WO2007058401A1 - Composition comprenant de l'acide okadaique pour la proliferation indifferenciee de cellules souches embryonnaires - Google Patents

Composition comprenant de l'acide okadaique pour la proliferation indifferenciee de cellules souches embryonnaires Download PDF

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Publication number
WO2007058401A1
WO2007058401A1 PCT/KR2005/003925 KR2005003925W WO2007058401A1 WO 2007058401 A1 WO2007058401 A1 WO 2007058401A1 KR 2005003925 W KR2005003925 W KR 2005003925W WO 2007058401 A1 WO2007058401 A1 WO 2007058401A1
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WIPO (PCT)
Prior art keywords
stem cells
embryonic stem
okadaic acid
feeder
undifferentiated
Prior art date
Application number
PCT/KR2005/003925
Other languages
English (en)
Inventor
Seungkwon You
Byung Sun Yoon
Ki Dong Kim
Eun Kyung Jun
Jai Hee Moon
Bona Kim
Gyuman Park
Seung Jun Yoo
Sung Sik Kwak
Issac Maeng
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Imgen Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Imgen Co., Ltd. filed Critical Imgen Co., Ltd.
Priority to JP2008529901A priority Critical patent/JP2009506785A/ja
Priority to EP05823886A priority patent/EP1948787A4/fr
Priority to CNA2005800496608A priority patent/CN101180388A/zh
Priority to PCT/KR2005/003925 priority patent/WO2007058401A1/fr
Publication of WO2007058401A1 publication Critical patent/WO2007058401A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates to a culture medium composition comprising okadaic acid for use in the undifferentiated proliferation of human embryonic stem cells, and to a method of inducing the undifferentiated proliferation of human embryonic stem cells in the culture medium composition.
  • hESCs Human embryonic stem cells
  • hESCs have been used as a source of cells for therapeutic treatment of diseases that had been regarded as incurable.
  • MEF mouse embryonic fibroblast
  • Another object of the present invention is to provide a method for inducing the undifferentiated proliferation of embryonic stem cells in the culture medium composition.
  • FIG. 1 shows the effects of okadaic acid on embryonic stem cells grown in a feeder-free condition.
  • Okadaic acid was added in an amount of 1 nM (A, B) and 0.1 nM (C), and was not added (D) .
  • FIG. 2 shows human embryonic stem cells grown on a feeder cell layer (A) and in the absence of feeder cells (B- D).
  • FIG. 3 shows human embryonic stem cells grown on a feeder cell layer in a bFGF-free culture system (A) and in a bFGF-free culture system containing 1 nM okadaic acid (B) .
  • FIG. 4 shows that the human embryonic stem cells grown in a feeder-free condition according to the present invention have the normal karyotype.
  • FIG. 5 shows that the human embryonic stem cells grown in a feeder-free condition according to the present invention have undifferentiated markers expressed thereon.
  • the present invention is directed to a culture medium composition comprising okadaic acid capable of inducing the undifferentiated proliferation of embryonic stem cells.
  • embryonic stem cell means a cell from an embryo which has the potential to proliferate while maintaining its pluripotency.
  • embryonic stem cells derived from any animal including humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rats, etc., with preference for mammals, and with further preference for humans .
  • proliferation means an increase in cell number, having the same meaning as “growth”.
  • undifferentiated proliferation it is meant that embryonic stem cells proliferate not into specific cells but into cells having the same properties as the embryonic stem cells, that is, into pluripotent cells. It will be obvious to practitioners of the art that undifferentiated cells can be readily discerned from differentiated cells. For instance, undifferentiated cells feature a high ratio of nucleus to cytoplasm and prominent nucleoli.
  • embryonic stem cells can proliferate in an undifferentiated state in a culture medium free of feeder cells in the presence of okadaic acid.
  • okadaic acid is known to inhibit the degradation of and increase the stability of c-myc (Yeh E et al. Nat Cell Biol 2004, 3:118).
  • okadaic acid is known to inhibit the degradation of and increase the stability of c-myc (Yeh E et al. Nat Cell Biol 2004, 3:118).
  • embryonic stem cells were proven to remain undifferentiated even after 15 passages in a culture medium containing okadaic acid through the detection of all of the undifferentiated ES markers nanog, oct-4, rexl and Tert, and the observation of a normal karyotype.
  • okadaic acid allows embryonic stem cells to undergo stable undifferentiated proliferation even in the absence of feeder cells.
  • okadaic acid can function in the place of the growth factor bFGF, which is typically used for the proliferation of embryonics stem cells. Therefore, okadaic acid can be used instead of or in combination with typical differentiation suppressors, such as bFGF, for conventional embryonic stem cell cultures employing feeder cells and matrices .
  • culture media means media which assure the growth and survival of stem cells in vitro, and which may include all of the pertinent media typically used in the art.
  • the culture media and conditions depend on the kind of stem cells.
  • a cell culture minimum medium CCMM
  • CCMM cell culture minimum medium
  • examples of the CCMM include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, ⁇ MEM ( ⁇ Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium.
  • an antibiotic such as penicillin, streptomycin, gentamicin, etc., may be added.
  • okadaic acid is added to an embryonic stem cell culture medium without restriction to as to medium kind or culture type.
  • okadaic acid may be used alone or in combination with one or more differentiation suppressors.
  • the culture medium must comprise an effective concentration of okadaic acid.
  • effective concentration means an amount sufficient for okadaic acid to induce the undifferentiated proliferation of okadaic acid. Because it shows no desirable effects at a concentration lower than the effective concentration and cytotoxicity at a concentration higher than the effective concentration, okadaic acid should be used within the effective concentration range. Factors determining the effective concentration of okadaic acid include the origin of the embryonic stem cells, the kind of culture media, the components of the culture media, differentiation suppressors used together therewith, etc. For instance, when okadaic acid is used as a sole differentiation suppressor in the culture of human embryonic stem cells, its effective concentration may be on the order of 0.5 to 1.5 nM.
  • the present invention is directed to a method for inducing the undifferentiated proliferation of embryonic stem cells, comprising culturing them in a culture medium containing okadaic acid.
  • embryonic stem cells can proliferate without spontaneous differentiation and remain undifferentiated until a desired time point.
  • Embryonic stem cells may be obtained using conventional methods. For instance, the preparation of human embryonic stem cells may be achieved according to the method of Thomson (U. S. Pat No.5843780; Science 282; 1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995).
  • the culture medium containing okadaic acid in accordance with the present invention assures that embryonic stem cells undergo undifferentiated proliferation without being contaminated by pathogens from the xenogeneic feeders, such as those from viruses and animals.
  • the hESC line HSF- ⁇ (University of California, San Francisco) was cultured while a 13.5 day-old CFl mouse fetus (ORIENT) was used as a feeder cell.
  • the feeder cell was incubated in a DMEM (high glucose, Invitrogen) supplemented with 10% FBS (HyClone) , 0.1 mM ⁇ -mercaptoethanol, and 0.1 mM non-essential amino acids and then treated with 10 ⁇ g/ml of mitomycin C (sigma) for 1.5 hrs to terminate the mitosis thereof.
  • DMEM high glucose, Invitrogen
  • FBS HyClone
  • 0.1 mM ⁇ -mercaptoethanol 0.1 mM non-essential amino acids
  • the feeder cell treated with mitomycin C was seeded in a 0.1% gelatin-coated four-well culture dish at a density of ⁇ .lxlO 4 cells per well.
  • the hESC was seeded, and subcultured at time intervals of 5-7 days in a mechanical manner .
  • the hESC was cultured in a Dulbecco's modified Eagle's medium/F12 (DMEM/F12, without pyruvate) supplemented with 20% knockout serum replacement (SR) (Gibco/BRL, Invitrogen, Carlsbad, CA), 0.1 mM ⁇ -mercaptoenthanol, 1% non-essential amino acids (Gibco/BRL) , 100 U/ml penicillin G, 100 g/ml streptomycin, and 4 ng/ml hr-bFGF (human recombinant basic fibroblast growth factor; Invitrogen) .
  • SR knockout serum replacement
  • FIG. 2A the hESC on a CFl mouse feeder cell layer is shown.
  • EXAPLE 2 Effect of Okadaic Acid on Human Embryonic Stem Cell
  • okadaic acid 0.1, 1, 10, 100 nM of okadaic acid in human embryonic stem cell culture media.
  • both the embryonic stem cells and the feeder cells underwent lysis in the culture media as a result of the cytotoxicity of okadaic acid.
  • EXAMPLE 3 Effect of Okadaic Acid on Human Embryonic Stem Cells in Feeder-Free Condition The effect of okadaic acid on hESCs was evaluated in the absence of feeder cells. 0.01 nM, 0.1 nM, and 1 nM of okadaic acid were added to culture systems using no CM. Without using matrices such as laminin and matrigel, in contrast to a previous culture system using such matrices, a four-well culture dish was coated at room temperature for 30 min with 0.1% gelatin, after which the embryonic stem cells were mechanically split. Thereafter, the embryonic stem cells were seeded in culture media containing 0.01 nM, 0.1 nM, and InM okadaic acid.
  • matrices such as laminin and matrigel
  • FIG. ID the human embryonic stem cells in the 1 mM okadaic acid culture media remained undifferentiated even after three passages (FIG. 2B-D) , without any sign of differentiation. It was also observed that the embryonic stem cells on the feeder cell layers had high ratios of nucleus to cytoplasm (FIG. 2D) .
  • EXAMPLE 4 Effect of Okadaic Acid on Embryonic Stem Cells Grown on Feeder Cell Layers in the Absence of bFGF
  • bFGF is usually used at a concentration of 4 ng/ml for the proliferation of undifferentiated embryonic stem cells.
  • Embryonic stem cells were analyzed for proliferation and differentiation in culture media containing 1 nM okadaic acid in the absence of bFGF.
  • culture media containing neither okadaic acid nor bFGF all of the embryonic stem cells grown on feeder cell layers were observed to differentiate after two or more passages (FIG. 3A) .
  • FGF bFGF
  • EXAMPLE 5 Expression of ES Marker on hESCs and Karyotype of hESCs in Feeder-Free Condition
  • RNA Reverse-transcription (RT) was carried out using 500 ng of the total RNA, oligo(dT) 12-18 mer primers
  • RNA samples 1 ⁇ l of cDNA, 10 pmol of each primer, and a PCR premix (IU Tag DNA polymerase, 250 ⁇ M dNTPs, 10 mM Tris-HCl, 40 mM KCl and 1.5 mM MgC12 Bioneer, Korea) were used.
  • IU Tag DNA polymerase 250 ⁇ M dNTPs, 10 mM Tris-HCl, 40 mM KCl and 1.5 mM MgC12 Bioneer, Korea
  • Oligonucleotides specific for undifferentiated ES markers nanog SEQ ID NOS.: 1 and 2), oct-4 (SEQ ID NOS.: 3 and 4) and rexl (SEQ ID NOS.: 5 and 6) and for hTert (SEQ ID NOS.: 7 and 8), expressed in ES, were used as primers in the RT-PCR.
  • Primers for ⁇ -actin SEQ ID NOS.: 9 and 10.
  • cDNA it was obtained from hESCs grown in the absence of feeder cells for 5, 10 and 15 passages, while hESCs grown on feeder cell layers were used as a positive control.
  • okadaic acid allows the hESCs grown in a feeder-free culture system in the presence of okadaic acid to have the same gene expression behavior and karyotype as in those grown on feeder cell layers (FIG. 4) .
  • the culture medium composition comprising okadaic acid in accordance with the present invention allows the undifferentiated proliferation of hESCs without the risk of cross-transfer of pathogens from xenogeneic feeders, such as those from viruses and other animals.

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Abstract

L'invention concerne une composition de milieu de culture comprenant de l'acide okadaïque destinée à être utilisée pour la prolifération indifférenciée de cellules souches embryonnaires humaines, ainsi qu'une méthode pour induire la prolifération indifférenciée de cellules souches embryonnaires humaines dans le milieu de culture.
PCT/KR2005/003925 2005-11-18 2005-11-18 Composition comprenant de l'acide okadaique pour la proliferation indifferenciee de cellules souches embryonnaires WO2007058401A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2008529901A JP2009506785A (ja) 2005-11-18 2005-11-18 胚芽幹細胞の未分化増殖を誘導するオカダ酸含有培地組成物
EP05823886A EP1948787A4 (fr) 2005-11-18 2005-11-18 Composition comprenant de l'acide okadaique pour la proliferation indifferenciee de cellules souches embryonnaires
CNA2005800496608A CN101180388A (zh) 2005-11-18 2005-11-18 用于胚胎干细胞未分化增殖的包括大田软海绵酸的组合物
PCT/KR2005/003925 WO2007058401A1 (fr) 2005-11-18 2005-11-18 Composition comprenant de l'acide okadaique pour la proliferation indifferenciee de cellules souches embryonnaires

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PCT/KR2005/003925 WO2007058401A1 (fr) 2005-11-18 2005-11-18 Composition comprenant de l'acide okadaique pour la proliferation indifferenciee de cellules souches embryonnaires

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KR101135636B1 (ko) * 2009-10-27 2012-04-17 서울대학교산학협력단 인간 만능줄기세포로부터 중배엽 줄기세포를 생산하는 방법 및 이에 의해 생성된 중배엽 줄기세포
EP2651973B1 (fr) * 2010-12-17 2016-08-31 Biolamina AB Laminine-521 recombinante

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2002051997A1 (fr) * 2000-12-22 2002-07-04 Aurox Llc Methodes de clonage de mammiferes a l'aide de chromatine donneuse reprogrammee ou de cellules donneuses reprogrammees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002051997A1 (fr) * 2000-12-22 2002-07-04 Aurox Llc Methodes de clonage de mammiferes a l'aide de chromatine donneuse reprogrammee ou de cellules donneuses reprogrammees

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
See also references of EP1948787A4 *
SONG H. ET AL.: "Transfection of mesenchymal stem cells with the FGF-2 gene improves their survival under hypoxic conditions", MOL. CELLS, vol. 19, no. 3, 2005, pages 402 - 407 *
XU C. ET AL.: "Basic fibroblast growth supports undifferentiated human embryonic stem cell growth without conditioned medium", STEM CELLS, vol. 23, no. 3, June 2005 (2005-06-01), pages 315 - 323, XP002409422 *
XU R.H. ET AL.: "Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells", NAT. METHODS, vol. 2, no. 3, March 2005 (2005-03-01), pages 185 - 190, XP002340999, DOI: doi:10.1038/nmeth744 *
YOU J, BIRD R.C.: "Selective induction of cell cycle regulatory genes cdk 1(p34cdc2), cyclins A/B, and the tumor suppressor gene Rb in transformed cells by okadaic acid", J. CELL PHYSIOL., vol. 164, no. 2, August 1995 (1995-08-01), pages 424 - 433 *

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EP1948787A1 (fr) 2008-07-30
JP2009506785A (ja) 2009-02-19
EP1948787A4 (fr) 2008-12-31
CN101180388A (zh) 2008-05-14

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