WO2006029197A1 - Medium and culture of embryonic stem cells - Google Patents

Medium and culture of embryonic stem cells Download PDF

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Publication number
WO2006029197A1
WO2006029197A1 PCT/US2005/031838 US2005031838W WO2006029197A1 WO 2006029197 A1 WO2006029197 A1 WO 2006029197A1 US 2005031838 W US2005031838 W US 2005031838W WO 2006029197 A1 WO2006029197 A1 WO 2006029197A1
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Prior art keywords
medium
cells
stem cells
culture
human
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Ceased
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PCT/US2005/031838
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English (en)
French (fr)
Inventor
James A. Thomson
Tenneille Ludwing
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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Filing date
Publication date
Priority to HK08103385.9A priority Critical patent/HK1113171B/xx
Priority to CN2005800299366A priority patent/CN101044235B/zh
Priority to ES05793904T priority patent/ES2383813T3/es
Priority to NZ553241A priority patent/NZ553241A/en
Priority to MX2007002390A priority patent/MX2007002390A/es
Priority to EP05793904A priority patent/EP1791948B1/en
Priority to CA2579643A priority patent/CA2579643C/en
Priority to BRPI0514778-6A priority patent/BRPI0514778B1/pt
Priority to JP2007531300A priority patent/JP5420837B2/ja
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Priority to AT05793904T priority patent/ATE549395T1/de
Priority to GB0706626A priority patent/GB2432846B/en
Priority to AU2005282510A priority patent/AU2005282510B2/en
Publication of WO2006029197A1 publication Critical patent/WO2006029197A1/en
Priority to IL181437A priority patent/IL181437A/en
Priority to IS8618A priority patent/IS8618A/is
Anticipated expiration legal-status Critical
Priority to NO20071576A priority patent/NO344695B1/no
Ceased legal-status Critical Current

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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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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
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
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    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
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    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/98—Xeno-free medium and culture conditions
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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
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    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/80—Neurotransmitters; Neurohormones
    • C12N2501/845—Gamma amino butyric acid [GABA]

Definitions

  • Stem cells are defined as cells that are capable of differentiation into many other differentiated cell types.
  • Embryonic stem cells are stem cells from embryos which are capable of differentiation into most, if not all, of the differentiated cell types of a mature body.
  • Stem cells are referred to as pluripotent, which describes this capability of differentiating into many cell types.
  • a category of pluripotent stem cell of high interest to the research community is the human embryonic stem cell, abbreviated here as human ES cell, which is an embryonic stem cell derived from a human embryonic source.
  • Human embryonic stem cells are of great scientific interest because they are capable of indefinite proliferation in culture and are thus capable, at least in principle, of supplying cells and tissues for replacement of failing or defective human tissue.
  • human embryonic stem cells offer the potential of unlimited amounts of human cells and tissues for use in a variety of therapeutic protocols to assist in human health. It is envisioned in the future human embryonic stem cells will be proliferated and directed to differentiate into specific lineages so as to develop differentiated cells or tissues which can be transplanted into human bodies for therapeutic purposes. Human embryonic stem cells and the differentiated cells that may be derived from them are also powerful scientific tools for the study of human cellular and developmental systems.
  • the fibroblast feeder cells acts, through some as yet incompletely understood mechanism, to encourage the stem cells to remain in an undifferentiated state. Later, it was discovered that the same phenomenon could be achieved if the stem cells were exposed to "conditioned media.”
  • Conditioned medium is a stem cell culture medium with which feeder cells, such as MEFs, had been previously been cultured. Either the feeder cells imparted some factor to the medium or removed some factor from the medium, but the result is that conditioned medium can be used to culture stem cells without differentiation. Either culture condition, the direct growth of human ES on murine feeder cells, or the use of conditioned media, raises the concern that one or more agents such as a virus could transmit from the mouse cells to the human ES cells.
  • a characteristic trait of human embryonic stem cells in culture is that if conditions are less than ideal, the cells have a tendency to differentiate. It is easy to induce human ES cells to differentiate while it is demanding to maintain the human ES cells in undifferentiated state in culture. Most culture conditions will results in some level of unwanted differentiation, particularly around the periphery of the growing ES cell colony. While ES cells can be cultured with some degree of unwanted differentiation, the objective is to define a culture condition that permits the culture to remain as undifferentiated as possible, i.e. with as few differentiated cells as possible. We believe that we have used particularly stringent standards to define conditions that will support the indefinite culture of undifferentiated ES cell cultures.
  • the state of differentiation of a stem cell culture can be assessed most easily by judging the morphological characteristics of the cells.
  • Undifferentiated stem cells have a characteristic morphology, i.e. small and compact cells with clearly defined cell borders, a morphology which can be easily seen by examination of a stem cell culture under a microscope.
  • cells which have differentiated appear larger and more diffuse, with indistinct borders.
  • the optimal stem cell culture is one that proliferates in the culture vessel with only minimal numbers of cells at the periphery of the culture appearing to be differentiated. With experience, one can judge the status of differentiation and health of human ES cell cultures visually with good accuracy.
  • a biochemical marker that is used to track the status of ES cells as undifferentiated is the presence of the transcription factor Oct4, which has come to be regarded as the most reliable marker of undifferentiated status of ES cells, and which is one of the first markers lost as undifferentiated cells begin to differentiate.
  • the present invention is summarized as a method for culturing human embryonic stem cells without the need for feeder cells or conditioned medium, the method including the step of culturing the human embryonic stem cells in a medium including salts, vitamins, amino acids, glucose, a fibroblast growth factor, gamma amino butyric acid, pipecholic acid, lithium and transforming growth factor beta, all in sufficient amount to maintain the stem cells in an undifferentiated state through multiple culture passages.
  • the present invention is also directed to an in vitro cell culture of human embryonic stem cells cultured in a medium including high levels of a fibroblast growth factor, gamma amino butyric acid, pipecholic acid, lithium and transforming growth factor beta so that the stem cells can be cultured indefinitely in an undifferentiated state without the need for fibroblast feeder cells or conditioned medium.
  • Fig. 1 presents data from experimental work described in the specification below showing that the components of the medium reduce the proportion of differentiated cells in the culture of human ES cells grown in it.
  • Fig. 2 presents a graphical presentation of data showing that the medium with human matrix proteins results in cultured human ES cells which do not exhibit a sialic acid residue of non-human origin.
  • Fig. 3 is a graphical presentation of data showing the high level of undifferentiated cells in the human stem cell culture.
  • Fig. 4 is a graphical representation of data showing that the medium described here results in robust growth of stem cell cultures.
  • a defined and humanized medium for the culture and proliferation of human ES cells typically includes salts, vitamins, a source of glucose, minerals and amino acids.
  • initially stem cell media included serum from one source or another.
  • the serum replacement can be a commercially available product sold for that purpose or can be a formulated mixture of protein, such as serum albumin, vitamins, salts, minerals, a transferrin or transferrin substitute, and insulin or an insulin substitute. This serum replacement component may also be supplemented with selenium.
  • TeSRl a defined serum replacement be used in lieu of serum from any source in culturing human ES cells, in order to avoid the issues of variation in serum constituents and to use media that are as defined as possible.
  • Table 1 lists all the components of our medium, designated TeSRl, by concentration of the constituents.
  • TeSRl medium is comprised of a DMEM/DF12 base, supplemented with human serum albumin, vitamins, antioxidants, trace minerals, specific lipids, and cloned growth factors.
  • one or more of these components can be deleted resulting in human ES cell cultures that will still grow at a loss of purity of undifferentiated status in the cultures. Such cultures may or may not remain stable over many passages. However, it is clear that the combination is sufficient to enable a variety of media that will support the long term culture and proliferation of undifferentiated human ES cells through an indefinite number of passages without feeder cells or conditioned medium.
  • Matrigel TM which is an artificial basement membrane of mouse cell origin, which is supplied as a commercial product free of mouse cells.
  • Another material of human origin also known now to serve a similar purpose is fibronectin, a human glycoprotein which is used in its insoluble form to create a fiber matrix also to serve as a basement membrane for ES cell culture. In our hands a matrix of fibronectin alone was not sufficient.
  • a human matrix material can be made of a combination of the human matrix proteins collagen IV, fibronectin, laminin, and vitronectin and this matrix suffices to support human ES cells indefinitely in an undifferentiated state in the TeSRl medium.
  • the GABA is believed to interact with the GABA receptor, and the scientific literature includes the identification of several molecules which are agonists of that same receptor and might be substituted for GABA in the medium as an equivalent. It is also believed that PA also interacts with the GABA receptor. While both PA and GABA were found to be helpful in the medium at the concentrations used here, it is also envisioned that one or the other of these constituents could be dramatically increased in concentration to obviate the need for the other. [00023] The fibroblast growth factor in higher concentrations (40 to 100 ng/ml) seems to obviated the need for feeder cells.
  • the preferred FGF is basic FGF, also referred to as bFGF and FGF2, but other FGFs including at least FGF4, FGF9, FGFl 7 and FGFl 8 will suffice for this purpose as well. Other FGFs may also work, even if at higher concentrations.
  • ES human embryonic stem
  • the three media defined below are defined, contain no animal cells, and permit the long term culture of undifferentiated human ES cells in an undifferentiated state.
  • An example is also presented of a medium in which the proteins in the medium are all human, to have a "humanized” medium and culture conditions to avoid any possible concerns about sub-cellular products of animal origin.
  • TeSRl medium The constituents of TeSRl medium, which was used for all cultures described here unless otherwise indicated, is set forth in Table 1 below.
  • Our preliminary experiments suggested that undifferentiated human ES cell proliferation was optimal at a pH of 7.2, an osmolality of 350 mOsMol, and an atmosphere of 10%CO 2 /5%O 2 . These conditions were used for all subsequent cultures described here.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Wood Science & Technology (AREA)
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  • Developmental Biology & Embryology (AREA)
  • Reproductive Health (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Transplantation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/US2005/031838 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells Ceased WO2006029197A1 (en)

Priority Applications (15)

Application Number Priority Date Filing Date Title
JP2007531300A JP5420837B2 (ja) 2004-09-08 2005-09-08 胚幹細胞の培地及び培養
ES05793904T ES2383813T3 (es) 2004-09-08 2005-09-08 Método de cultivo y cultivo de células madre embrionarias
AT05793904T ATE549395T1 (de) 2004-09-08 2005-09-08 Medium und kultur von embryonalen stammzellen
MX2007002390A MX2007002390A (es) 2004-09-08 2005-09-08 Medio y cultivo de celulas progenitoras embrionarias.
EP05793904A EP1791948B1 (en) 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells
CA2579643A CA2579643C (en) 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells
BRPI0514778-6A BRPI0514778B1 (pt) 2004-09-08 2005-09-08 Meio para cultivar células tronco embrionárias
HK08103385.9A HK1113171B (en) 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells
NZ553241A NZ553241A (en) 2004-09-08 2005-09-08 Medium and culture of pluripotent stem cells
CN2005800299366A CN101044235B (zh) 2004-09-08 2005-09-08 胚胎干细胞的培养基和培养
GB0706626A GB2432846B (en) 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells
AU2005282510A AU2005282510B2 (en) 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells
IL181437A IL181437A (en) 2004-09-08 2007-02-19 Medium and method for culturing undifferentiated stem cells
IS8618A IS8618A (is) 2004-09-08 2007-03-06 Æti og rækt fósturstofnfrumna
NO20071576A NO344695B1 (no) 2004-09-08 2007-03-26 Fremgangsmåte for dyrking av humane embryonale stamceller i en udifferensiert tilstand, in vitro cellekultur og medium for dyrkning av embryonale stamceller.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US60804004P 2004-09-08 2004-09-08
US60/608,040 2004-09-08

Publications (1)

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WO2006029197A1 true WO2006029197A1 (en) 2006-03-16

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PCT/US2005/031838 Ceased WO2006029197A1 (en) 2004-09-08 2005-09-08 Medium and culture of embryonic stem cells

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US (5) US7449334B2 (enExample)
EP (1) EP1791948B1 (enExample)
JP (3) JP5420837B2 (enExample)
KR (1) KR101264940B1 (enExample)
CN (2) CN101052712A (enExample)
AT (1) ATE549395T1 (enExample)
AU (1) AU2005282510B2 (enExample)
BR (1) BRPI0514778B1 (enExample)
CA (1) CA2579643C (enExample)
ES (1) ES2383813T3 (enExample)
GB (1) GB2432846B (enExample)
IL (1) IL181437A (enExample)
IS (1) IS8618A (enExample)
MX (1) MX2007002390A (enExample)
NO (1) NO344695B1 (enExample)
NZ (1) NZ553241A (enExample)
WO (1) WO2006029197A1 (enExample)

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008007082A3 (en) * 2006-07-12 2008-03-06 Univ Sheffield Cell growth medium
WO2009084662A1 (ja) * 2007-12-28 2009-07-09 Fujirebio Inc. 哺乳動物体細胞用培地及びそのための添加剤
JP2010532173A (ja) * 2007-06-29 2010-10-07 セルラー ダイナミクス インターナショナル， インコーポレイテッド 胚性幹細胞培養のための自動化された方法および装置
JP2011206064A (ja) * 2004-09-08 2011-10-20 Wisconsin Alumni Research Foundation 胚幹細胞の培地及び培養
WO2014006379A1 (en) * 2012-07-04 2014-01-09 University Of Edinburgh Stem cell culture with modulators of the g protein signal transduction pathway
EP2823037A4 (en) * 2012-03-07 2015-09-16 Janssen Biotech Inc DEFINED MEDIA FOR THE EXPANSION AND CARE OF PLURIPOTENTAL STEM CELLS
US9388387B2 (en) 2008-10-31 2016-07-12 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9506036B2 (en) 2010-08-31 2016-11-29 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
EP3098307A1 (en) * 2015-05-28 2016-11-30 Shenzhen Fulixin Health Industry Development Limited A culture medium for hematopoietic stem cells and the applications thereof as well as the stem cells cultire method
US9528090B2 (en) 2010-08-31 2016-12-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9593310B2 (en) 2009-12-23 2017-03-14 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9593305B2 (en) 2008-06-30 2017-03-14 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US9752125B2 (en) 2010-05-12 2017-09-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9752126B2 (en) 2008-10-31 2017-09-05 Janssen Biotech, Inc. Differentiation of human pluripotent stem cells
US9969972B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Pluripotent stem cell culture on micro-carriers
US9969973B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Methods and compositions for cell attachment and cultivation on planar substrates
US9969981B2 (en) 2010-03-01 2018-05-15 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9969982B2 (en) 2007-11-27 2018-05-15 Lifescan, Inc. Differentiation of human embryonic stem cells
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