WO2003035852A1 - Melange pour differencier des cellules souches ou des cellules souches neuronales dans des cellules neuronales - Google Patents

Melange pour differencier des cellules souches ou des cellules souches neuronales dans des cellules neuronales Download PDF

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Publication number
WO2003035852A1
WO2003035852A1 PCT/EP2002/011512 EP0211512W WO03035852A1 WO 2003035852 A1 WO2003035852 A1 WO 2003035852A1 EP 0211512 W EP0211512 W EP 0211512W WO 03035852 A1 WO03035852 A1 WO 03035852A1
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WIPO (PCT)
Prior art keywords
mixture according
cells
mixture
further factor
factor
Prior art date
Application number
PCT/EP2002/011512
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German (de)
English (en)
Inventor
Hans Werner MÜLLER
Claudia Rosenbaum
Original Assignee
Kourion Therapeutics Ag
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.)
Filing date
Publication date
Application filed by Kourion Therapeutics Ag filed Critical Kourion Therapeutics Ag
Priority to EP02774714A priority Critical patent/EP1438391A1/fr
Publication of WO2003035852A1 publication Critical patent/WO2003035852A1/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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • 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
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]

Definitions

  • the present invention relates to a mixture for differentiating stem cells or neural progenitor cells into neuronal cells.
  • the development of the nervous system is a finely tuned process that results in the maturation of cellular elements, neurons, astrocytes and oligodendrocytes. All of these cell types arise from initially multipotent precursors, which lose their potential in the course of differentiation.
  • Neural progenitor cells and functional postmitotic neurons can be derived in vitro from embryonic stem cells (Okabe et al., 1996), neuronal progenitor cells such as the human teratocarcinoma cell line NT2 (Stratagene) or neural stem cells from adult central nervous system tissue (H.
  • the differentiation protocols are time-consuming (several weeks to months) and comprise several stages, each of which is characterized by specific mitogenic factors and / or growth factors (Lee et al., Nature Biotechnology 18, 675 (2000), Stratagene, Instruction Manual, S. Okabe et al., Mechanisms of Development 59, 89 (1996)).
  • Some protocols even require the stem cells to be grown on so-called “feeder layers”, which preferably consist of fibroblasts (T. Wakayama et al., Science 292, 740 (2001)).
  • feeder layers which preferably consist of fibroblasts (T. Wakayama et al., Science 292, 740 (2001)).
  • stem cells or neural progenitor cells are to be differentiated into neuronal cells in a shorter time with less effort.
  • a medium for differentiating stem cells in neurons is disclosed.
  • This medium which can also be DMEM, contains 10 ⁇ M retinoic acid, 0.25 mM IBMX, 100 ng / ml FGF1 as well as 200 nM TPA and 50 ⁇ M forskulin.
  • Brain Research 912, (2001), pp. 99-104 describes HAM's F12 and ETS as part of the media. This are not essential differentiating factors found according to the present invention.
  • the technical problem on which the present invention is based is the provision of a mixture which makes it possible to differentiate stem cells or neural progenitor cells into neuronal cells.
  • the figure shows immunostaining on differentiated NT2 cells. Neurons were generated from the progenitor cells in the mixture described within approximately 14 days. Neurons were identified using specific marker expression (neurofilament), the detection of neurotransmitters (GABA) or neurotransmitter-synthesizing enzymes. (Tyrosine hydroxylase) as well as proteins responsible for synaptic transmission (synaptophysin).
  • NT2 cells in the mixture according to the invention already differentiate after 6 to 14 days and not only, as in the Stratagene protocol, after a 5-week pre-differentiation in retinoic acid and subsequent cultivation with antimitotic agents for 10 days .
  • TH thyrosine hydroxylase
  • the mixture according to the invention preferably contains, as a further factor (d), at least one activator of adenylate cyclase, cAMP and / or at least one derivative of cAMP which has improved cell uptake.
  • At least one nerve growth factor is present in the mixture as a further factor (s).
  • Nerve growth factors serve to support neurite growth and cell maturation.
  • the mixture according to the invention is particularly suitable for differentiating embryonic and adult stem cells, neural precursor cells and tumor cells.
  • the mixture according to the invention can also be used for the differentiation of neural progenitor cells of the NT2, SHSY-5Y type.
  • Human progenitor cells that have emerged from tumor cells can also be differentiated into neuronal cells.
  • the base medium is preferably DMEM (Dulbecco's modified Eagle Medium).
  • the supplementary medium is preferably FCS, or BCS, or HS, or NGS, in a concentration of 0.5% to 25.
  • the mixture according to the invention can advantageously be used to produce a nutrient medium for differentiating stem cells or neural progenitor cells.
  • the mixture according to the invention is dissolved or taken up in buffer solution in order to obtain an essentially aqueous solution.
  • the mixture according to the invention preferably contains all-trans retinoic acid as a further factor (a).
  • the further factor (b) in the mixture according to the invention is preferably 3-isobutyl-1-methylxanthine (IBMX). This substance serves to inhibit phosphodiesterase and thus to an increase in the intracellular cAMP level.
  • IBMX 3-isobutyl-1-methylxanthine
  • the further factor is in particular (d) dibutyryladenosine cyclomonophosphate, forskolin, 8-bromo-cAMP.
  • the further factor (s) in the mixture according to the invention is in particular ⁇ -NGF, BDNF, NT3, NT4 / 5, CNTF, GDNF, HGF, BMP4 or activin.
  • Mixtures consisting of a mixture of components (a) and (b) and (c) and / or (b) and (c) and (e) can be used as an intermediate product for the preparation of the mixture according to the invention.
  • the other factors in the mixture according to the invention are preferably present in the following concentrations in the aqueous phase: the further factor (a) in a concentration of 0.01 to 100 ⁇ M, the further factor (b) in a concentration of 0.001 to 10 mM, the further factor (c) in a concentration of 1 to 50 ng / ml, the further factor (d) in a concentration of 1 ⁇ M to 10 mM, the other Factor (s) in a concentration of 5 to 500 ng / ml.
  • the mixture according to the invention further contains sonic hedgehoc (SHH) and / or dopamine and / or TPA (phorbol myristate acetate).
  • the figure shows immunostaining on differentiated NT2 cells:
  • a / B 20x / 40x magnification of a stain with an anti-GABA antibody
  • C / D 20x / 40x magnification of a color with an anti-neurofilament antibody cocktail
  • E / F 20-fold magnification of a staining with an anti-synaptophysin antibody
  • G / H 20x / 40x magnification of a stain with an anti-tyrosine hydroxylase antibody.
  • NT2 Neuronal Precursor Cells Human NT2 precursor cells (NT2 Neuronal Precursor Cells, Stratagene) were grown exactly according to the protocol of the Stratagene company and kept in culture. For the differentiation of the cells these were with a density of
  • Fixation was carried out for 15 minutes in 4% paraformaldehyde (except for GABA / tyrosine hydroxylase staining: 5 minutes in 4% paraformaldehyde / 0.05% glutaraldehyde (Merck), 20 minutes in ethanolamine (Sigma)). This was followed by blocking in 10% NGS (Sigma) / 0.03% Triton X 100 (Merck) and incubation at 4 ° C.

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Neurology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurosurgery (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un mélange servant à différencier des cellules souches ou des cellules souches neuronales dans des cellules neuronales et comprenant des substances d'un agent de base, d'un agent complémentaire et d'autres facteurs. L'invention est caractérisée en ce que les autres facteurs contenus dans le mélange sont sélectionnés dans le groupe constitué par (a) de l'acide rétinoïque et (b) au moins un inhibiteur de la phosphodiestérase cAMP combiné à des (c) facteurs de croissance de fibroblaste.
PCT/EP2002/011512 2001-10-20 2002-10-15 Melange pour differencier des cellules souches ou des cellules souches neuronales dans des cellules neuronales WO2003035852A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02774714A EP1438391A1 (fr) 2001-10-20 2002-10-15 Melange pour differencier des cellules souches ou des cellules souches neuronales dans des cellules neuronales

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10152264 2001-10-20
DE10152264.9 2001-10-20

Publications (1)

Publication Number Publication Date
WO2003035852A1 true WO2003035852A1 (fr) 2003-05-01

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PCT/EP2002/011512 WO2003035852A1 (fr) 2001-10-20 2002-10-15 Melange pour differencier des cellules souches ou des cellules souches neuronales dans des cellules neuronales

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EP (1) EP1438391A1 (fr)
WO (1) WO2003035852A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1619244A1 (fr) * 2004-06-23 2006-01-25 Henrich Cheng Méthode pour l'induction de la différentiation neurale

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053465A1 (fr) * 2000-01-21 2001-07-26 The Johns Hopkins University School Of Medicine Cellules issues de corps embryoides humains
US6294346B1 (en) * 1991-07-08 2001-09-25 Neurospheres Holdings, Ltd. Use of multipotent neural stem cells and their progeny for the screening of drugs and other biological agents

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294346B1 (en) * 1991-07-08 2001-09-25 Neurospheres Holdings, Ltd. Use of multipotent neural stem cells and their progeny for the screening of drugs and other biological agents
WO2001053465A1 (fr) * 2000-01-21 2001-07-26 The Johns Hopkins University School Of Medicine Cellules issues de corps embryoides humains

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
IACOVITTE L ET AL: "DIFFERENTIATION OF HUMAN DOPAMINE NEURONS FROM AN EMBRYONIC CARCINOMAL STEM CELL LINE", BRAIN RESEARCH, AMSTERDAM, NL, vol. 912, 2001, pages 99 - 104, XP002902584, ISSN: 0006-8993 *
IACOVITTI L ET AL: "The differentiation of dopamine neurons from stem/ precursor cells in culture and in vivo.", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 26, no. 1-2, 2000, 30th Annual Meeting of the Society of Neuroscience;New Orleans, LA, USA; November 04-09, 2000, pages Abstract No. - 312.21, XP001040605, ISSN: 0190-5295 *
WAGNER J ET AL: "Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes", NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, vol. 17, July 1999 (1999-07-01), pages 653 - 659, XP002154887, ISSN: 1087-0156 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1619244A1 (fr) * 2004-06-23 2006-01-25 Henrich Cheng Méthode pour l'induction de la différentiation neurale

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Publication number Publication date
EP1438391A1 (fr) 2004-07-21

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