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 PDFInfo
- 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
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.
Abstract
Description
Claims
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 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)
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)
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 |
-
2002
- 2002-10-15 EP EP02774714A patent/EP1438391A1/en not_active Withdrawn
- 2002-10-15 WO PCT/EP2002/011512 patent/WO2003035852A1/en not_active Application Discontinuation
Patent Citations (2)
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)
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)
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 |
---|---|---|
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 | |
DE69332759T3 (en) | BIOLOGICAL FACTORS AND NEURONAL STEM CELLS | |
DE19756864C1 (en) | Production of neuronal or glial progenitor cells | |
US7250294B2 (en) | Screening small molecule drugs using neural cells differentiated from human embryonic stem cells | |
Whittemore et al. | Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations | |
CN103396993B (en) | The oligodendroglia of myelinization again and the treatment for spinal cord injury of derived from primate embryonic stem cells | |
DiCicco-Bloom et al. | NT-3 stimulates sympathetic neuroblast proliferation by promoting precursor survival | |
Kennea et al. | Neural stem cells | |
Engele et al. | Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia | |
Sortwell et al. | Oligodendrocyte‐type 2 astrocyte‐derived trophic factors increase survival of developing dopamine neurons through the inhibition of apoptotic cell death | |
JP5529561B2 (en) | Methods for in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells | |
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 | |
DE60123937T2 (en) | TREATMENT OF NEURO-DEGENERATIVE GASTROINTESTINAL DISEASES BY IMPANTATION OF NEURONAL STEM CELLS AND / OR THEIR DEPARTMENT IN GASTROINTESTINAL ORGANS | |
CN110396500B (en) | Composition for inducing fibroblast to directly transdifferentiate towards neuron 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 | |
Di Porzio et al. | Positive control of target cerebellar cells on norepinephrine uptake in embryonic brainstem cultures in serum-free medium | |
DE19928210B4 (en) | Neuronal cell material and method for its production | |
Tucker et al. | Spinal interneurons and cell transplantation | |
Maxwell et al. | Adrenergic development of neural crest cells grown in a defined medium under a reconstituted basement-membrane-like matrix |
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 |