胎千细胞 ESCs或者 iPSCs (统称 PSC， 全能千细胞） 培养方法 需要成纤维饲养细胞或者接触成纤维细胞饲养细胞的培养基以将千细胞维持在未 分化状态。 现在我们用可以促进细胞黏附的多肽的用于全能千细胞的培养， 全能 千细胞能在多次传代中无限期的维持未分化状态。
在系统地测试了超过 200种多肽片段后， 得到了可以支持 ESCs/iPSCs细胞生 长粘附的基底多肽。 这些多肽的一种或多种 （包含衍生物） 也被证明可以支持 ESCs/iPSCs的生长， 并处于未分化状态。 只有连续多次 （超过 10次传代） 并能 继续千细胞处于未分化状态的基底覆盖物才能定义形成本发明中所描述的核心多 肽序列。 这种基质足以支持 ESCs/iPSCs在培养基中永远处于未分化状态。 这些培 养基底己被证实能支持未分化的 ES细胞增殖多代， 至少 6代， 这是这些培养基底 将支持这种无限期地培养的有力证据。 这些数据中的一些示于图 1和 2。 0ct4和 Alkaline phosphatase是胚胎千细胞特异表达的蛋白， 在本发明中是分别用于表 征千细胞未分化状态和定量千细胞的扩增的倍数。
将以上能明显支持全能千细胞 （PSCs) 粘附扩增的多肽通过化学键合到培养 表面以后， 进一步测试了传代 5次后 （第 35天） 的全能千细胞标记物的表达。
为检测 ES细胞集落的条件和长期维持人 ES细胞培养物的培养适用性， 将 TeSRl 培养基与之前最好的培养条件 (就我们掌握的情况是使用调理培养基)进行 比较。 发现 TeSRI培养基能够维持人 ES细胞处于这样一种未分化状态， 即超过 90%的细胞即使在长期培养后仍继续呈 0ct4阳性。 该检测结果示于图 3。
在传代维持过程中， 类似在其他培养体系下一样， 明显分化的克隆被人工剔 除。
图 3是未分化概率和加入 ROCK inhibitor量的关系。 ROCK inhibitor Y27632 能够降低人类全能千细胞对于细胞外基质的依赖性。 我们发现 l-5mM的 Y27632可 以进一步提高全能千细胞在以上多肽物质上的未分化程度。
实施例 1 : 人类胚胎干细胞系在本培养体系下的生长， 传代和分化
作为细胞外基质的多肽序列为： NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) ， 其多肽水溶液浓度为 lmg/ml。 用移液枪吸取 lmL的多肽水溶液， 移入面积为 10cm2培养皿中， 缓慢晃动， 摇匀， 使之均匀分布于培养皿底部， 然后将多肽水溶 液覆盖的培养皿在 37°C ± 1 °C下放置 3小时使其能够充分的吸附或者反应完毕。 多肽通过共价键、 静电、 疏水作用等吸附方式固定于培养用表面上。
步骤 2 ) 中， 接种前的人类胚胎千细胞经过以下处理步骤： 1 ) 解冻。 已经在 连续的培养之中的胚胎千细胞不需要解冻。 2 ) 消化。 使用剪切酶或 EDTA使细胞 脱离培养表面。 然后接种在新的在附着有上述多肽的培养表面上。 处理后的胚胎 千细胞团接种于前述步骤 1 ) 的培养皿中。
步骤 3 ) 中的无饲养层培养基， 包括以下组分： 水、 矿物质、 维生素、 氨基
4 酸、 葡萄糖、 成纤维细胞生长因子、 2-哌啶酸、 TGF- i5、 转铁蛋白、 胰岛素或胰 岛素样生长因子 （insulin-like growth factor) 。 胚胎千细胞培养环境为 37 °C， 5% C02的恒温箱。 每天更换培养基 1次， 5-7天传代一次。
证实细胞系 CN hESCs8/22在 5代之后在此多肽的培养表面上核型正常。 以上 细胞系在经过 10代在多肽表面培养以后， 可形成畸胎瘤， 证明经过长期培养的人 类胚胎千细胞仍然保持全能性。 不含任何饲养层细胞且不含调理培养基的培养基 能维持人 ES细胞的未分化生长水平至培养中 80%以上的的细胞在所有阶段维持未 分化的程度。 凋亡抑制剂 Y27632的加入 ( 2. 5mM) 使未分化 (用 0ct4阳性标记) 胚胎千细胞数量提高到 90%以上。
实施例 2: 诱导的人类全能干细胞系在本培养体系下的生长， 传代和分化
作为细胞外基质的多肽序列为： NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) 。 其多肽水溶液浓度为 2mg/ml。 用移液枪吸取 lmL的多肽水溶液， 移入面积为 10cm2培养皿中， 缓慢晃动， 摇匀， 使之均匀分布于培养皿底部， 然后将多肽水溶 液覆盖的培养皿在 37°C ± 1 °C下放置 12小时。 多肽通过共价键、 静电、 疏水作用 等吸附方式固定于培养用表面上。
步骤 2 ) 中， 接种前的诱导全能千细胞经过以下处理步骤： 1 ) 解冻。 已经在 连续的培养之中的诱导全能千细胞不需要解冻。 2 ) 消化。 使用剪切酶或 EDTA使 细胞脱离培养表面。 然后接种在新的在附着有上述多肽的培养表面上。
处理后的诱导全能千细胞团接种于前述步骤 1 ) 的培养皿中。
步骤 3 ) 中的培养基， 包括以下组分： 水、 矿物质、 维生素、 氨基酸、 葡萄糖、 成纤维细胞生长因子、 2-哌啶酸、 TGF- i5、 转铁蛋白、 胰岛素或胰岛素样生长因 子 （insulin-like growth factor) 。 诱导全能千细胞培养温度为 37°C， 每天更 换培养基 1次， 7天传代一次。
证实细胞系 MMHPS-iPSCs-001在此培养体系扩增 5代之后核型正常。 证实细 胞系 MMHPS-iPSCs-001在 6代后形成畸胎瘤。 不含任何饲养层细胞且不含调理培 养基的培养基能维持人 ES细胞的未分化生长水平至培养中 80%以上的的细胞在所 有阶段维持未分化的程度。 凋亡抑制剂 Y27632的加入 （2. 5mM) 使未分化 （用 0ct4阳性标记） iPSCs千细胞数量提高到 85%以上。

Claims

权 利 要 求 书
1、 一种全能干细胞培养方法， 包括以下步骤：
1 ) 用人工合成的包含氨基酸序列为 NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) 的 多肽水溶液作为细胞外基质， 铺于细胞培养皿表面；
2 ) 将全能干细胞接种于所述的细胞培养皿表面；
3 ) 加入无饲养层培养基， 进行全能干细胞的培养传代。
2、 如权利要求 1所述的方法， 其特征在于， 步骤 1 ) 中所述的多肽的氨基酸序列 为 NCKHQCTCIDGAVGCIPLCP ( SEQ ID NO: 1 ) 。
3、 如权利要求 1 所述的方法， 其特征在于， 所述的多肽水溶液浓度为 0. 01mg/ml〜20mg/ml ; 优选的浓度为 0. 1-lOmg/ml ， 进一步更为优选的浓度为 0. 2- 5mg/ml， 再进一步更为优选的浓度为 0. 5- 2mg/ml。
4、 如权利要求 1所述的方法， 其特征在于， 所述的多肽水溶液作为细胞外基质， 铺于细胞培养皿表面的方法为， 用移液枪吸取 0. lmL 的所述的多肽水溶液， 移入 细胞培养皿中， 晃动， 摇匀， 均匀分布于培养皿底部， 然后将所述的多肽水溶液 覆盖的细胞培养皿在 37°C ± 1 °C下放置 3〜12小时。
5、 如权利要求 1所述的方法， 其特征在于， 步骤 2 ) 中， 全能干细胞接种前经过 以下处理步骤：
1 ) 解冻； 或已经在连续的培养之中的全能干细胞不需要解冻；
2 ) 使用剪切酶或 EDTA使全能干细胞脱离原培养表面。
6、 如权利要求 1 所述的方法， 其特征在于， 步骤 3 ) 中的所述的无饲养层培养 基， 包括以下组分： 水、 矿物质、 维生素、 氨基酸、 葡萄糖、 成纤维细胞生长因 子、 TGF- i5、 转铁蛋白、 胰岛素或胰岛素样生长因子。
7、 如权利要求 6 所述的方法， 其特征在于， 全能干细胞培养温度为 36°C〜37. 5 °C， 每天更换所述的无饲养层培养基 1次， 7天传代一次。
8、 如权利要求 1所述的方法， 其特征在于， 所述的全能干细胞是胚胎干细胞或诱 导全能干细胞。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11080706B2 (en) 2016-03-07 2021-08-03 International Business Machines Corporation Blocking fraudulent transactions in an NFC device

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1222193A (zh) * 1996-03-15 1999-07-07 穆尼恩公司 胞外基质信号分子
CN101008002A (zh) * 2006-01-28 2007-08-01 肖磊 Activin A 用于人胚胎干细胞无滋养层细胞培养的应用
CN102586176A (zh) * 2012-01-11 2012-07-18 中国科学院生物物理研究所 一种新型的无动物源、无饲养层的人多能干细胞培养系统

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1222193A (zh) * 1996-03-15 1999-07-07 穆尼恩公司 胞外基质信号分子
CN101008002A (zh) * 2006-01-28 2007-08-01 肖磊 Activin A 用于人胚胎干细胞无滋养层细胞培养的应用
CN102586176A (zh) * 2012-01-11 2012-07-18 中国科学院生物物理研究所 一种新型的无动物源、无饲养层的人多能干细胞培养系统

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN, GUOHUI ET AL.: "Progress of Cyr61 and Breast Cancer.", MEDICAL RECAPITULATE, vol. 13, no. 5, 31 March 2007 (2007-03-31) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11080706B2 (en) 2016-03-07 2021-08-03 International Business Machines Corporation Blocking fraudulent transactions in an NFC device

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