[go: up one dir, main page]

WO2003035852A1 - Mixture used to differentiate stem cells or neuronal progenitor cells in neuronal cells - Google Patents

Mixture used to differentiate stem cells or neuronal progenitor cells in neuronal cells Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
mixture according
cells
mixture
further factor
factor
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2002/011512
Other languages
German (de)
French (fr)
Inventor
Hans Werner MÜLLER
Claudia Rosenbaum
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KOURION THERAPEUTICS AG
PerkinElmer Chemagen Technologie GmbH
Original Assignee
KOURION THERAPEUTICS AG
Kourion Therapeutics GmbH
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, Kourion Therapeutics GmbH filed Critical KOURION THERAPEUTICS AG
Priority to EP02774714A priority Critical patent/EP1438391A1/en
Publication of WO2003035852A1 publication Critical patent/WO2003035852A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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.

Landscapes

  • 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

The invention relates to a mixture that is used to differentiate stem cells or neuronal progenitor cells in neuronal cells. The mixture contains the constituents of a base medium, a supplementary medium and other factors. The factors contained in the mixture are selected from the group including (a) retinoic acid and (b) at least one inhibitor of the cAMP phosphodiesterase combined with (c) fibroblast growth factors.

Description

Mischung zur Differenzierung von Stammzellen oder neuralen Progenitorzellen in neuronale ZellenMixture for differentiating stem cells or neural progenitor cells into neuronal cells

Die vorliegende Erfindung betrifft eine Mischung zur Differenzierung von Stammzellen oder neuralen Progenitorzellen in neuronale Zellen. Die Entwicklung des Nervensystems ist ein fein abgestimmter Prozess, der in der Reifung der zellulären Elemente, Neurone, Astrozyten und Oligodendrozyten, resultiert. All diese Zelltypen entstehen aus zunächst multipotenten Vorläufern, die im Laufe der Differenzierung ihr Potenzial verlieren. Neuronale Vorläuferzellen und funktionelle postmitotische Neuronen können in vitro aus embryonalen Stammzellen (Okabe et al., 1996), neuronalen Vorläuferzellen wie z.B. der humanen Teratocarzinomzellinie NT2 (Stratagene) oder neuralen Stammzellen aus adultem Gewebe des Zentralnervensystems (H. Toda et al., Experimental Neurology 165, 66 (2000)) gewonnen werden. Die Differenzierungsprotokolle sind zeitaufwendig (mehrere Wochen bis Monate) und umfassen mehrere Stadien, die jeweils durch spezifische mitogene Faktoren und/oder Wachstumsfaktoren gekennzeichnet sind (Lee et al., Nature Biotechnology 18, 675 (2000), Stratagene, Instruction Manual, S. Okabe et al., Mechanisms of Development 59, 89 (1996)). Einige Protokolle erfordern sogar eine Anzucht der Stammzellen auf sogenannten "feeder layern", die vorzugsweise aus Fibroblasten bestehen (T. Wakayama et al., Science 292,740 (2001)). Mit Hilfe der vorliegenden Erfindung sollen Stammzellen bzw. neurale Vorläuferzellen in kürzerer Zeit mit weniger Aufwand in neuronale Zellen differenziert werden. In Society for Neuroscience Abstracts (2000) Vol. 26, No. 1 - 2, Abstract No. 312.21 sowie Brain Research 912, (2001), S. 99 - 104 ist ein Medium zur Differenzierung von Stammzellen in Neuronen offenbart. Dieses Medium, das auch DMEM sein kann, enthält 10 μM Retinsäure, 0,25 mM IBMX, 100 ng/ml FGF1 sowie 200 nM TPA und 50 μM Forskulin. Brain Research 912, (2001), S. 99 - 104 beschreibt als Bestandteil der Medien HAM 's F12 sowie ETS. Dies sind keine wesentlichen Differenzierungsfaktoren, die gemäß vorliegender Erfindung gefunden wurde.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. Toda et al., Experimental Neurology 165, 66 (2000)). 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)). With the help of the present invention, stem cells or neural progenitor cells are to be differentiated into neuronal cells in a shorter time with less effort. In Society for Neuroscience Abstracts (2000) Vol. 26, No. 1 - 2, Abstract No. 312.21 and Brain Research 912, (2001), pp. 99-104, a medium for differentiating stem cells in neurons is disclosed. This medium, which can also be DMEM, contains 10 μM retinoic acid, 0.25 mM IBMX, 100 ng / ml FGF1 as well as 200 nM TPA and 50 μM 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.

Das der vorliegenden Erfindung zu Grunde liegende technische Problem ist die Bereitstellung einer Mischung, die es erlaubt, Stammzellen oder neurale Progenitorzellen in neuronale Zellen zu differenzieren.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.

Gelöst wird dieses technische Problem durch eine Mischung mit Inhaltsstoffen eines Basismediums, Ergänzungsmediums sowie weiterer Faktoren, die dadurch gekennzeichnet ist, dass die weiteren Faktoren ausgewählt sind aus der Gruppe bestehend aus (a) Retinsäure undThis technical problem is solved by a mixture with ingredients of a basic medium, supplementary medium and other factors, which is characterized in that the other factors are selected from the group consisting of (a) retinoic acid and

(b) mindestens einem Inhibitor der cAMP-Phosphodiesterase in Kombination mit (c) Fibroblastenwachstumsfaktoren in der Mischung vorhanden sind.(b) at least one inhibitor of cAMP phosphodiesterase in combination with (c) fibroblast growth factors are present in the mixture.

Die Figur zeigt Immunfärbungen an differenzierten NT2 Zellen. Aus den Vorläuferzellen sind innerhalb von ca. 14 Tagen in der beschriebenen Mischung Neurone generiert worden. Neurone wurden mittels spezifischer Markerexpression (Neurofilament), dem Nachweis von Neurotransmitter (GABA) bzw. Neurotransmitter-synthetisierenden Enzymen -. (Tyrosinhydroxylase) sowie Proteinen, die für die synaptische Transmission (Synaptophysin) verantwortlich sind, identifiziert.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).

Des weiteren wird erfindungsgemäß beobachtet, dass im Gegensatz zu dem Stratagene Protokoll NT2 Zellen in der erfindungsgemäßen Mischung bereits nach 6 bis 14 Tagen differenzieren und nicht erst wie gemäß Stratagene Protokoll nach einer 5-wöchigen Vordifferenzierung in Retinsäure und anschließender 10-tägiger Kultivierung mit antimitotischen Agenzien. In dem Abstract Society for Neuroscience Abstracts (2000) Vol. 26, No. 1 - 2, Abstract No. 312.21 wird angegeben, dass die dort benutzten Zellen zur Differenzierung in Thyrosin-Hydroxylase (TH) exprimierende Zellen zunächst in NSE+ Neurone und danach mit einem weiteren Medium TH-positive dopaminergen Neurone differenzieren. Danach ist ein Zweistufenverfahren erforderlich, um zu TH positiven dopaminergen Zellen zu gelangen. Erfindungsgemäß hingegen werden Differenzierungen in einem einheitlichen Verfahren innerhalb von 6 bis 14 Tagen erreicht.Furthermore, it is observed according to the invention that, in contrast to the Stratagene protocol, 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 , In the Abstract Society for Neuroscience Abstracts (2000) Vol. 26, No. 1 - 2, Abstract No. 312.21 it is stated that the cells used there for differentiation into thyrosine hydroxylase (TH) expressing cells first differentiate into NSE + neurons and then with another medium TH-positive dopaminergic neurons. After that, a two-step process is required to get to TH positive dopaminergic cells. In contrast, according to the invention, differentiations are achieved within 6 to 14 days in a uniform process.

Vorzugsweise enthält die erfindungsgemäße Mischung als weiteren Faktor (d) mindestens einen Aktivator der Adenylatcyclase, cAMP und/oder mindestens ein Derivat des cAMP, das eine verbesserte Zellaufnahme besitzt.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.

Diese Faktoren dienen der Aktivierung von Proteinkinasen und somit einer Stimulierung von Signalkaskaden.These factors serve to activate protein kinases and thus stimulate signal cascades.

In einer weiteren Ausführungsform der Erfindung ist in der Mischung als weiterer Faktor (e) mindestens ein Nervenwachstumsfaktor vorhanden.In a further embodiment of the invention, at least one nerve growth factor is present in the mixture as a further factor (s).

Nervenwachstumsfaktoren dienen der Unterstützung des Neuritenwachstums und der Zellreifung.Nerve growth factors serve to support neurite growth and cell maturation.

Die erfindungsgemäße Mischung ist insbesondere zur Differenzierung von embryonalen und adulten Stammzellen, neuralen Vorläuferzellen und Tumorzellen geeignet.The mixture according to the invention is particularly suitable for differentiating embryonic and adult stem cells, neural precursor cells and tumor cells.

Es handelt sich hierbei um eine generelle Mischung mit einem breiten Spektrum an Zielzellen, die spezifisch auf den neuronalen Phänotyp ausgerichtet ist.It is a general mixture with a broad spectrum of target cells that is specifically geared towards the neuronal phenotype.

Die erfindungsgemäße Mischung kann auch zur Differenzierung von neuralen Progenitorzellen des Typs NT2, SHSY-5Y verwendet werden.The mixture according to the invention can also be used for the differentiation of neural progenitor cells of the NT2, SHSY-5Y type.

Auch Humane Progenitorzellen, die aus Tumorzellen hervorgegangen sind, lassen sich in neuronale Zellen differenzieren.Human progenitor cells that have emerged from tumor cells can also be differentiated into neuronal cells.

Vorzugsweise ist das Basismedium DMEM (Dulbecco's modified Eagle Medium).The base medium is preferably DMEM (Dulbecco's modified Eagle Medium).

In der erfindungsgemäßen Mischung ist das Ergänzungsmedium vorzugsweise FCS, oder BCS, oder HS, oder NGS, in einer Konzentration von 0,5 % bis 25 .In the mixture according to the invention, the supplementary medium is preferably FCS, or BCS, or HS, or NGS, in a concentration of 0.5% to 25.

Im Serum finden sich wichtige niedermolekulare Substanzen und Wachstumsfaktoren, die dem Überleben der Zellen dienen. Die erfindungsgemäße Mischung kann vorteilhafterweise zur Herstellung eines Nährmediums zur Differenzierung von Stammzellen oder neuralen Progenitorzellen verwendet werden. Dazu wird die erfindungsgemäße Mischung in Pufferlösung gelöst oder aufgenommen, um eine im wesentlichen wäßrige Lösung zu erhalten.Important low-molecular substances and growth factors are found in the serum, which serve the survival of the cells. The mixture according to the invention can advantageously be used to produce a nutrient medium for differentiating stem cells or neural progenitor cells. For this purpose, the mixture according to the invention is dissolved or taken up in buffer solution in order to obtain an essentially aqueous solution.

- Vorzugsweise enthält die erfindungsgemäße Mischung all-trans Retinsäure als weiteren Faktor (a).- The mixture according to the invention preferably contains all-trans retinoic acid as a further factor (a).

Der weitere Faktor (b) in der erfindungsgemäßen Mischung ist vorzugsweise 3- Isobutyl-1-methylxanthin (IBMX) Diese Substanz dient der Inhibierung der Phosphodiesterase und somit zu einem Anstieg des intrazellulären cAMP- Spiegels.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.

Der weitere Faktor (c) in der erfindungsgemäßen Mischung ist vorzugsweise Fibroblast growth factor 2 (FGF2 = bFGF), FGF 1 (=aFGF), FGF 8, FGF4. Diese Faktoren sind als neurotrophe Faktoren mit teilweise mitogenen Funktionen beschrieben.The further factor (c) in the mixture according to the invention is preferably fibroblast growth factor 2 (FGF2 = bFGF), FGF 1 (= aFGF), FGF 8, FGF4. These factors are described as neurotrophic factors with partially mitogenic functions.

Der weitere Faktor ist insbesondere (d) Dibutyryladenosin-cyclo- monophosphat, Forskolin, 8-Brom-cAMP.The further factor is in particular (d) dibutyryladenosine cyclomonophosphate, forskolin, 8-bromo-cAMP.

Diese Substanzen dienen der Anhebung des intrazellulären cAMP-Spiegels.These substances serve to raise the intracellular cAMP level.

Der weitere Faktor (e) in der erfindungsgemäßen Mischung ist insbesondere ß- NGF, BDNF, NT3, NT4/5 , CNTF, GDNF, HGF, BMP4 oder Activin.The further factor (s) in the mixture according to the invention is in particular β-NGF, BDNF, NT3, NT4 / 5, CNTF, GDNF, HGF, BMP4 or activin.

Diese Substanzen dienen u. a. dem Überleben und der Differenzierung der Zellen.These substances serve a. the survival and differentiation of cells.

Als Zwischenprodukt zur Herstellung der erfindungsgemäßen Mischung können Mischungen bestehend aus einem Gemisch der Komponenten, (a) und (b) und (c) und/oder (b) und (c) und (e) eingesetzt werden.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.

Die weiteren Faktoren liegen in der erfindungsgemäßen Mischung in der wässrigen Phase vorzugsweise in den folgenden Konzentrationen vor: der weitere Faktor (a) in einer Konzentration von 0,01 bis 100 μM, der weitere Faktor (b) in einer Konzentration von 0,001 bis 10 mM, der weitere Faktor (c) in einer Konzentration von 1 bis 50 ng/ml, der weitere Faktor (d) in einer Konzentration von 1 μM bis 10 mM, der weitere Faktor (e) in einer Konzentration von 5 bis 500 ng/ml. In einer bevorzugten Ausführungsform enthält die erfindungsgemäße Mischung weiterhin Sonic hedgehoc (SHH) und/oder Dopamin und/oder TPA (Phorbol myristate acetate).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 μM, 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 μM to 10 mM, the other Factor (s) in a concentration of 5 to 500 ng / ml. In a preferred embodiment, the mixture according to the invention further contains sonic hedgehoc (SHH) and / or dopamine and / or TPA (phorbol myristate acetate).

Diese Substanzen dienen der Erzeugung neuronaler Subtypen, wie z.B. dopaminerge Neurone. Die Figur zeigt Immunfärbungen an differenzierten NT2 Zellen:These substances are used to create neuronal subtypes, e.g. dopaminergic neurons. The figure shows immunostaining on differentiated NT2 cells:

A/B: 20-fache/40-fache Vergrößerung einer Färbung mit einem anti-GABA Antikörper; C/D: 20-fache/40-fache Vergrößerung einer Färbung mit einem anti-Neurofilament Antikörpercocktail; E/F: 20-fache Vergrößerung einer Färbung mit einem anti-Synaptophysin Antikörper; G/H: 20-fache/40-fache Vergrößerung einer Färbung mit einem anti-Tyrosinhydroxylase Antikörper.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.

Die Erfindung wird an Hand der folgenden Beispiele näher erläutert:The invention is illustrated by the following examples:

Beispielexample

Humane NT2 Vorläuferzellen (NT2 Neuronal Precursor Cells, Stratagene) wurden genau nach dem Protokoll der Fa. Stratagene angezogen und in Kultur gehalten. Für die Differenzierung der Zellen wurden diese mit einer Dichte vonHuman 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

15 000 Zellen/cm 2 auf mit PDL (Sigma, 10 μg/ml) und Laminin (Cell Systems, 13 μg/ml) beschichteten Glas-Coverslips in 24-well-Platten ausgesät. Die15,000 cells / cm 2 are seeded on glass cover slip coated with PDL (Sigma, 10 μg / ml) and laminin (Cell Systems, 13 μg / ml) in 24-well plates. The

Inkubation erfolgte 6-14 Tage lang in XXL-Medium: (DMEM high Glucose, Glutamax (Gibco), 15 % FCS (hitzeinaktiviert) (PAA), Penicillin/StreptomycinIncubation took place for 6-14 days in XXL medium: (DMEM high glucose, Glutamax (Gibco), 15% FCS (heat-inactivated) (PAA), penicillin / streptomycin

(Gibco), 10 μM all trans Retinsäure (Sigma), 0,5 mM IBMX (Sigma), ImM(Gibco), 10 µM all trans retinoic acid (Sigma), 0.5 mM IBMX (Sigma), ImM

Dibutyryladenosin cyclisches Monophosphat (Sigma), 50ng/ml ßNGF (CellDibutyryladenosine cyclic monophosphate (Sigma), 50ng / ml ßNGF (Cell

Systems), 20ng/ml bFGF (Cell Systems) in einem 5 % CO2-Inkubator. Ein Mediumwechsel erfolgte alle zwei Tage. Anschließend wurden die Zellen zur Immuncytochemie verwendet.Systems), 20ng / ml bFGF (Cell Systems) in a 5% CO 2 incubator. On Medium change took place every two days. The cells were then used for immunocytochemistry.

Die Fixierung erfolgte für 15 Minuten in 4 % Paraformaldehyd (außer für die GABA/Tyrosinhydroxylase-Färbung : 5 Minuten in 4 % Paraformaldehyd / 0,05 % Glutaraldehyd (Merck), 20 Minuten IM Ethanolamin (Sigma)). Es folgte eine Blockierung in 10 % NGS (Sigma) / 0.03 % Triton X 100 (Merck) und eine 24- stündige Inkubation bei 4 °C mit dem ersten Antikörper (polyklonaler anti- GABA: (Sigma, 1:1000), monoklonaler anti-Tyrosin Hydroxylase (Sigma, 1: 100), monoklonaler anti-Synaptophysin (Sigma, 1: 100), polyklonaler anti- Neurofilamentcocktail (Bio Trend, 1:1000). Als Zweit-Antikörper wurden Rhodamin-RedX-goat anti-rabbit ( MoBiTec, 1: 1000) bzw FITC-konjugiertem goat-anti-mouse (Southern Biotechnology Associates, 1: 100) verwendet und ebenfalls für 24 Stunden im Dunkeln bei 4° C inkubiert. Die Glascoverslips wurden mit Citifluor (Citifluor Ltd.) auf Glas-Objektträger montiert. Mikroskopie und Bildgebung erfolgten an einem Nikon-Mikroskop. 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. for 24 hours with the first antibody (polyclonal anti-GABA: (Sigma, 1: 1000), monoclonal anti -Tyrosine hydroxylase (Sigma, 1: 100), monoclonal anti-synaptophysin (Sigma, 1: 100), polyclonal anti-neurofilament cocktail (Bio Trend, 1: 1000). Rhodamine-RedX-goat anti-rabbit ( MoBiTec, 1: 1000) or FITC-conjugated goat-anti-mouse (Southern Biotechnology Associates, 1: 100) was used and also incubated for 24 hours in the dark at 4 ° C. The glass coverslips were applied to glass with Citifluor (Citifluor Ltd.) - Slide mounted, microscopy and imaging performed on a Nikon microscope.

Claims

Patentansprüche claims 1. Mischung zur Differenzierung von Stammzellen oder neuralen Progenitorzellen in neurale Zellen mit Inhaltsstoffen eines Basismediums, Ergänzungsmediums sowie weiterer Faktoren, dadurch gekennzeichnet, dass die weiteren Faktoren ausgewählt sind aus der Gruppe bestehend aus1. Mixture for differentiating stem cells or neural progenitor cells into neural cells with ingredients of a basic medium, supplementary medium and further factors, characterized in that the further factors are selected from the group consisting of (a) Retinsäure und(a) retinoic acid and (b) mindestens einem Inhibitor der cAMP-Phosphodiesterase in Kombination mit (c) Fibroblastenwachstumsfaktoren in der Mischung vorhanden sind.(b) at least one inhibitor of cAMP phosphodiesterase in combination with (c) fibroblast growth factors are present in the mixture. 2. Mischung nach Anspruch 1, dadurch gekennzeichnet, dass als weiterer Faktor (d) mindestens ein Aktivator der Adenylatcyclase, cAMP und/oder mindestens einem Derivat des cAMP, das eine verbesserte Zellaufnahme besitzt vorhanden ist. 2. Mixture according to claim 1, characterized in that at least one activator of adenylate cyclase, cAMP and / or at least one derivative of cAMP, which has an improved cell uptake, is present as a further factor (d). 3. Mischung nach Anspruch 1 und/oder 2, dadurch gekennzeichnet, dass als weiterer Faktor (e) mindestens ein Nervenwachstumsfaktor vorhanden ist.3. Mixture according to claim 1 and / or 2, characterized in that at least one nerve growth factor is present as a further factor (s). 4. Mischung nach mindestens einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die Stammzellen embryonale Stammzellen oder adulte Stammzellen sind.4. Mixture according to at least one of claims 1 to 3, characterized in that the stem cells are embryonic stem cells or adult stem cells. 5. Mischung nach mindestens einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die neuralen Progenitorzellen Primärzellen oder Zelllinien sind5. Mixture according to at least one of claims 1 to 3, characterized in that the neural progenitor cells are primary cells or cell lines 6. Mischung • nach mindestens einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass das Basismedium DMEM ist.6. Mixture • according to at least one of claims 1 to 5, characterized in that the base medium is DMEM. 7. Mischung nach mindestens einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass das Ergänzungsmedium FCS, BCS, HS (Humane Seren), NGS ist. 7. Mixture according to at least one of claims 1 to 6, characterized in that the supplementary medium is FCS, BCS, HS (human sera), NGS. 8. Mischung nach mindestens einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass die Mischung in wässriger Lösung vorliegt.8. Mixture according to at least one of claims 1 to 7, characterized in that the mixture is in aqueous solution. 9. Mischung nach mindestens einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass die Retinsäure in der all-trans Konstitution vorliegt. 10. Mischung nach mindestens einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, dass der weitere Faktor (b) 3-Isobutyl-l-methylxanthin (IBMX) ist.9. Mixture according to at least one of claims 1 to 8, characterized in that the retinoic acid is present in the all-trans constitution. 10. Mixture according to at least one of claims 1 to 9, characterized in that the further factor (b) is 3-isobutyl-1-methylxanthine (IBMX). 11. Mischung nach mindestens einem der Ansprüche 1 bis 10, dadurch gekennzeichnet, dass der weitere Faktor (c) Fibroblast growth factor 2 (bFGF),FGF 1 (aFGF), FGF4, FGF 8 ist.11. Mixture according to at least one of claims 1 to 10, characterized in that the further factor (c) is fibroblast growth factor 2 (bFGF), FGF 1 (aFGF), FGF4, FGF 8. 12. Mischung nach mindestens einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, dass der weitere Faktor (d) Dibutyryladenosin-cyclo- monophosphat, Forskolin, 8-Brom-cAMP ist.12. Mixture according to at least one of claims 1 to 11, characterized in that the further factor (d) is dibutyryladenosine cyclo-monophosphate, forskolin, 8-bromo-cAMP. 13. Mischung nach mindestens einem der Ansprüche 1 bis 12, dadurch gekennzeichnet, dass der weitere Faktor (e) ß-NGF, BDNF, NT3, NT4/5,13. Mixture according to at least one of claims 1 to 12, characterized in that the further factor (s) β-NGF, BDNF, NT3, NT4 / 5, CNTF, GDNF, HGF, BMP4 oder Activin ist.CNTF, GDNF, HGF, BMP4 or Activin. 14. Mischung bestehend aus einem Gemisch der Komponenten (a) und (b) und (c) und/oder (b) und (c) und (e) zur Herstellung einer Mischung nach einem der Ansprüche 1 bis 14. 15. Mischung gemäß Anspruch 8 bis 14, dadurch gekennzeichnet, dass der weitere Faktor (a) in einer Konzentration von 0,01 bis 100 μM, der weitere Faktor (b) in einer Konzentration von 0,001 bis 10- mM, der weitere Faktor (c) in einer Konzentration von 1 bis 50 ng/ml, der weitere Faktor (d) in einer Konzentration von lμM bis 10 mM, der weitere Faktor' (e) in einer Konzentration von 5 bis 500 ng/ml in der wässrigen Phase vorliegt.14. Mixture consisting of a mixture of components (a) and (b) and (c) and / or (b) and (c) and (e) for the preparation of a mixture according to one of claims 1 to 14. 15. Mixture according to Claims 8 to 14, characterized in that the further factor (a) in a concentration of 0.01 to 100 μM, the further factor (b) in a concentration of 0.001 to 10 mM, the further factor (c) in one Concentration of 1 to 50 ng / ml, the further factor (d) in a concentration of 1 μm to 10 mM, the further factor '(e) in a concentration of 5 to 500 ng / ml in the aqueous phase. 16. Mischung nach mindestens einem der Ansprüche 1 bis 15, dadurch gekennzeichnet, dass weiterhin Sonic hedgehoc (SHH), und/oder Dopamin, und/oder TPA (Phorbol myristate acetate) vorhanden ist. 16. Mixture according to at least one of claims 1 to 15, characterized in that Sonic hedgehoc (SHH), and / or dopamine, and / or TPA (Phorbol myristate acetate) is also present. 7. Verwendung einer Mischung nach mindestens einem der Ansprüche 1 bis 16 für ein Medium zur Kultivierung von Stammzellen und neuralen Progenitorzellen. 7. Use of a mixture according to at least one of claims 1 to 16 for a medium for the cultivation of stem cells and neural progenitor cells.
PCT/EP2002/011512 2001-10-20 2002-10-15 Mixture used to differentiate stem cells or neuronal progenitor cells in neuronal cells Ceased WO2003035852A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02774714A EP1438391A1 (en) 2001-10-20 2002-10-15 Mixture used to differentiate stem cells or neuronal progenitor cells in neuronal cells

Applications Claiming Priority (2)

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

Publications (1)

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

Family

ID=7703431

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/011512 Ceased WO2003035852A1 (en) 2001-10-20 2002-10-15 Mixture used to differentiate stem cells or neuronal progenitor cells in neuronal cells

Country Status (2)

Country Link
EP (1) EP1438391A1 (en)
WO (1) WO2003035852A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1619244A1 (en) * 2004-06-23 2006-01-25 Henrich Cheng Method for inducing neural differentiation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053465A1 (en) * 2000-01-21 2001-07-26 The Johns Hopkins University School Of Medicine Human embryoid body-derived cells
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 (en) * 2000-01-21 2001-07-26 The Johns Hopkins University School Of Medicine Human embryoid body-derived cells

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 (en) * 2004-06-23 2006-01-25 Henrich Cheng Method for inducing neural differentiation

Also Published As

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

Similar Documents

Publication Publication Date Title
DE69332759T2 (en) BIOLOGICAL FACTORS AND NEURONAL STEM CELLS
DE60129943T2 (en) DIFFERENTIATION OF BONE MARROW CELLS IN NEURONAL CELLS AND THEIR USES
Studer et al. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats
DE69737949T3 (en) ISOLATION, REPRODUCTION AND TARGETED DIFFERENTIATION OF MAMMALIAN CELLS OF THE CENTRAL NERVOUS SYSTEM
DE19756864C1 (en) Production of neuronal or glial progenitor cells
DE69332147T2 (en) REMYELINATION USING NEURONAL STEM CELLS
US7250294B2 (en) Screening small molecule drugs using neural cells differentiated from human embryonic stem cells
Xuan et al. BDNF improves the effects of neural stem cells on the rat model of Alzheimer's disease with unilateral lesion of fimbria-fornix
DiCicco-Bloom et al. NT-3 stimulates sympathetic neuroblast proliferation by promoting precursor survival
Whittemore et al. Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations
Sortwell et al. Oligodendrocyte‐type 2 astrocyte‐derived trophic factors increase survival of developing dopamine neurons through the inhibition of apoptotic cell death
Mujtaba et al. Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells
JP5529561B2 (en) Methods for in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells
Choi et al. Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration
Riaz et al. The differentiation potential of human foetal neuronal progenitor cells in vitro
AU2004257000B2 (en) Oligodendrocyte precursor cells and methods of obtaining and culturing the same
DE60035191T2 (en) MATERIALS AND METHODS FOR THE DEVELOPMENT OF DOPAMINERGEN NEURONES
DE60319599T2 (en) METHOD FOR DIFFERENTIATING A MESENCHYME STEM CELL TO A NERVENZELLE AND THE NERVENZELLE CONTAINING PHARMACEUTICAL COMPOSITION AGAINST A NEURODEGENERATIVE DISEASE
CN110396500B (en) Composition for inducing direct transdifferentiation of fibroblasts to neurons and application thereof
EP1438391A1 (en) Mixture used to differentiate stem cells or neuronal progenitor cells in neuronal cells
Schumm et al. Enhanced viability and neuronal differentiation of neural progenitors by chromaffin cell co-culture
WO2001076507A2 (en) Use of oxygen carriers to improve grafted cell survival in neural transplantation
CN115463158B (en) Uses of induced pluripotent stem cell-derived midbrain organoids
Xu et al. The isoflavone puerarin promotes generation of human iPSC‐derived pre‐oligodendrocytes and enhances endogenous remyelination in rodent models
DE69501165T2 (en) Serum-free medium for the culture of postnatal central neurons

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2002774714

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2002774714

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2002774714

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP